Ros-responsive selenium-cerium composite nanoszyme and preparation and application thereof

Abstract

The application discloses a ROS response type selenium-cerium composite nanometer enzyme and a preparation and application thereof, and comprises the following steps: S1, preparation of an organic silicon source double-[3-(triethoxy)silyl propyl] diselenide silane; S2, preparation of a mesoporous organic silicon nanometer material bridged by a diselenide bond; S3, preparation of a hyaluronic acid coated cerium dioxide nanometer enzyme; and S4, preparation of the ROS response type selenium-cerium composite nanometer enzyme. Through the three core innovations of embedding a diselenide bond skeleton, preloading HA-CeO2 in a channel and multi-enzyme simulation cooperation, the application systematically solves the key bottlenecks of stability, functionality, targeting and antioxidant efficiency of the existing nanometer platform, and provides a high-efficiency, safe and intelligent drug-free nanometer treatment new strategy for acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a ROS-responsive selenium-cerium composite nanozyme and its preparation and application. Background Technology

[0002] In recent years, nanozymes have attracted widespread attention in the fields of antioxidant and organ protection due to their natural enzyme-like catalytic activity and good stability. Among them, cerium dioxide (CeO2) nanoparticles have gained attention for their reversible CeO2 catalytic activity. 3+ / Ce 4+ Redox pairs exhibit dual mimicry activities of superoxide dismutase (SOD) and catalase (CAT), effectively scavenging various reactive oxygen species (ROS) and are used to treat oxidative stress-related diseases such as acute kidney injury (AKI). However, naked CeO2 tends to accumulate in vivo, has a short circulation time, and lacks selective enrichment ability in damaged kidney tissue, limiting its therapeutic efficacy. To improve targeting, researchers often use hyaluronic acid (HA) to modify nanocarriers, utilizing its specific binding to the CD44 receptor, which is highly expressed on the surface of damaged renal tubular epithelial cells, to achieve active targeting. Meanwhile, mesoporous organosilica nanoparticles (MONs) are widely used as carriers for functional units such as CeO2 due to their high specific surface area, good biocompatibility, and ease of functionalization.

[0003] In existing technologies, HA-modified CeO2 loaded onto mesoporous silica has been reported for AKI treatment. However, this system uses a traditional silane cross-linked framework, lacking responsiveness to ROS in the lesion microenvironment. Drug release relies on passive diffusion, making on-demand drug release difficult. Other studies have introduced disulfide bonds to construct ROS-responsive carriers, but these bonds are not sensitive enough to physiologically relevant concentrations of ROS, typically requiring millimolecular-level oxidants to trigger breakage, resulting in low response efficiency in the real AKI microenvironment. Although selenium (Se) contains glutathione peroxidase (GP... x While it exhibits similar activity and can synergistically enhance antioxidant capacity with CeO2, there is currently no strategy to design the ROS-responsive carrier backbone itself as a functional selenium source. More importantly, most HA-modified systems treat HA as a static coating layer, without linking it to the carrier degradation process. This can lead to the target unit being prematurely exposed or shielded in the bloodstream, affecting CD44 recognition efficiency and causing a disconnect between targeting and therapeutic functions.

[0004] Although some progress has been made in nanozyme delivery and kidney targeting, existing technologies still have certain shortcomings: (1) Insufficient ROS responsiveness: Most mesoporous silica carriers rely on pH, enzymes or low-sensitivity disulfide bonds to achieve responsive drug release. In the typical moderate-intensity ROS microenvironment of acute kidney injury, it is difficult to effectively trigger degradation, resulting in delayed or insufficient release of active ingredients, which cannot match the dynamic changes of lesions; (2) Single carrier function: Traditional mesoporous silica frameworks are only used as inert drug delivery platforms. After degradation, no substances with therapeutic activity are produced. The material itself is not endowed with antioxidant function, resulting in resource waste and a single treatment mechanism; (3) Disconnection between targeting and drug release mechanism: Hyaluronic acid (HA) is usually used as a static surface modification layer. It is continuously exposed regardless of whether the carrier reaches the lesion. It is easily degraded by hyaluronidase in vivo or causes non-specific adsorption. It cannot coordinate with the carrier degradation process, affecting the CD44-mediated targeting efficiency; (4) Limited antioxidant capacity: Single CeO2 or exogenous selenium agents are difficult to cover the complex ROS spectrum (such as ·OH, O2) in AKI. - (e.g., H2O2), lacking multi-enzyme mimicry (SOD / CAT / GP) x The cascading synergistic effect of these substances leads to low clearance efficiency and short duration of action.

[0005] The aforementioned shortcomings collectively result in existing nanoplatforms exhibiting low targeting precision, slow response speed, poor antioxidant durability, and limited therapeutic efficacy in the treatment of acute kidney injury, making it difficult to effectively inhibit ferroptosis and subsequent inflammatory responses. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a ROS-responsive selenium-cerium composite nanozyme, its preparation, and its application. By embedding diselenyl bonds as organic bridging groups into a mesoporous organosilicon framework and loading hyaluronic acid (HA)-modified cerium dioxide (CeO2) nanospheres within the mesopores, a kidney protection platform with intelligent responsiveness, targeting, and multi-enzyme mimicry activity is constructed. This solves the problems of insufficient responsiveness, single function, low targeting efficiency, and poor antioxidant durability of existing nanocarriers in the treatment of acute kidney injury, enabling on-demand release of active ingredients from kidney injury lesions.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing a ROS-responsive selenium-cerium composite nanozyme includes the following steps:

[0009] S1. Prepare sodium diselenide solution. Add 3-chloropropyltriethoxysilane to sodium diselenide solution and stir to react. After the reaction is completed, extract, dry and purify to obtain organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane.

[0010] S2. Add hexadecyltrimethyl-p-toluenesulfonate ammonium and triethanolamine to deionized water, stir until homogeneous to obtain a solution, then add organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane and tetraethyl orthosilicate to the solution, carry out a constant temperature reaction, centrifuge after the reaction is completed, wash and dry the precipitate to obtain diselenide bond-bridged mesoporous organosilicon nanomaterials.

[0011] S3. Mix cerium nitrate hexahydrate solution with hyaluronic acid solution, then add ammonia water and carry out a constant temperature reaction. After the reaction is completed, dialyze and wash the obtained solid product to obtain hyaluronic acid-coated cerium dioxide nanozyme.

[0012] S4. Disperse diselenide-bridged mesoporous organosilicon nanomaterials in anhydrous ethanol, then add 3-aminopropyltriethoxysilane and reflux to obtain aminated diselenide-bridged mesoporous organosilicon nanomaterials; add EDC, NHS, and hyaluronic acid-coated cerium dioxide nanoenzymes to deionized water, stir to activate, then add aminated diselenide-bridged mesoporous organosilicon nanomaterials, adjust pH, stir to react, centrifuge after reaction, wash and dry the solid product to obtain the ROS-responsive selenium-cerium composite nanoenzyme.

[0013] Preferably, the sodium diselenide solution in step S1 is prepared as follows: half of the selenium powder is added to a flask, and a sodium borohydride aqueous solution with a mass concentration of 5-10% is added under a nitrogen atmosphere and in an ice-water bath. The mixture is stirred until the selenium powder is completely dissolved and the reaction solution is colorless. The other half of the selenium powder is then added to the reaction solution, the reaction temperature is adjusted to 100°C, and the mixture is stirred until the reaction solution turns reddish-brown. The molar ratio of the selenium powder to sodium borohydride is 1:1.

[0014] Preferably, in step S1, the mass ratio of 3-chloropropyltriethoxysilane to sodium diselenide solution is 10-15:25-35, the stirring reaction temperature is 20-30°C, and the time is 12-15 h.

[0015] Preferably, the mass ratio of hexadecyltrimethyl-p-toluenesulfonate ammonium, triethanolamine, deionized water, organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane, and tetraethyl orthosilicate in step S2 is 0.5-0.8:0.1-0.2:40-60:1-1.5:4-5.

[0016] Preferably, the isothermal reaction in step S2 is carried out at a temperature of 75-85°C for 4-5 hours.

[0017] Preferably, in step S3, the concentration of the cerium nitrate hexahydrate solution is 5-50 mg / mL, the concentration of the hyaluronic acid solution is 1-10 mg / mL, the mass concentration of the ammonia solution is 5-10%, the mass ratio of the cerium nitrate hexahydrate solution, the hyaluronic acid solution, and the ammonia solution is 1-5:1-4:0.5-2, the temperature of the isothermal reaction is 35-40℃, and the time is 2-3 hours.

[0018] Preferably, in step S4, the mass ratio of the diselenylene-bridged mesoporous organosilicon nanomaterial and 3-aminopropyltriethoxysilane is 1:0.1-0.5, the reflux reaction temperature is 80°C, and the time is 10-15 h.

[0019] Preferably, in step S4, the mass ratio of EDC, NHS, hyaluronic acid-coated cerium dioxide nanozyme, and amino-diselenide-bridged mesoporous organosilicon nanomaterial is 0.1-0.5:0.1-0.5:1-3:1; the pH is 9-10; the stirring reaction temperature is 35-40℃; and the reaction time is 12-16h.

[0020] This invention also protects a ROS-responsive selenium-cerium composite nanozyme prepared by the method described above.

[0021] This invention also protects the use of a ROS-responsive selenium-cerium composite nanozyme as described above in the preparation of drugs for the prevention or treatment of acute kidney injury.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The ROS-responsive selenium-cerium composite nanozyme provided by the present invention directly embeds the diselenyl bond as an organic bridging group into the mesoporous organosilicon framework. Utilizing its high sensitivity to moderate oxidative stress (H2O2 10–100 μM), the material remains stable in normal blood circulation (H2O2 < 100 nM). Se–Se bond breakage occurs only in the locally elevated ROS microenvironment of acute kidney injury lesions, triggering controllable degradation of the framework. This design effectively avoids premature leakage of the contents before reaching the target site and significantly improves the spatiotemporal accuracy of drug delivery.

[0024] (2) The ROS-responsive selenium-cerium composite nanozyme provided by this invention releases glutathione peroxidase (GP) simultaneously after the diselenyl bond backbone is degraded. x ), the Se 2- It can directly participate in the intracellular antioxidant cycle and clear lipid peroxides. Therefore, the carrier itself becomes an active therapeutic unit without the need for additional loading of small molecule drugs. This not only simplifies the formulation process but also significantly improves the bioefficacy per unit mass of material, maximizing the utilization of material functions.

[0025] (3) The ROS-responsive selenium-cerium composite nanozyme provided by this invention enhances the renal tubular targeting efficiency through a degradation-triggered targeting mechanism. Existing HA modification strategies often expose the ligands on the outer surface of the carrier, which are easily degraded by hyaluronidase or cause non-specific adsorption during circulation, leading to targeting failure. This invention innovatively preloads HA-CeO2 nanospheres into the mesopores, so that HA is in a shielded state when the carrier is intact. Only when the material reaches the high ROS lesion and undergoes skeleton degradation is HA exposed and immediately bind to the CD44 receptor. This lock-and-key design significantly prolongs the in vivo stability of HA, while ensuring that the targeting effect is activated only in the lesion area, thereby greatly improving the uptake efficiency and treatment specificity of damaged renal tubular epithelial cells.

[0026] (4) The ROS-responsive selenium-cerium composite nanozyme provided by this invention is used to construct SOD / CAT / GP x A triple enzyme cascade network enables broad-spectrum and long-lasting antioxidant activity; single CeO2 or selenium agents are insufficient to address the complex ROS profile (such as ·OH, O2) in AKI. - (H2O2, lipid peroxides). This invention utilizes the SOD / CAT activity of CeO2 and Se... 2- GP x Synergistic effects of activity form a cascade catalytic cycle: CeO2 first removes O2 - And H2O2, reduce the formation of ·OH; Se 2- It further reduces the already formed lipid peroxides and blocks the ferroptosis pathway. The two are co-delivered in space and released synchronously in time, producing a synergistic effect of 1+1>2, which is significantly better than a single nanozyme system, and the antioxidant effect is more comprehensive and longer lasting.

[0027] (5) All active components of this invention (mesoporous organosilicon framework, CeO2, HA) are inorganic / natural polymer materials with well-defined chemical structures. They do not require encapsulation of small molecule drugs or complex modifications, thus avoiding problems such as drug leakage, batch differences and toxicological uncertainties. The preparation process only involves sol-gel self-assembly, hydrothermal synthesis and physical adsorption / electrostatic loading. It does not require high temperature, high pressure or toxic reagents. The process conditions are mild and have strong scalability, which is beneficial for subsequent GMP production and clinical translation.

[0028] In summary, this invention systematically solves the key bottlenecks of existing nanoplatforms in terms of stability, functionality, targeting, and antioxidant efficacy through three core innovations: embedded diselenide bond framework, pore preloading with HA-CeO2, and multi-enzyme synergistic simulation. It provides a highly efficient, safe, and intelligent drug-free nanotherapy strategy for acute kidney injury. Attached Figure Description

[0029] Figure 1The images shown are TEM and SEM images of MON and MON@HA-CeO2 prepared in Example 1 of this invention.

[0030] Figure 2 The images show the zeta potential and infrared spectra of MON, MON-NH2, and MON@HA-CeO2 prepared in Example 1 of this invention.

[0031] Figure 3 The TEM images show the degradation of MON and MON@HA-CeO2 prepared in Example 1 of this invention after reacting with H2O2.

[0032] Figure 4 This is a graph showing the release rate of selenium in MON@HA-CeO2 prepared in Example 1 of the present invention under oxidation conditions.

[0033] Figure 5 The graph shows the hemolysis rate of erythrocytes and the activity of HK-2 cells of MON@HA-CeO2 prepared in Example 1 of this invention.

[0034] Figure 6 Figures showing serum creatinine and blood urea nitrogen levels in different groups of mice.

[0035] Figure 7 This is a diagram showing the results of H&E staining of mouse kidney tissue. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0038] Example 1

[0039] A method for preparing a ROS-responsive selenium-cerium composite nanozyme includes the following steps:

[0040] S1. Add 2.37g of selenium powder to a flask, and add 22.7g of 10% sodium borohydride aqueous solution under a nitrogen atmosphere and in an ice-water bath. Stir until the selenium powder is completely dissolved and the reaction solution is colorless. Continue to add 2.37g of selenium powder to the reaction solution, adjust the reaction temperature to 100℃, and stir until the reaction solution turns reddish-brown to obtain a sodium diselenide solution. Add 12g of 3-chloropropyltriethoxysilane to 27.44g of sodium diselenide solution, stir the reaction at 25℃ for 12h, add ice water to terminate the reaction, extract with dichloromethane, dry the organic layer with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and purify the crude product by silica gel column chromatography (petroleum ether: dichloromethane = 10:1-1:1) to obtain the organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane.

[0041] S2. 0.6 g of hexadecyltrimethyl-p-toluenesulfonate ammonium and 0.15 g of triethanolamine were added to 50 g of deionized water and stirred at 80 °C for 0.5 h to obtain a solution. Then, 1.2 g of organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane and 4.5 g of tetraethyl orthosilicate were added to the solution and reacted at 80 °C and 1000 rpm for 4.5 h. After the reaction was completed, the mixture was centrifuged at 10000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in anhydrous ethanol and mixed. The centrifugation was repeated, and the washing operation with anhydrous ethanol was repeated twice. The product was refluxed in anhydrous ethanol solution of 1% (w / v) ammonium nitrate for 12 h to further remove residual template agent. The mixture was centrifuged again at 10000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in anhydrous ethanol and mixed. The centrifugation was repeated three times to obtain diselenide-bridged mesoporous organosilicon nanomaterials, denoted as MON.

[0042] S3. Mix 3g of cerium nitrate hexahydrate solution with 30mg / mL and 3g of hyaluronic acid solution with 5mg / mL, stir vigorously at 37℃ for 0.5h, then add 1.5g of 8% ammonia solution, and react at 37℃ for 2.5h. After the reaction is complete, dialyze the obtained product in deionized water for 24h using a dialysis bag (Thermo, 10kDa), then place it in an ultrafiltration tube (Milipore, 30kD), centrifuge and wash several times with deionized water to obtain hyaluronic acid-coated cerium dioxide nanozyme, denoted as HA-CeO2.

[0043] S4. Disperse 1g MON in 50mL anhydrous ethanol, stir at 80℃ for 3h, cool to room temperature, then add 0.3g 3-aminopropyltriethoxysilane, reflux at 80℃ for 12h, centrifuge the product at 10000rpm for 15min, wash three times with deionized water and collect the precipitate to obtain amino-substituted diselenylene-bridged mesoporous organosilicon nanomaterials, denoted as MON-NH2; Add 0.3g EDC, 0.3g NHS, and 2g HA-CeO2 to 50mL deionized water, stir and activate at room temperature in the dark for 0.5h, then add 1g MON-NH2, add triethylamine to adjust the pH to 9, stir and react at 37℃ for 14h, after the reaction is complete, centrifuge at 10000rpm for 15min, discard the supernatant, wash three times with deionized water and collect the precipitate to obtain ROS-responsive selenium-cerium composite nanozyme, denoted as MON@HA-CeO2.

[0044] Example 2

[0045] A method for preparing a ROS-responsive selenium-cerium composite nanozyme includes the following steps:

[0046] S1. Add 2.37g of selenium powder to a flask, and add 22.7g of 10% sodium borohydride aqueous solution under a nitrogen atmosphere and in an ice-water bath. Stir until the selenium powder is completely dissolved and the reaction solution is colorless. Continue to add 2.37g of selenium powder to the reaction solution, adjust the reaction temperature to 100℃, and stir until the reaction solution turns reddish-brown to obtain a sodium diselenide solution. Add 12g of 3-chloropropyltriethoxysilane to 27.44g of sodium diselenide solution, stir the reaction at 20℃ for 15h, add ice water to terminate the reaction, extract with dichloromethane, dry the organic layer with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and purify the crude product by silica gel column chromatography (petroleum ether: dichloromethane = 10:1-1:1) to obtain the organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane.

[0047] S2. 0.5 g of hexadecyltrimethyl-p-toluenesulfonate ammonium and 0.1 g of triethanolamine were added to 40 g of deionized water and stirred at 80 °C for 0.5 h to obtain a solution. Then, 1 g of organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane and 4 g of tetraethyl orthosilicate were added to the solution and reacted at 75 °C and 1000 rpm for 5 h. After the reaction was completed, the mixture was centrifuged at 10000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in anhydrous ethanol and mixed. The centrifugation was repeated, and the washing operation with anhydrous ethanol was repeated twice. The product was refluxed in anhydrous ethanol solution of 1% (w / v) ammonium nitrate for 12 h to further remove residual template agent. The mixture was centrifuged again at 10000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in anhydrous ethanol and mixed. The centrifugation was repeated three times to obtain diselenide-bridged mesoporous organosilicon nanomaterials, denoted as MON.

[0048] S3. Mix 1g of 50mg / mL cerium nitrate hexahydrate solution with 1g of 10mg / mL hyaluronic acid solution and stir vigorously at 37℃ for 0.5h. Then add 0.5g of 10% ammonia solution and react at 35℃ for 3h. After the reaction is complete, dialyze the obtained product in deionized water for 24h using a dialysis bag (Thermo, 10kDa). Then place it in an ultrafiltration tube (Milipore, 30kD), centrifuge and wash several times with deionized water to obtain hyaluronic acid-coated cerium dioxide nanozyme, denoted as HA-CeO2.

[0049] S4. Disperse 1g MON in 50mL anhydrous ethanol, stir at 80℃ for 3h, cool to room temperature, then add 0.1g 3-aminopropyltriethoxysilane, reflux at 80℃ for 10h, centrifuge the product at 10000rpm for 15min, wash three times with deionized water and collect the precipitate to obtain amino-substituted diselenylene-bridged mesoporous organosilicon nanomaterials, denoted as MON-NH2; Add 0.1g EDC, 0.1g NHS and 1g HA-CeO2 to 50mL deionized water, stir and activate at room temperature in the dark for 0.5h, then add 1g MON-NH2, add triethylamine to adjust pH to 10, stir and react at 35℃ for 16h, after the reaction is complete, centrifuge at 10000rpm for 15min, discard the supernatant, wash three times with deionized water and collect the precipitate to obtain ROS-responsive selenium-cerium composite nanozyme, denoted as MON@HA-CeO2.

[0050] Example 3

[0051] A method for preparing a ROS-responsive selenium-cerium composite nanozyme includes the following steps:

[0052] S1. Add 2.37g of selenium powder to a flask, and add 22.7g of 10% sodium borohydride aqueous solution under a nitrogen atmosphere and in an ice-water bath. Stir until the selenium powder is completely dissolved and the reaction solution is colorless. Continue to add 2.37g of selenium powder to the reaction solution, adjust the reaction temperature to 100℃, and stir until the reaction solution turns reddish-brown to obtain a sodium diselenide solution. Add 12g of 3-chloropropyltriethoxysilane to 27.44g of sodium diselenide solution, stir the reaction at 25℃ for 12h, add ice water to terminate the reaction, extract with dichloromethane, dry the organic layer with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and purify the crude product by silica gel column chromatography (petroleum ether: dichloromethane = 10:1-1:1) to obtain the organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane.

[0053] S2. 0.8 g of hexadecyltrimethyl-p-toluenesulfonate ammonium and 0.2 g of triethanolamine were added to 60 g of deionized water and stirred at 80 °C for 0.5 h to obtain a solution. Then, 5 g of organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane and 1.5 g of tetraethyl orthosilicate were added to the solution and reacted at 85 °C and 1000 rpm for 4 h. After the reaction was completed, the mixture was centrifuged at 10000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in anhydrous ethanol and mixed. The centrifugation was repeated, and the washing operation with anhydrous ethanol was repeated twice. The product was refluxed in anhydrous ethanol solution of 1% (w / v) ammonium nitrate for 12 h to further remove residual template agent. The mixture was centrifuged again at 10000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in anhydrous ethanol and mixed. The centrifugation was repeated three times to obtain diselenide-bridged mesoporous organosilicon nanomaterials, denoted as MON.

[0054] S3. Mix 5g of cerium nitrate hexahydrate solution with 4g of hyaluronic acid solution with 1mg / mL, stir vigorously at 37℃ for 0.5h, then add 2g of 5% ammonia solution, and react at 40℃ for 2h. After the reaction is complete, dialyze the obtained product in deionized water for 24h using a dialysis bag (Thermo, 10kDa), then place it in an ultrafiltration tube (Milipore, 30kD), centrifuge and wash several times with deionized water to obtain hyaluronic acid-coated cerium dioxide nanozyme, denoted as HA-CeO2.

[0055] S4. Disperse 1g MON in 50mL anhydrous ethanol, stir at 80℃ for 3h, cool to room temperature, then add 0.5g 3-aminopropyltriethoxysilane, reflux at 80℃ for 15h, centrifuge the product at 10000rpm for 15min, wash three times with deionized water and collect the precipitate to obtain amino-substituted diselenylene-bridged mesoporous organosilicon nanomaterials, denoted as MON-NH2; Add 0.5g EDC, 0.5g NHS, and 3g HA-CeO2 to 50mL deionized water, stir and activate at room temperature in the dark for 0.5h, then add 1g MON-NH2, add triethylamine to adjust the pH to 10, stir and react at 40℃ for 12h, after the reaction is complete, centrifuge at 10000rpm for 15min, discard the supernatant, wash three times with deionized water and collect the precipitate to obtain ROS-responsive selenium-cerium composite nanozyme, denoted as MON@HA-CeO2.

[0056] The sample prepared in Example 1 was tested, as follows:

[0057] Transmission electron microscopy (TEM) sample preparation: Appropriate concentrations of MON, MON@HA-CeO2 anhydrous ethanol, or ultrapure water dispersions were dropped onto a copper mesh, dried at room temperature in a desiccator, and then stored in a desiccator. The copper mesh containing the sample was fixed in the sample holder, and observed and photographed using a Talos F200X field emission transmission electron microscope. Figure 1 As shown.

[0058] SEM Sample Preparation: Dispense appropriate concentrations of MON, MON@HA-CeO2 anhydrous ethanol, or ultrapure water dispersion onto the silicon wafer, dry at room temperature in a desiccator, and then store in a desiccator. Carefully mount the silicon wafer onto a clean SEM system platform coated with conductive adhesive, spray platinum, and then observe and photograph it. Figure 1 As shown.

[0059] Zeta potential diagram: Appropriate amounts of MON, MON-NH2 and MON@HA-CeO2 nanomaterials were dispersed in ultrapure water or 10 mM phosphate buffer (PBS, pH=7.4) to prepare a homogeneous suspension with a concentration of approximately 0.1 mg / mL. The suspension was sonicated for 10 min to ensure full dispersion. The dispersion was then injected into a cuvette for a zeta potential analyzer, taking care to avoid generating air bubbles. The zeta potential value of the sample was measured at 25 °C using a nanoparticle size and zeta potential analyzer.

[0060] FTIR Sample Preparation: 1 mg of the sample powder to be tested was mixed with approximately 100 mg of potassium bromide powder, carefully ground into a fine powder, and then pressed into a transparent circular film of appropriate thickness for FTIR analysis. The absorption spectra of each sample in the 400–4000 cm⁻¹ wavelength region were obtained as follows: Figure 2 As shown in b.

[0061] from Figure 2 As can be seen, this invention uses the zeta potential and FTIR of MON, MON-NH2, and MON@HA-CeO2 for detection and analysis to further verify the success of MON amination modification and HA-CeO2 loading. The zeta potential of MON-NH2 is 21.87±0.98 mV; the zeta potential of MON@HA-CeO2 is -21.33±1.00 mV. Figure 2 a) After APTES amination, the zeta potential of MON nanoparticles significantly increased due to the introduction of surface amino groups, and the charge behavior of the nanoparticles in aqueous solution changed from negative to positive. After loading HA-CeO2, the HA coating on the surface of MON-NH2 caused the charge behavior of the nanoparticles to revert to negative in aqueous solution, while also improving the dispersion of the nanoparticles. FTIR spectra were observed in the 650-750 cm⁻¹ range. -1The specific absorption observed at this site is attributed to the Se-Se bond, confirming the presence of the Se-Se bond structure in MON, which is retained in subsequent synthesis. Figure 2 b). After amino modification, MON was 1561 cm. -1 The absorption peak at 517 cm⁻¹ corresponds to the introduction of an NH bond. MON@HA-CeO₂ at 517 cm⁻¹ -1 The newly appearing absorption peak is due to the Ce-O bond in HA-CeO2.

[0062] To test the reactive oxygen species-responsive degradation of MON and MON@HA-CeO2, 1 mg of MON and MON@HA-CeO2 were added to an aqueous solution containing 200 μM H2O2, respectively. After ultrasonic dispersion, the mixture was continuously stirred at 37°C. Samples were collected after one day and three days, prepared on a copper mesh, and observed under TEM for morphological changes. Figure 3 As shown in the image, after treatment with a solution containing 200 μM H2O2 for one day, both MON and MON@HA-CeO2 underwent significant structural degradation. Their surface and overall structures were disrupted, mesoporous pores increased, and some fragments broke down into irregularly shaped pieces. TEM images of MON@HA-CeO2 after treatment with an aqueous solution containing 200 μM H2O2 for three days reveal that MON@HA-CeO2 was completely degraded into extremely small particles coated with organic polymers. The darker particles are cerium dioxide nanozyme quantum dots, while the lighter gray particles are organic mesoporous silica fragments. The irregular light-colored regions exhibited by the organic polymer HA are highly distributed around the dark black CeO2 particles. HA can be biodegraded in vivo, while both the organic mesoporous silica fragments and CeO2 nanozyme quantum dots have extremely small particle sizes (<5 nm), making them easily eliminated by the kidneys.

[0063] The sample prepared in Example 1 was subjected to a hydroxyl radical scavenging experiment, as follows:

[0064] Using an aqueous system, the Fento reaction system is used to generate hydroxyl radicals, i.e., H₂O₂, in ferrous sulfate. 2+Under the influence of [the substance], hydroxyl radicals with strong oxidizing power are generated. Using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a hydroxyl radical scavenger, the electron spin resonance (ESR) spectra of different nanoparticles at the same concentration were measured by electron paramagnetic resonance spectroscopy to evaluate the hydroxyl radical scavenging efficiency of MON@HA-CeO2. The basic procedure is as follows: Ferrous sulfate hexahydrate powder was weighed and dissolved in deionized water to prepare a fresh 1 mM FeSO4 solution; a 30% hydrogen peroxide solution was taken and prepared to prepare a 10 mM hydrogen peroxide solution; HA-CeO2, MON, and MON@HA-CeO2 were taken and prepared to prepare a 200 μg / mL solution. Prepare a 100 μL system: Add 10 μL of DMPO to 50 μL of the sample to be tested, mix thoroughly by pipetting, then add 20 μL of FeSO4 solution and 20 μL of hydrogen peroxide solution, mix thoroughly by pipetting, and after 3 minutes, use a capillary pipette to aspirate the liquid, seal the bottom of the capillary pipette with paraffin, and test the ESR of the sample to characterize its free radical scavenging efficiency. Figure 4 As shown, the release of Se in MON@HA-CeO2 under oxidation conditions was studied by measuring the content of Se in the supernatant of MON@HA-CeO2 under the treatment of a solution containing 200 μM H2O2 at different time points. The results showed that the Se inside the composite nanoplatform structure can be released into the solution as the composite nanoplatform structure disintegrates.

[0065] Hemolysis testing is the most commonly used method for assessing the biocompatibility of erythrocytes. Different concentrations of MON@HA-CeO2 solution were co-incubated with erythrocyte suspensions for 3 hours. No obvious hemolysis was observed in the supernatants obtained from any concentration group, and the calculated hemolysis rate for each concentration group was below 5% (e.g., ...). Figure 5 As shown in a), this meets the hemolytic safety range for the bioapplication of nanomaterials. The hemolysis experiment results showed that even after exposure to a high concentration of MON@HA-CeO2 (200 μg / mL), erythrocytes were not significantly threatened, indicating that MON@HA-CeO2 has excellent biocompatibility with erythrocytes, providing a basis for its application in the treatment of kidney diseases via blood circulation. The CCK-8 assay is the most commonly used method for assessing cell viability. HK-2 cells were co-incubated with different concentrations of MON@HA-CeO2 solution for 24 hours. Cell viability in different treatment groups was calculated by detecting cell metabolic levels. The calculated cell viability in each treatment group was more than 90% of that in the control group. Figure 5(as shown in b). The CCK-8 experiment results showed that when the concentration of MON@HA-CeO2 was in the range of 0-100 μg / mL, it did not have a significant impact on the safety of HK-2 cells, indicating that MON@HA-CeO2 has excellent biocompatibility in this dose range and meets the safety requirements as a kidney-targeting nanomedicine.

[0066] The sample obtained in Example 1 of the present invention was subjected to mouse experiments, as follows:

[0067] Construction of a mouse model of rhabdomyolysis

[0068] An acute kidney injury model caused by rhabdomyolysis was established by intramuscular injection of 50% glycerol into the hind limbs of 8-week-old male C57BL / 6J mice. The specific procedure was as follows: the mice were acclimatized under standard conditions for 1 week. Fifteen hours before model establishment, the mice were deprived of water but allowed free access to food. After the water deprivation was completed, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution. After anesthesia, the leg hair was removed, and the injection area was disinfected with iodine. 50% glycerol was injected intramuscularly into both hind limbs at a total injection dose of 8 mg. After the injection was completed, the mice were allowed free access to food and water.

[0069] Grouping and intervention of laboratory animals

[0070] Eight-week-old male C57BL / 6J mice were randomly divided into five groups: Control group, AKI group (acute kidney injury), AKI+HA-CeO2 group, AKI+MON group, and AKI+MON@HA-CeO2 group. Interventions were administered 2 hours after model establishment. Mice were fixed to a tail vein injection device. The tail was held in place by the thumb, index, and middle fingers of one hand, while the tail was gently wiped with an alcohol swab to make the veins more visible. A sterile insulin syringe was then inserted parallel to the tail vein. After blood was aspirated, the corresponding intervention fluid was injected into the mouse's tail vein.

[0071] The mice were administered sterile PBS via tail vein injection in the Control group, AKI group via tail vein injection, AKI+HA-CeO2 group via tail vein injection of HA-CeO2 suspension prepared with sterile PBS, AKI+MON group via tail vein injection of MON suspension prepared with sterile PBS, and AKI+MON@HA-CeO2 group via tail vein injection of MON@HA-CeO2 suspension prepared with sterile PBS. Twenty-four hours after intervention, the mice were euthanized and their tissues were collected.

[0072] Serum creatinine and blood urea nitrogen, as core clinical indicators for assessing renal function, can be used to diagnose the success of glycerol-induced AKI model construction and to evaluate the renal function improvement effects of various nano-preparations. Figure 6As can be seen, the levels of CRE and BUN in the glycerol-induced AKI model group were significantly higher than those in the control group, indicating severe kidney damage in the mice and successful establishment of the AKI model. Figure 6 (ab). After 24 hours of intervention with different nano-preparations, the serum creatinine and blood urea nitrogen levels in all treatment groups improved, and the MON@HA-CeO2 composite nano-platform group showed the best renal function recovery effect, indicating that the MON@HA-CeO2 composite nano-platform can achieve efficient treatment of glycerol-induced AKI.

[0073] from Figure 7 As can be seen, the control group mice had intact kidney structure, regular glomerular morphology, clear Bowman's capsule, good proximal tubular brush border structure, and normal morphology of tubular epithelial cells and glomerular cells. No pathological changes such as cast formation or cell degeneration were observed. In stark contrast, the AKI model group exhibited typical pathological features, with numerous casts in the mouse kidney tissue; edema of the renal tubular basement membrane, and local formation of bare basement membrane; and damaged cells detaching into the renal tubular lumen, pathologically confirming the successful establishment of the glycerol-induced AKI model. After intervention with HA-CeO2, MON, or MON@HA-CeO2, the kidney damage in AKI mice was improved to varying degrees, manifested as a reduction in casts, less shedding of renal tubular epithelial cells, and reduced necrosis. Among them, the kidney tissue condition of the MON@HA-CeO2 treatment group was closest to that of the normal group, indicating that the MON@HA-CeO2 composite nanoplatform can effectively inhibit the pathological changes in the kidneys caused by glycerol-induced AKI.

[0074] The above description is a further detailed explanation of the present invention in conjunction with specific implementation examples. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the protection scope of the present invention.

[0075] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a ROS-responsive selenium-cerium composite nanozyme, characterized in that, Includes the following steps: S1. Prepare sodium diselenide solution. Add 3-chloropropyltriethoxysilane to sodium diselenide solution and stir to react. After the reaction is completed, extract, dry and purify to obtain organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane. S2. Add hexadecyltrimethyl-p-toluenesulfonate ammonium and triethanolamine to deionized water, stir until homogeneous to obtain a solution, then add organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane and tetraethyl orthosilicate to the solution, carry out a constant temperature reaction, centrifuge after the reaction is completed, wash and dry the precipitate to obtain diselenide bond-bridged mesoporous organosilicon nanomaterials. S3. Mix cerium nitrate hexahydrate solution with hyaluronic acid solution, then add ammonia water and carry out a constant temperature reaction. After the reaction is completed, dialyze and wash the obtained solid product to obtain hyaluronic acid-coated cerium dioxide nanozyme. S4. Disperse diselenide-bridged mesoporous organosilicon nanomaterials in anhydrous ethanol, then add 3-aminopropyltriethoxysilane and reflux to obtain aminated diselenide-bridged mesoporous organosilicon nanomaterials; add EDC, NHS, and hyaluronic acid-coated cerium dioxide nanoenzymes to deionized water, stir to activate, then add aminated diselenide-bridged mesoporous organosilicon nanomaterials, adjust pH, stir to react, centrifuge after reaction, wash and dry the solid product to obtain the ROS-responsive selenium-cerium composite nanoenzyme.

2. The preparation method according to claim 1, characterized in that, The sodium diselenide solution in step S1 is prepared as follows: half of the selenium powder is added to a flask, and a sodium borohydride aqueous solution with a mass concentration of 5-10% is added under a nitrogen atmosphere and in an ice-water bath. The mixture is stirred until the selenium powder is completely dissolved and the reaction solution is colorless. The other half of the selenium powder is then added to the reaction solution, the reaction temperature is adjusted to 100°C, and the mixture is stirred until the reaction solution turns reddish-brown. The molar ratio of the selenium powder to sodium borohydride is 1:

1.

3. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of 3-chloropropyltriethoxysilane to sodium diselenide solution is 10-15:25-35, and the stirring reaction is carried out at a temperature of 20-30°C for 12-15 hours.

4. The preparation method according to claim 1, characterized in that, The mass ratio of hexadecyltrimethyl-p-toluenesulfonate ammonium, triethanolamine, deionized water, organosilicon source bis-[3-(triethoxy)silylpropyl]diselenide silane, and tetraethyl orthosilicate in step S2 is 0.5-0.8:0.1-0.2:40-60:1-1.5:4-5.

5. The preparation method according to claim 1, characterized in that, The isothermal reaction in step S2 is carried out at a temperature of 75-85℃ for 4-5 hours.

6. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the cerium nitrate hexahydrate solution is 5-50 mg / mL, the concentration of the hyaluronic acid solution is 1-10 mg / mL, the mass concentration of the ammonia solution is 5-10%, the mass ratio of the cerium nitrate hexahydrate solution, the hyaluronic acid solution, and the ammonia solution is 1-5:1-4:0.5-2, and the isothermal reaction is carried out at a temperature of 35-40℃ for 2-3 hours.

7. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of the diselenylene-bridged mesoporous organosilicon nanomaterial and 3-aminopropyltriethoxysilane is 1:0.1-0.5, and the reflux reaction temperature is 80℃ for 10-15 hours.

8. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of EDC, NHS, hyaluronic acid-coated cerium dioxide nanozyme, and amino-diselenide-bridged mesoporous organosilicon nanomaterial is 0.1-0.5:0.1-0.5:1-3:1; the pH is 9-10; the stirring reaction temperature is 35-40℃; and the reaction time is 12-16h.

9. A ROS-responsive selenium-cerium composite nanozyme prepared by the method according to any one of claims 1-8.

10. The use of the ROS-responsive selenium-cerium composite nanozyme as described in claim 9 in the preparation of a drug for the prevention or treatment of acute kidney injury.

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