Drug-loaded nanoscale enzyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury and preparation method thereof

Abstract

This invention discloses a drug-loaded nanozyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury. Its structure is CeO2@ZIF-8@Taxifolin encapsulated in a neutrophil membrane, wherein ZIF-8 is a metal-organic framework structure, with embedded CeO2 nanoparticles as the enzyme core, resulting in porous CeO2@ZIF-8. Taxifolin is loaded into the pores of the porous CeO2@ZIF-8 to obtain CeO2@ZIF-8@Taxifolin; and CeO2@ZIF-8@Taxifolin is then encapsulated in neutrophils. This drug-loaded nanozyme can cross the blood-brain barrier and target damaged brain tissue. The neutrophil membrane enables it to actively chemotact with inflammatory signals. After reaching the site of injury, the drug-loaded nanozyme exerts a synergistic effect: CeO2 scavenges reactive oxygen species (ROS), while Taxifolin exerts an antioxidant effect and inhibits the NLRP3 inflammasome pathway, reducing caspase-1 activation, GSDMD cleavage, and subsequent release of pro-inflammatory cytokines IL-1β and IL-18. This dual action transforms the state of "exacerbating secondary damage" into the state of "alleviating secondary damage," thereby protecting neurons and promoting functional recovery.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a drug-loaded nanozyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury and its preparation method. Background Technology

[0002] The statements in this section are merely background information related to the present invention and do not necessarily constitute prior art.

[0003] Nanozymes, as a class of nanomaterials with enzyme-like catalytic activity, have shown great potential in the biomedical field in recent years, particularly in the treatment of central nervous system diseases such as traumatic brain injury (TBI). The pathophysiological process of TBI is extremely complex, with a cascade of secondary damage following the primary injury, including reactive oxygen species (ROS) bursts, neuroinflammation, calcium overload, ferroptosis, and mitochondrial dysfunction, forming the core mechanisms leading to neuronal death and dysfunction. Traditional antioxidant drugs often suffer from limitations such as poor stability, low bioavailability in the brain, and difficulty in penetrating the blood-brain barrier (BBB). Nanozymes, with their unique physicochemical properties, high catalytic efficiency, multi-enzyme activity, and designability, offer a new strategy for regulating the complex microenvironment following TBI.

[0004] Although nanozymes have shown great potential in the treatment of diseases such as traumatic brain injury, current research still faces many problems and limitations. Most technologies are still far from clinical translation. The main problems currently include: (1) Insufficient blood-brain barrier penetration and targeting The blood-brain barrier, as the most important protective barrier for the central nervous system, has tight junctions and active efflux mechanisms that severely limit the efficiency of nanoparticles entering the brain. Although some ultrasmall nanozymes (such as 2.1 nm Cu) can still achieve this, their absorption into the brain is limited by these mechanisms. 5.4 O) has been shown to passively penetrate the blood-brain barrier, but this process still lacks active targeting capabilities, resulting in limited enrichment efficiency of nanozymes in the brain injury area. Existing strategies mostly rely on passive diffusion, making it difficult to actively identify the microenvironment of the injury site (such as inflammatory chemokines and damaged vascular endothelium), thereby reducing the specificity and efficacy of treatment.

[0005] (2) Limited ability to comprehensively intervene in the complex pathological mechanisms of traumatic brain injury Most existing nanozymes target only the single mechanism of oxidative stress, while secondary damage from total brain injury (TBI) is a dynamic process involving multiple intertwined mechanisms, including ROS bursts, calcium overload, ferroptosis, neuroinflammation, and mitochondrial dysfunction. Single-function nanozymes cannot comprehensively block this multi-factor cascade reaction, thus limiting their overall therapeutic efficacy.

[0006] (3) The integration of synergistic treatment strategies is still immature. To overcome the limitations of monotherapy, some studies have attempted to combine nanozymes with traditional drugs (such as cerium-zirconium nanozymes with nimodipine) to simultaneously address oxidative stress and calcium overload. However, key issues in this synergistic strategy, such as formulation optimization, dosing timing, interaction mechanisms, and potential antagonistic effects, still lack systematic research, hindering the establishment of highly effective multi-target treatment regimens.

[0007] (4) Challenges of large-scale production and quality control As a novel type of nanomedicine, nanozymes face challenges in large-scale production, including poor batch consistency, lack of quality control standards, and difficulty in ensuring storage stability. These delays in production and quality control not only affect the reliability of experimental results but also become significant obstacles to their clinical translation.

[0008] In conclusion, although nanozymes show great promise in the treatment of TBI, breakthroughs are needed in areas such as brain-targeted delivery, multi-mechanism synergistic intervention, and industrial quality control to advance their clinical translation. Summary of the Invention

[0009] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide a drug-loaded nanozyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury and its preparation method. This drug-loaded nanozyme uses porous ZIF-8 as a carrier to simultaneously encapsulate CeO2 nanozyme and Taxifolin, achieving a multi-synergistic mechanism by which CeO2 and Taxifolin simultaneously intervene in three pathways: oxidative stress, pyroptosis, and ferroptosis.

[0010] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a drug-loaded nanozyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury, characterized in that its structure is CeO2@ZIF-8@Taxifolin encapsulated in a neutrophil membrane, wherein ZIF-8 is a metal-organic framework structure, and embedded CeO2 nanoparticles are used as the enzyme core to obtain porous CeO2@ZIF-8; Taxifolin small molecule compound is loaded into the pores of porous CeO2@ZIF-8 to obtain CeO2@ZIF-8@Taxifolin; and CeO2@ZIF-8@Taxifolin is encapsulated in neutrophils.

[0011] In some specific embodiments, the CeO2 nanoparticles have a particle size of 100-150 nm, the CeO2@ZIF-8 has a particle size of 160-180 nm, and the neutrophil membrane has a thickness of 5-25 nm.

[0012] In some specific embodiments, the CeO2 nanoparticles have a particle size of 133.42±2.4 nm, the CeO2@ZIF-8 has a particle size of 171.7±3.67 nm, and the neutrophil membrane has a thickness of 5-25 nm.

[0013] In some specific embodiments, the loading of the Taxifolin small molecule compound is 5-25%.

[0014] As part of the same inventive concept, this invention also provides a method for preparing the aforementioned drug-loaded nanozyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury, comprising the following steps: 1) Synthesis of CeO2 nanoparticles; 2) During the synthesis of ZIF8, CeO2 nanoparticles are embedded to form porous CeO2@ZIF8 composite nanoparticles; 3) Taxifolin small molecule compound was loaded into the pores of porous CeO2@ZIF8 nanoparticles to finally obtain CeO2@ZIF-8@Taxifolin; 4) Neutrophil membranes were coated onto the surface of CeO2@ZIF-8@Taxifolin to obtain CeO2@ZIF-8@Taxifolin coated with neutrophil membranes.

[0015] In some specific embodiments, step 2) of embedding CeO2 nanoparticles during the synthesis of ZIF8 specifically involves: 1) Weigh 10-30 mg of CeO2 nanoparticles and ultrasonically disperse them in 5-15 mL of methanol. Add 0.1-0.4 g of Zn(NO3)2·6H2O, stir to dissolve, and obtain CeO2 / Zn 2+ Mixture; 2) Dissolve 0.3-1.0 g of 2-methylimidazole in 5-15 mL of methanol to obtain a 2-methylimidazole solution; 3) Under vigorous stirring, slowly add the 2-methylimidazole solution dropwise to CeO2 / Zn. 2+ In the mixture, continue stirring at room temperature for 1-3 hours, and the reaction solution will change from clear to milky white; 4) Collect the precipitate by centrifugation, wash it 2-5 times with methanol, and dry it under vacuum at 50-70°C for 10-15 h to obtain porous CeO2@ZIF-8 composite nanoparticles.

[0016] In some specific embodiments, the loading of the Taxifolin small molecule compound in the pores of porous CeO2@ZIF8 nanoparticles in step 3) specifically refers to: 1) Weigh 40-60 mg of CeO2@ZIF-8 composite nanoparticles and disperse them in 5-15 mL of Taxifolin methanol solution. Stir at room temperature in the dark for 20-30 h. 2) Collect the taxifolin-loaded nanoparticles by centrifugation, wash them quickly with methanol 1-3 times to remove unadsorbed drug on the surface, and dry them under vacuum at 30-50°C to obtain taxifolin-loaded CeO2@ZIF-8 nanoparticles.

[0017] In some specific embodiments, the method for coating the neutrophil membrane onto the CeO2@ZIF-8@Taxifolin surface in step 4) is either ultrasonication or liposome extrusion.

[0018] As part of the same inventive concept, this invention also provides the application of the aforementioned drug-loaded nanozyme in the preparation of a drug to alleviate cognitive impairment following traumatic brain injury.

[0019] Description of the pharmaceutical composition: Preferably, the drug further includes a pharmaceutically acceptable carrier, which includes (but is not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorbents.

[0020] The drug of the present invention can be prepared into various dosage forms as needed, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0021] The administration route of the drug of the present invention is not limited, as long as it can achieve the desired therapeutic or preventive effect, including but not limited to oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, local or systemic transdermal administration.

[0022] Compared with the prior art, the present invention has at least the following advantages: 1) The preparation method provided by the present invention first synthesizes Ce(OH)3 nanoparticles, then converts them into CeO2 nanoparticles; then encapsulates the CeO2 nanoparticles in a porous ZIF-8 framework, and loads the natural compound Taxifolin into the pores of ZIF-8; finally, the entire core (CeO2@ZIF-8@Taxifolin) is coated with a neutrophil membrane, thereby obtaining an injectable drug-loaded nanozyme. 2) The drug-loaded nanozyme of the present invention, after intravenous injection, can cross the blood-brain barrier and target damaged brain tissue. The neutrophil membrane enables it to actively chemotact with inflammatory signals. After reaching the site of injury, the drug-loaded nanozyme exerts a synergistic effect: CeO2 scavenges reactive oxygen species (ROS), while Taxifolin, while exerting an antioxidant effect, can also inhibit the NLRP3 inflammasome pathway, reduce the activation of caspase-1 (CASP-1), the cleavage of GSDMD, and the subsequent release of pro-inflammatory cytokines IL-1β and IL-18. This dual effect transforms the state of "exacerbating secondary damage" (characterized by ROS burst, pyroptosis, and inflammation) into the state of "alleviating secondary damage," thereby protecting neurons and promoting functional recovery. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0024] Figure 1 The physicochemical characterization of CeO2@ZIF-8@NM provided by this invention includes: (A) a schematic diagram of the stepwise construction of CeO2@ZIF-8@NM nanoparticles from the Ce(OH)3 / PVP precursor; (B) representative transmission electron microscopy (TEM) images of CeO2 and CeO2@ZIF-8 nanoparticles, showing the characteristic particle morphology after ZIF-8 coating; the inset shows a magnified view of the boxed area, scale bar: 100 nm; (C) energy dispersive X-ray spectroscopy (EDS) of CeO2@ZIF-8@NM (2C), illustrating the quantitative elemental composition of the nanocomposite material, expressed as atomic percentage (%) and mass percentage (%). Figure 2 Physicochemical characterization of CeO2@ZIF-8@NM provided by this invention; Figure 3This invention relates to the enzyme activity of CeO2@ZIF-8@NM, specifically: A) UV-Vis absorption spectra of CeO2@ZIF-8@NM (nano) at different concentrations (0-320 µg / mL); B) Comparison of absorption spectra of the control group, free CeO2, and CeO2@ZIF-8@NM; C) Electron paramagnetic resonance (EPR) spectra of ROS in the presence of the control group (ROS only), ROS+CeO2, and ROS+CeO2@ZIF-8@NM; D) Comparison of UV-Vis absorption spectra of the control group, CeO2, and CeO2@ZIF-8@NM over a wider wavelength range (500-840 nm); E) Extended absorption measurement (500-840 nm); F) EPR spectrum; G) Control group, CeO2, and CeO2@ZIF-8@NM. Absorption spectra of @ZIF-8@NM in the 0-380 nm range; H, absorption spectra in the 380-540 nm range; I, quantitative analysis of the POD activity fold change of the control group, CeO2, and CeO2@ZIF-8; J, changes in dissolved oxygen generation of CeO2@ZIF-8@NM over time at different concentrations (0-300 µg / mL); K, comparison of dissolved oxygen generation over time of the control group, CeO2, and CeO2@ZIF-8; L, quantitative analysis of the CAT activity fold change of the control group, CeO2, and CeO2@ZIF-8; Data in Figures I and L are mean ± standard deviation (n=3, 3 independent experiments). Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test, ***P<0.001. All experiments were repeated at least twice, and the results were similar. Figure 4 To illustrate the anti-inflammatory and antioxidant effects of CeO2@ZIF-8@NM in TBI mice, the following images are presented: A) Representative DCFH-DA (DCFA) staining, used to detect intracellular ROS in brain tissue sections from sham-operated mice (control group), PBS-treated group, free CeO2-treated group, or Nano-treated group (scale bar: 100 μm); B) Quantitative analysis of the relative fluorescence intensity of DCFA; C) TUNEL staining, used to detect apoptotic cells (scale bar: 100 μm); D) Quantitative analysis of TUNEL-positive cells in each field of view; E) PI / Calcein-AM double staining, showing dead cells (PI... + (red) and live cells (Calcein) +F, Quantitative analysis of PI-positive dead cells in each field of view; G, C11-BODIPY581 / 591 staining for detecting lipid peroxidation (increased green signal indicates oxidation), scale bar: 100 μm; H, Quantitative analysis of relative C11 oxidation intensity, data are expressed as mean ± standard error (mean ± sem); each group contained 5 biologically independent experimental mice; statistical significance was determined by one-way ANOVA and Tukey's post-hoc test, *P<0.05, **P<0.01, ***P<0.001. All experiments were repeated at least three times, and the results were similar; Figure 5 The images show the polarization of microglia / macrophages in TBI mice after treatment with CeO2@ZIF-8@NM. A, representative immunofluorescence images showing the co-staining of Tuj1 (green) and iNOS (red) in brain tissue sections from TBI mice in the sham-operated control group, PBS-treated group, free CeO2-treated group, or CeO2@ZIF-8@NM (Nano)-treated group (scale bar: 100 μm); b, line contour analysis of iNOS fluorescence intensity along the white line shown in image A; C, representative immunofluorescence images showing the co-staining of NFκB (red) and Tuj1 (green) (scale bar: 100 μm); D, quantitative analysis of NFκB fluorescence intensity (data are mean ± standard error, n=5, 5 mice per group); E, CD206. + (M2) Representative flow cytometry image of macrophages / microglia; F, CD206 + Quantitative analysis of cells; G, CD163 + Representative image of (M2) cells; H, CD163 + Quantitative analysis of cells; I, CD80 + Representative image of (M1) cells; J, CD80 + Quantitative analysis of cells; data in Figures F, H, and J are expressed as mean ± standard error (mean ± sem). Statistical significance was determined using one-way ANOVA and Tukey's post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were repeated at least three times, and the results were similar. Figure 6This image shows the microglial pyroptosis in TBI mice treated with CeO2@ZIF-8@NM. A) Representative immunofluorescence images of Tuj1 (a neuronal marker) and GSDMD co-staining in brain tissue sections from LPS-treated positive control, sham-operated (Control), PBS-treated, free CeO2-treated, and Nano-treated TBI mice (scale bar: 100 μm); B) Quantitative analysis of the relative fluorescence intensity of GSDMD in the sham-operated, TBI, free CeO2-treated, and Nano (CeO2@ZIF-8@NM) groups; C) Representative images of Tuj1 and GSDMD co-staining in the LPS-treated, Control, PBS-treated, free CeO2-treated, and Nano-treated groups. Image, scale bar: 100 μm; D, Quantitative analysis of the relative intensity of GSDMD in the experiments shown in C; E, Representative images of co-stained Tuj1 and HMGB1, scale bar, 100 μm; F, Quantitative analysis of the relative intensity of HMGB1; G, Representative images of co-stained Tuj1 and caspase-1 (CASP1), scale bar, 100 μm; H, Quantitative analysis of the relative intensity of CASP1. Data are expressed as mean ± standard error (mean ± sem), (n=5 biologically independent experimental mice in each group). Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test. *P<0.05, **P<0.01, ***P<0.001. All experiments were repeated at least three times, and the results were similar. Figure 7 To assess the cellular uptake and Transwell migration abilities of CeO2@ZIF-8@NM in TBI mice after treatment; A, Schematic diagram of the experimental setup: bEnd.3 (brain endothelial cells) and BV2 (microglia) cells were incubated with fluorescently labeled nanoparticles (CeO2@ZIF-8@NM) to evaluate cellular uptake; B, Changes in nanoparticle uptake over time in nEnd.3 cells (top) and BV2 cells (bottom) compared to the untreated control group (Ctrl) at 0.5, 1, 2, and 4 hours; C, Nanoparticles. Biodistribution in sham-operated and traumatic brain injury (TBI) mice: Top figure: Time course of EPI-FLU in brain at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-injection; Bottom figure: Ex vivo fluorescence imaging of anatomical organs (brain, lung, heart, kidney, liver, and spleen) 4 hours post-injection; Quantitative analysis (right figure) confirmed that the brain fluorescence intensity of TBI mice was approximately five times that of sham-operated mice. Data are mean ± standard error (n=4, 4 biologically independent mice per group). Statistical significance was determined using two-way ANOVA. Post-hoc testing confirmed that, for example, P < 0.001, all experiments were repeated at least twice, and the results were similar. Figure 8 The molecular mechanism of CeO2@ZIF-8@NM's therapeutic effect on TBI mice is illustrated in the volcano plot of differentially expressed genes / proteins between the TBI group and the sham-operated group. The plot shows upregulation features (red) and downregulation features (blue), with log2 (fold change) as the ordinate and -log2 as the indices. 10 (P-value) is plotted on the x-axis, with the dashed line representing the significance threshold; B, Volcano plot of differentially expressed genes / proteins between the CeO2@ZIF-8@NM treated TBI group and the untreated TBI group, note the significant downregulation of inflammation-related genes (blue); C, Bar chart showing the number of upregulated (red) and downregulated (blue) genes / proteins in the two comparisons (TBI vs. sham surgery; Nano vs. TBI); D, Summary of upregulated and downregulated proteins in the two comparisons; E, Gene Ontology (GO) enrichment analysis of upregulated genes / proteins compared to the sham surgery group in the TBI group, the top ten enriched items are all related to... Pyroptosis, inflammasome assembly, interleukin-1 (IL-1) and IL-18 signaling pathways, and pyroptosis-related factors; F, visualization of the same GO item confirms the consistency of enrichment; G, GO enrichment analysis of downregulated genes / proteins compared to the untreated group; H, representative enrichment plot of the GO item "Pyroptosis Inflammatory Response", showing extremely high enrichment (P=0.0016), n=3, 3 biologically independent samples per group, statistical significance of GO analysis was performed using Fisher's exact test, and false discovery rate (FDR) correction was applied; Figure 9 This study assesses the functional recovery of TBI mice after treatment with CeO2@ZIF-8@NM. A represents the representative swimming trajectories of mice in the Morris water maze probe test, including the sham control group (Sham) and mice with brain injury treated with saline, free cerium dioxide (CeO2), or nanoparticles (CeO2@ZIF-8@NM); B represents the quantification of platform crossings; C represents the representative trajectory in the Y-maze test; D represents the representative swimming paths during the water maze learning phase; and E represents the swimming paths observed in the Y-maze test. Spontaneous alternation rate; F, typical open field or water maze exploration trajectory; G, escape latency during the Morris water maze training phase; Figure H shows representative behavioral trajectories (or other indicators, such as swimming speed or distance) of mice in each group. Data are expressed as mean ± standard error (mean ± sem) (n=10, 10 mice per group). Statistical significance was determined by one-way ANOVA and Tukey post-hoc test. *P<0.05, **P<0.01, ***P<0.001; Figure 10To assess the biosafety of CeO2@ZIF-8@NM in TBI mice, serum biochemical parameters were measured in the following groups: A, Alanine aminotransferase (ALT); B, Aspartate aminotransferase (AST); C, Total bilirubin (TBIL); D, Alkaline phosphatase (ALP); E, Triglycerides (TG); F, Glucose (GLU); G, Creatinine (CREA); and H, Uric acid. (ULA); there were no significant differences in any indicators among the groups; data are expressed as mean ± standard error (mean ± sem) (n=8, 8 mice per group). Figure I shows representative hematoxylin-eosin (H&E) stained sections of major organs (possibly including liver, kidney and other organs, see figure) of mice in the sham-operated group, TBI group, free CeO2 treatment group and nanotherapy group. Scale bar: 100 μm; statistical significance was determined by one-way ANOVA and Tukey post-hoc test; all comparisons were not statistically significant (ns), and the experiment was repeated three times with similar results. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the scope of protection of this invention. Any formal equivalent modifications made based on the concept of this invention should be considered within the scope of this invention.

[0026] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0027] The raw materials or reagents used in the following embodiments are specifically as follows: Cerium nitrate hexahydrate Ce(NO3)3·6H2O, zinc nitrate hexahydrate Zn(NO3)2·6H2O (analytical grade), sodium hydroxide (analytical grade), polyvinylpyrrolidone PVP (K30), 2-methylimidazole (2-Melm), Taxifolin (purity ≥98%, extracted from Siberian larch, Chengdu Manster Biotechnology), erythrocyte lysis buffer (BD Biosciences), and a mixture of protease inhibitors (Roche). Example 1: Preparation of CeO2@ZIF-8@Taxifolin encapsulated in neutrophil membranes This embodiment provides a method for preparing CeO2@ZIF-8@Taxifolin encapsulated in neutrophil membranes (the process flow is as follows). Figure 1 As shown in A), it includes the following steps: 1. Preparation of CeO2 nanoparticles (high-temperature calcination method) (1) Dissolve 0.434 g Ce(NO3)3·6H2O in 50 mL of deionized water, and slowly add 1 M NaOH solution with stirring until pH=10 to form a white Ce(OH)3 precipitate; continue stirring for 2 h, centrifuge (10,000 rpm, 10 min), wash 3 times each with deionized water and ethanol, and vacuum dry at 60°C for 12 h to obtain Ce(OH)3 powder; (2) The Ce(OH)3 powder was placed in a muffle furnace and heated to 400°C at a heating rate of 5°C / min. It was calcined in air for 4 hours and then naturally cooled to room temperature to obtain CeO2 nanoparticles. 2. Synthesis of porous CeO2@ZIF-8 composite nanoparticles (room temperature stirring method) (1) Weigh 20 mg of CeO2 nanoparticles and ultrasonically disperse them in 10 mL of methanol. Add 0.297 g of Zn(NO3)2·6H2O and stir to dissolve to obtain CeO2 / Zn 2+ Mixture; (2) Dissolve 0.656 g of 2-methylimidazole in 10 mL of methanol to obtain a 2-methylimidazole solution; (3) Under vigorous stirring, the 2-methylimidazole solution was slowly added dropwise to CeO2 / Zn. 2+ In the mixture, the reaction was continued to be stirred at room temperature for 2 hours, and the reaction solution changed from clear to milky white. (4) The precipitate was collected by centrifugation (12,000 rpm, 15 min), washed three times with methanol, and dried under vacuum at 60°C for 12 h to obtain porous CeO2@ZIF-8 composite nanoparticles. Figure 1 B); 3. Taxifolin load (1) Weigh 50 mg of porous CeO2@ZIF-8 composite nanoparticles and disperse them in 10 mL of Taxifolin methanol solution (5 mg / mL). Stir at room temperature in the dark for 24 h. (2) The taxifolin-loaded nanoparticles were collected by centrifugation (12,000 rpm, 15 min), washed twice with methanol to remove unadsorbed drug from the surface, and dried under vacuum at 40°C to obtain taxifolin-loaded CeO2@ZIF-8 nanoparticles; the nanoparticles were analyzed by energy-dispersive X-ray spectroscopy (EDS). Figure 1C) The elemental composition data of the synthesized CeO2@ZIF-8 nanoparticles (CeO2 NPs) were obtained. Analysis results showed that the nanoparticles mainly contained four elements: cerium (Ce), oxygen (O), carbon (C), and trace amounts of nitrogen (N). Among them, zinc (Zn) accounted for an absolute majority of the mass percentage, reaching 73.35% (corresponding to an atomic percentage of 63.45%); the mass percentage of cerium (Ce) was 21.17% (atomic percentage of 8.65%). The detection of Ce and O elements effectively confirmed the chemical composition of cerium oxide.

[0028] 4. Extraction of neutrophil membranes (1) Take peripheral blood from 6-8 week old male C57BL / 6 mice, collect about 1 mL of blood per mouse via the heart, and collect it in a centrifuge tube containing heparin; (2) Add an equal volume of PBS to dilute, slowly spread on lymphocyte separation medium (Ficoll-Paque), centrifuge at 400 g for 30 min, and collect the neutrophil layer; (3) Wash neutrophils twice with PBS (300 g, 10 min), count the cells, and resuspend them in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2 mM PMSF) containing protease inhibitor (1×) and incubate on ice for 30 min. (4) Homogenize 30 times with a glass homogenizer (close fit), centrifuge at 10,000 g for 20 min at 4°C to remove cell nuclei and large fragments, take the supernatant and centrifuge at 100,000 g for 1 h at 4°C (Optima XPN-100, Beckman), and collect the precipitate as neutrophil membrane. (5) Resuspend the membrane precipitate in PBS, determine the protein concentration by BCA method, adjust to 1 mg / mL, and store at -80°C for later use; 5. Neutrophil membrane encapsulation (co-extrusion method) (1) The above-mentioned CeO2@ZIF-8 nanoparticles loaded with Taxifolin were resuspended in PBS and the concentration was adjusted to 2 mg / mL; then 1 mL of CeO2@ZIF-8 nanoparticle suspension loaded with Taxifolin (2 mg / mL) was mixed with 1 mL of neutrophil membrane suspension (1 mg / mL) and premixed by sonication in an ice bath (40 W, 30 s); (2) Using an Avanti micro extruder, the mixture was extruded through 400 nm, 200 nm and 100 nm polycarbonate films 11 times each (each round trip is counted as 1 time). (3) The extruded solution was centrifuged at 50,000 g for 30 min at 4°C to remove uncoated free membrane fragments. The precipitate was washed twice with PBS and finally resuspended in PBS to obtain CeO2@ZIF-8@Taxifolin nanozyme (denoted as "Nano" or "CeO2@ZIF-8@NM") coated with neutrophil membrane.

[0029] Test Example 1: Physicochemical Characterization of CeO2@ZIF-8@NM This test case provides a performance characterization of CeO2@ZIF-8@Taxifolin encapsulated in a neutrophil membrane, specifically: 1) Physical property characterization This test series characterized the physical properties of the neutrophil membrane-coated CeO2@ZIF-8@Taxifolin nanozyme (abbreviated as Nano or CeO2@ZIF-8@NM) and its intermediates in this application. The results are as follows: Figure 2 As shown; the elemental surface mapping of Zn, O, N, C and Ce in CeO2@ZIF-8@NM, and its corresponding HAADF image, demonstrate the spatial distribution of each component in the nanocomposite material. Figure 2 A); The superimposed diagram of the elemental surface distribution of the same particle confirms its complete core-shell structure. Figure 2 B), scale bar: 200 nm; X-ray diffraction (XRD) analysis of CeO2@ZIF-8 showed characteristic diffraction peaks corresponding to the CeO2 fluorite structure and the ZIF-8 crystal framework, confirming the successful formation of the composite material. Figure 2 C); X-ray photoelectron spectroscopy (XPS) full-spectrum analysis showed that the composite material contained Ce, Zn, O, N and C elements ( Figure 2 D); High-resolution Ce3d spectra show the presence of Ce. 3+ and Ce 4+ A mixture of two oxidation states is crucial for mimicking the activities of catalase and superoxide dismutase. Figure 2 E); Zn 2p spectrum at 1044.8 eV (Zn 2p 1 / 2 ) and 1021.7eV (Zn 2p 3 / 2 Two characteristic peaks are shown at the position, which correspond to the Zn content in the ZIF-8 framework. 2+ Consistency Figure 2 F); N1s spectrum ( Figure 2 G) and O1s spectrum ( Figure 2 H) further confirmed the presence of imidazole bonds and surface oxygen species; C 1s XPS spectrum (2I) confirmed the presence of surface N oxygen species; dynamic light scattering (DLS) measurement results are as follows: Figure 2As shown in Figure J, the average hydrodynamic diameter of CeO2 nanoparticles is approximately 100 nm. After coating with ZIF-8 (CeO2@ZIF-8), the particle size increases to approximately 150 nm. Subsequently, after encapsulation with neutrophil membranes (CeO2@ZIF-8@NM), the particle size further increases slightly to approximately 180 nm, and the particle size distribution is relatively narrow. Figure 2 G); Zeta potential analysis showed that the CeO2 nanoparticles had a slightly negative charge on their surface, while CeO2@ZIF-8 was rich in Zn on its surface. 2+ The presence of a positive potential (2K) reflects the gradual evolution of surface charge during the assembly of nanoparticles; after film coating, the zeta potential becomes close to neutral or slightly negative, indicating that the film coating was successful.

[0030] 2) In vitro evaluation of CeO2@ZIF-8@NM enzyme activity This application also evaluated the enzyme mimicry activities of CeO2@ZIF-8@NM and its components, specifically: Test methods: 1. Peroxidase (POD)-like activity assay (1) Reaction system: 50 μL of acetate buffer (0.2 M, pH 4.0), 10 μL of TMB (10 mg / mL, dissolved in DMSO), 10 μL of H2O2 (10 mM) and different concentrations (0, 20, 40, 80, 160, 320 μg / mL) of the test sample (CeO2, CeO2@ZIF-8, Nano) were added to the 96-well plate in sequence, and finally deionized water was added to bring the total volume to 200 μL; (2) Reaction conditions: After incubation at 37°C for 15 min, add 50 μL of 2 M H2SO4 to terminate the reaction; (3) Detection: The absorbance at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader; the absorbance was plotted against the sample concentration to calculate the specific activity; the results showed that the POD activity of CeO2@ZIF-8 was about 2.5 times that of free CeO2. 2. Assay for catalase (CAT)-like activity (1) Reaction system: Dissolved oxygen was monitored using a YSI Pro20 instrument. Samples of different concentrations (0, 20, 40, 80, 100, 200, 300 μg / mL) were added to PBS buffer (pH 7.4) containing 10 mM H2O2, and the change in dissolved oxygen concentration (mg / L) over time (0-720 s) was recorded in real time. (2) Results: The oxygen production rate and oxygen production of the CeO2@ZIF-8 group were significantly higher than those of the free CeO2 group (about 3 times), and the results were concentration-dependent. 3. Determination of reactive oxygen species (ROS) scavenging capacity (EPR) (1) System: A hydroxyl radical (·OH) capture system was used. 0.1 M DMPO (spin capture agent), 0.1 mM FeSO4, 0.1 mM H2O2 and the sample to be tested (50 μg / mL) were added to the EPR tube and mixed evenly. (2) Detection: Bruker EMXplus EPR spectrometer, parameters: central magnetic field 338 mT, scan width 10 mT, microwave power 20 mW, modulation amplitude 0.1 mT; (3) Results: Both CeO2 and Nano significantly reduced the characteristic quartet signal of DMPO-OH, with clearance rates of 65% and 82%, respectively; Test results are as follows Figure 3 As shown, the UV-Vis absorption spectra reveal that both CeO2 and CeO2@ZIF-8@NM exhibit concentration-dependent absorbance changes, characteristic of peroxidase (POD)-like activity. The signal intensity of the nanoparticles is slightly higher than that of free CeO2. Figure 3 A, B); Electron paramagnetic resonance (EPR) spectroscopy using spin traps confirmed that both CeO2 and nanoparticles effectively scavenged hydroxyl radicals (·OH), and the characteristic reactive oxygen species (ROS) signal was significantly reduced compared to the control group. Figure 3 C, F); other absorption spectral measurements (C, F); Figure 3 (D, E, G, H) further confirmed the ROS scavenging ability of this nanozyme; quantitative analysis of peroxidase (POD) activity ( Figure 3 (I) This indicates that the activity of CeO2@ZIF-8 is approximately 2.5 times higher than that of free CeO2, suggesting that the ZIF-8 framework coating enhances catalytic efficiency. Regarding catalase-like (CAT) activity, this application monitored the change in dissolved oxygen production over time. The oxygen production of nano-CeO2@ZIF-8@NM showed a concentration-dependent effect; the higher the concentration (300 µg / mL), the faster the oxygen production rate and the higher the yield. Figure 3 J); Compared with free CeO2, CeO2@ZIF-8 showed significantly enhanced catalase-like activity, manifested as a steeper increase in dissolved oxygen over time (J). Figure 3 K); a fold increase in catalase-like activity ( Figure 3 L) confirmed that the ZIF-8 composite structure increased catalase-like activity by approximately three times compared to naked CeO2. These data collectively demonstrate that the CeO2@ZIF-8 structure not only retains the inherent ROS scavenging and oxygen generation capabilities of CeO2 but also enhances these capabilities, making it an effective nanozyme for TBI therapy.

[0031] Test Example 2: Application of CeO2@ZIF-8@NM in a mouse model of traumatic brain injury This test case demonstrates the application of CeO2@ZIF-8@NM in a mouse model of traumatic brain injury, specifically: 1. Establishment of a model of traumatic brain injury (TBI) A mouse TBI model was established using the controlled cortical impaction (CCI) method. (1) Animals: Male C57BL / 6 mice, 8-10 weeks old, weighing 20-25 g, raised in an SPF-grade environment; (2) Anesthesia: Inhalation anesthesia with isoflurane (induction 4%, maintenance 1.5%), and the mice were fixed on a stereotaxic apparatus; (3) Craniotomy: The scalp is cut along the midline to expose the right skull. A 3 mm diameter bone window is drilled 1.5 mm behind the anterior fontanelle and 2.0 mm beside the midline with a dental drill, keeping the dura mater intact. (4) Impact: Using the PinPoint™ precision cortical impactor (Hatteras Instruments), the impact head diameter is 3 mm, the impact speed is 3.5 m / s, the dwell time is 150 ms, the impact depth is 1.5 mm, and the bone window is sealed with bone wax immediately after impact and the scalp is sutured. (5) The sham surgery group (Sham) underwent craniotomy without impact. 2. Dosing regimen (1) Grouping: The TBI model mice were randomly divided into the following groups (n=12 per group): PBS group: An equal volume of PBS was injected via the tail vein; Free CeO2 group: CeO2 nanoparticles (10 mg / kg) were injected via tail vein. Nano group: Tail vein injection of the composite assembled nanozyme prepared in Example 1 (5 mg / kg based on Taxifolin and 10 mg / kg based on CeO2). Sham group: PBS was injected using the same method; (2) Administration time: Tail vein injection once at 1 h, 24 h and 48 h after TBI modeling, for a total of 3 times; 3. Tissue sampling and testing (1) Neurobehavioral tests: Morris water maze (orientation navigation for 5 days, spatial exploration on day 6) and Y maze (spontaneous alternation rate) tests were performed on day 7 after drug administration; (2) Immunofluorescence staining: On day 7, some animals (n=5 per group) were sacrificed, and frozen sections of brain tissue were taken for staining with Tuj1 / iNOS, NFκB, GSDMD, CASP1, HMGB1, etc., and observed under a confocal microscope. (3) Detection of oxidative stress and cell death: brain slices were stained with DCFH-DA (ROS), TUNEL (apoptosis), PI / Calcein-AM (necrosis), and C11-BODIPY (lipid peroxidation); (4) Flow cytometry: Isolate mononuclear cells from brain tissue, label them with CD80, CD206, and CD163 antibodies, and detect the polarization of microglia / macrophages; (5) Transcriptome / proteome analysis: RNA or protein was extracted from the surrounding cortex of the lesion and analyzed by RNA-seq or mass spectrometry. (6) Safety assessment: Blood samples were collected on day 7 to test ALT, AST, TBIL, ALP, TG, GLU, CREA, and ULA; heart, liver, spleen, lung, and kidney samples were taken for H&E staining; Test Example 3: Anti-inflammatory and antioxidant effects of CeO2@ZIF-8@NM on mice with traumatic brain injury after treatment This test case examined the oxidative stress and cell death in mice with traumatic brain injury after treatment with CeO2@ZIF-8@NM, specifically: To evaluate the therapeutic effect of neutrophil membrane-coated CeO2@ZIF-8@NM nanozyme (Nano) in a mouse model of traumatic brain injury (TBI), this application performed fluorescent staining of brain tissue sections (7 days after drug administration) from mice in the sham-operated control group, PBS-treated group, free CeO2-treated group, and Nano-treated group for reactive oxygen species (ROS, DCFA), apoptosis (TUNEL), cell death (PI / Calcein), and lipid peroxidation (C11-BODIPY). Figure 4 AH). The results show as follows: Figure 4 As shown, TBI significantly increased ROS levels, TUNEL-positive cells, PI-positive dead cells, and C11 oxidation (lipid peroxidation) levels in the PBS-treated group; the free CeO2-treated group partially reduced these signals, but these indicators were still significantly elevated compared to the control group; in stark contrast, the Nano-treated group reduced all four parameters to near-control levels, with a significant decrease in ROS fluorescence, a reduction in the number of TUNEL and PI-positive cells, and a significant reduction in C11 oxidation compared to both the PBS and free CeO2-treated groups. Figure 4 (B, D, F, H). These results indicate that Nano effectively reduces oxidative stress, apoptosis, necrosis, ferroptosis, and lipid peroxidation, thereby providing superior neuroprotective effects in TBI.

[0032] Test Example 4: CeO2@ZIF-8@NM can promote the transformation of microglia in mice with traumatic brain injury to an anti-inflammatory phenotype. In this application, the polarization of microglia / macrophages in mice with traumatic brain injury treated with CeO2@ZIF-8@NM is as follows: To evaluate the anti-inflammatory and immunomodulatory effects of neutrophil membrane-coated CeO2@ZIF-8@NM nanozyme (hereinafter referred to as "Nano") in a mouse model of traumatic brain injury (TBI), this application performed immunofluorescence staining of Tuj1 (neuronal marker) and iNOS (pro-inflammatory marker) on brain tissue sections (7 days after drug administration) from the sham-operated control group, PBS-treated group, free CeO2-treated group, or Nano (CeO2@ZIF-8@NM)-treated group. The results are as follows Figure 5 As shown, TBI resulted in strong co-localization of Tuj1 and iNOS in the PBS group, indicating the presence of neuronal oxidative stress. Free CeO2 treatment partially reduced this co-localization, while Nano treatment almost completely eliminated it. Figure 5 A); Linear profile analysis of iNOS fluorescence intensity confirmed that the Nano-treated group had the lowest signal intensity ( Figure 5 B); Similarly, NFκB immunostaining showed that NFκB expression was significantly upregulated in the PBS group, CeO2 could attenuate this upregulation, while Nano further inhibited this upregulation (B). Figure 5 C, D); Flow cytometry analysis showed that TBI shifted the microglia / macrophage phenotype towards the pro-inflammatory M1 type, with CD80 levels significantly higher in the PBS group compared to the control group (0.39%). + The proportion of (M1) cells was significantly increased (69.37%); CeO2 could reduce it to 15.27%, while Nano could reduce it to 8.75%. Figure 5 In contrast, the anti-inflammatory M2 markers CD206 and CD163 showed only slight increases in the PBS group (8.25% and 7.33%, respectively), but were significantly enhanced after CeO2 treatment (20.08% and 14.63%, respectively), and the enhancement was even more significant after Nano treatment (35.21% and 21.19%, respectively). Figure 5 These data indicate that the Nano platform not only inhibits the pro-inflammatory NFκB / iNOS signaling pathway, but also effectively promotes the transformation of microglia from the M1 phenotype to the M2 phenotype, thereby alleviating neuroinflammation after TBI.

[0033] Test Example 5: CeO2@ZIF-8@NM can inhibit microglia pyroptosis in mice with traumatic brain injury. To determine whether neutrophil membrane-coated CeO2@ZIF-8@NM nanozymes (Nano) can inhibit pyroptosis after TBI, some mice (n=5 per group) were sacrificed on day 7 of drug administration, and frozen sections of brain tissue were collected. The expression of key pyroptosis-related proteins—GSDMD, caspase-1 (CASP1), and HMGB1—in the brain tissue sections was detected by immunofluorescence. Lipopolysaccharide (LPS)-treated samples were used as positive controls for inflammasome activation.

[0034] The results are as follows Figure 6 As shown in the figure, TBI significantly enhanced the fluorescence intensity of GSDMD and was highly colocalized with the neuronal marker Tuj1. Figure 6 A); Quantitative analysis showed that the relative intensity of GSDMD in the PBS-treated TBI group was approximately five times that of the sham-operated control group (Sham). Free CeO2 partially inhibited GSDMD expression, while Nano (CeO2@ZIF-8@NM) reduced it to a level comparable to that in the Sham group. Figure 6 B); In another independent experiment, GSDMD obtained similar results when comparing the LPS group, control group, PBS group, free CeO2 treatment group, and Nano group. Figure 6 C, D); HMGB1 is another pyroptosis-inflammatory mediator, which was significantly upregulated in the PBS group and significantly inhibited by Nano ( Figure 6 E, F). The fluorescence intensity of Caspase-1 (CASP1) also exhibits the same pattern: significantly induced by TBI, partially inhibited by free CeO2, and almost completely restored to normal by Nano. Figure 6 (G, H). In summary, these data indicate that the Nano platform can effectively inhibit the classical pyroptosis pathway (CASP1-GSDMD) and HMGB1-mediated neuroinflammation after TBI.

[0035] Test Example 6: Targeting of CeO2@ZIF-8@NM on the lesion area in mice with traumatic brain injury This test case evaluated the cell uptake and Transwell migration capabilities of CeO2@ZIF-8@NM. The test results are as follows: Figure 7 As shown: This application involves incubating bEnd.3 (brain endothelial cells) and BV2 (microglia) with fluorescently labeled nanoparticles. Figure 7 A); Time-series imaging showed that fluorescence intensity steadily increased in both cell lines, peaking at 2 hours and remaining detectable at 4 hours. Figure 7 (B, both figures above and below); Quantitative analysis showed that the cell fluorescence signal reached approximately 2.5 × 10⁻⁶ after 2 hours.8 The signal of the control group (Ctrl) is negligible, while the signal of the control group (Ctrl) is negligible. Figure 7 B, right figure); Next, the ability of nanoparticles to cross the blood-brain barrier (BBB) ​​was evaluated using a Transwell system containing a bEnd.3 monolayer; the number of nanoparticles migrating to the lower chamber increased over time, with significant transport observed as early as 0.5 hours and reaching maximum accumulation at 4 hours (B). Figure 7 B, lower half and quantitative analysis); To investigate in vivo biodistribution and brain targeting efficiency, fluorescently labeled nanoparticles were intravenously injected into sham-operated or traumatic brain injury (TBI) mice, and major organs were collected at different time points (0.5-24 hours). In TBI mice, strong fluorescent signals were detected in the brain as early as 0.5 hours, gradually increasing over 4 hours and remaining at a high level at 24 hours. In contrast, no significant fluorescent signals were detected in the brain of sham-operated mice at any time point. Figure 7 C, Upper part and quantitative analysis); Ex vivo imaging of anatomical organs 4 hours after injection showed that Nano mainly accumulated in the brain of TBI mice, with low signal in the liver, spleen, lungs and kidneys, while the signal in the heart was negligible. Figure 7 C, lower half); quantitative analysis confirmed that the fluorescence intensity in the brains of TBI mice was approximately five times that of the sham-operated group mice ( Figure 7 (C, right figure). These results indicate that Nano can effectively cross the blood-brain barrier, actively target damaged brain tissue, and exhibits good biodistribution characteristics, making it suitable for TBI treatment.

[0036] Test Example 7: Effects of CeO2@ZIF-8@NM on inflammation in mice with traumatic brain injury (TBI) This test case elucidates the molecular mechanism of the therapeutic effect of CeO2@ZIF-8@NM in traumatic brain injury (TBI), specifically: Transcriptomic (or proteomic) analysis was performed on brain tissue from the sham surgery group, the TBI group, and the Nano treatment group (7 days after drug administration). The results are as follows: Figure 8 As shown, volcano plot analysis revealed a large number of differentially expressed genes / proteins in the TBI group compared to the Sham group. Figure 8 A), among which, genes involved in inflammation and pyroptosis pathways were significantly upregulated ( Figure 8 A (right red dot); Conversely, compared with the TBI group that did not receive Nano treatment, most TBI-induced changes were reversed in the TBI group mice that received Nano treatment, and the expression of pro-inflammatory and pyroptosis-related genes was significantly downregulated ( Figure 8 B); gene counting analysis further confirmed the distribution of upregulation and downregulation features in the two comparisons ( Figure 8 C, D); Gene Ontology (GO) enrichment analysis was performed on upregulated proteins / genes in the TBI and sham-operated groups, identifying a specific feature highly correlated with pyroptosis and inflammasome pathways. The terms with the highest enrichment levels included "positive regulation of inflammatory response", "regulation of interleukin-1 production", "interleukin-1β production", "positive regulation of interleukin-1β production", "pyroptosis-inflammatory response", "assembly of NLRP3 inflammasome complex", "interleukin-18-mediated signaling pathway", "positive regulation of pyroptosis-inflammatory response", "pyroptosis", and "assembly of pyroptosis body complex". Figure 8 E); In the comparison between the TBI group and the sham-operated group, the upregulated features were also significantly enriched with the same set of GO terms, confirming the activation of the classic pyroptosis cascade ( Figure 8 F); Notably, GO analysis of downregulated genes / proteins in TBI mice compared to the untreated group in the nanotherapy group revealed that terms associated with pyroptosis were the most significantly downregulated pathways. Figure 8 G); for example, the enrichment of "pyroptosis-inflammatory response" was very significant, with a P-value of 0.0016 ( Figure 8 These data indicate that nanotherapy can effectively inhibit pyroptosis-related transcription and translation programs that are strongly activated after TBI.

[0037] Test Example 8: Functional recovery of TBI in mice after treatment with CeO2@ZIF-8@NM This test case evaluated the functional recovery of traumatic brain injury (TBI) in mice treated with CeO2@ZIF-8@NM, specifically: This application conducted Morris water maze (orientation navigation for 5 days, spatial exploration on day 6) and Y maze (spontaneous alternation rate) tests on TBI model mice on day 7 after drug administration. The results are as follows: Figure 9 As shown, in the Morris water maze probe test, compared with the sham control group (Sham), the number of platform crossings in saline-treated TBI mice was significantly reduced. Free CeO2 treatment partially improved this indicator, while the number of platform crossings in the Nano treatment group was comparable to that in the Sham group. Figure 9 B); Mice in the TBI group treated with saline showed a significantly prolonged escape latency during the training phase, indicating impaired spatial learning ability. CeO2 alone only slightly shortened the latency, while the escape latency in the Nano treatment group was not significantly different from that in the Sham group. Figure 9G); In the Y-maze test assessing working memory, TBI mice receiving saline injections showed a significantly reduced spontaneous alternation frequency compared to the sham-operated group; CeO2 partially salvaged this deficit, while Nano treatment restored the alternation frequency to near the level of the sham-operated group. Figure 9 E); Figure 9 A, C, D, F, and H show representative motion trajectory diagrams in open fields or water mazes, indicating that the exploratory and swimming behavior patterns of mice in the Nano treatment group were improved. In summary, these data show that Nano can effectively improve cognitive and behavioral impairments caused by TBI, and its effect is better than that of free CeO2.

[0038] Test Example 8: Biocompatibility of CeO2@ZIF-8@NM This test case evaluated the biosafety of CeO2@ZIF-8@NM in TBI mice, specifically: This application examined serum liver and kidney function markers, as well as glucose and lipid metabolism indicators, in TBI mice (administered for 7 days) in a sham-operated control group, mice receiving saline (TBI), mice treated with free CeO2, or mice treated with Nano, respectively. The results are as follows: Figure 10 As shown; compared with the sham-operated control group, TBI alone did not cause a decrease in serum alanine aminotransferase (ALT). Figure 10 A) Aspartate aminotransferase (AST), Figure 10 B) Total bilirubin (TBIL), Figure 10 C) Alkaline phosphatase (ALP) Figure 10 D) Triglycerides (TG) Figure 10 E), glucose (GLU, Figure 10 F), Creatinine (CREA), Figure 10 G) or uric acid (ULA, Figure 10 Significant changes in H); no detectable effects were observed on these parameters after treatment with free CeO2 or Nano, all values ​​remained within the normal range, and there were no statistically significant differences between groups; histological examination of major organs ( Figure 10 (I) The results show that after treatment with Nano nanoparticles, no significant pathological changes, tissue necrosis, or inflammatory infiltration were observed in the liver, kidneys, or other tissues (representative images shown). The structure of the liver parenchyma, glomeruli, and renal tubules remained normal, similar to the sham-operated group and untreated TBI mice. In summary, these results indicate that under experimental conditions, Nano nanoparticles do not cause measurable hepatotoxicity, nephrotoxicity, or metabolic disturbances, supporting their good safety profile in TBI treatment.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A drug-loaded nanozyme for targeting pyroptosis to alleviate cognitive impairment after traumatic brain injury, characterized in that, Its structure is CeO2@ZIF-8@Taxifolin encapsulated in neutrophil membranes, where ZIF-8 is a metal-organic framework structure and embedded CeO2 nanoparticles are used as the enzyme core to obtain porous CeO2@ZIF-8; Taxifolin small molecule compound is loaded into the pores of porous CeO2@ZIF-8 to obtain CeO2@ZIF-8@Taxifolin; and CeO2@ZIF-8@Taxifolin is encapsulated in neutrophils.

2. The drug-loaded nanoenzyme for targeting pyroptosis and alleviating cognitive impairment after traumatic brain injury according to claim 1, characterized in that, The CeO2 nanoparticles have a particle size of 100-150 nm, the CeO2@ZIF-8 has a particle size of 160-180 nm, and the neutrophil membrane has a thickness of 5-25 nm.

3. A method for preparing a drug-loaded nanozyme for targeting pyroptosis and alleviating cognitive impairment after traumatic brain injury, as described in claim 1 or 2, characterized in that, Includes the following steps: 1) Synthesis of CeO2 nanoparticles; 2) During the synthesis of ZIF8, CeO2 nanoparticles are embedded to form porous CeO2@ZIF8 composite nanoparticles; 3) Taxifolin small molecule compound was loaded into the pores of porous CeO2@ZIF8 nanoparticles to finally obtain CeO2@ZIF-8@Taxifolin; 4) Neutrophil membranes were coated onto the surface of CeO2@ZIF-8@Taxifolin to obtain CeO2@ZIF-8@Taxifolin coated with neutrophil membranes.

4. The method for preparing drug-loaded nanozymes for targeting pyroptosis and alleviating cognitive impairment after traumatic brain injury according to claim 3, characterized in that, In step 2), the embedding of CeO2 nanoparticles during the synthesis of ZIF8 specifically involves: 1) Weigh 10-30 mg of CeO2 nanoparticles and ultrasonically disperse them in 5-15 mL of methanol. Add 0.1-0.4 g of Zn(NO3)2·6H2O, stir to dissolve, and obtain CeO2 / Zn 2+ Mixture; 2) Dissolve 0.3-1.0 g of 2-methylimidazole in 5-15 mL of methanol to obtain a 2-methylimidazole solution; 3) Under vigorous stirring, slowly add the 2-methylimidazole solution dropwise to CeO2 / Zn. 2+ In the mixture, continue stirring at room temperature for 1-3 hours, and the reaction solution will change from clear to milky white; 4) Collect the precipitate by centrifugation, wash it 2-5 times with methanol, and dry it under vacuum at 50-70°C for 10-15 h to obtain porous CeO2@ZIF-8 composite nanoparticles.

5. The method for preparing drug-loaded nanozymes for targeting pyroptosis and alleviating cognitive impairment after traumatic brain injury according to claim 3, characterized in that, Step 3) describes the loading of the Taxifolin small molecule compound into the pores of porous CeO2@ZIF8 nanoparticles. 1) Weigh 40-60 mg of CeO2@ZIF-8 composite nanoparticles and disperse them in 5-15 mL of Taxifolin methanol solution. Stir at room temperature in the dark for 20-30 h. 2) Collect the taxifolin-loaded nanoparticles by centrifugation, wash them quickly with methanol 1-3 times to remove unadsorbed drug on the surface, and dry them under vacuum at 30-50°C to obtain taxifolin-loaded CeO2@ZIF-8 nanoparticles.

6. The method for preparing drug-loaded nanozymes for targeting pyroptosis and alleviating cognitive impairment after traumatic brain injury according to claim 3, characterized in that, In step 4), the method for coating the neutrophil membrane onto the CeO2@ZIF-8@Taxifolin surface is either ultrasound or liposome extrusion.

7. The use of the drug-loaded nanozyme according to claim 1 or 2, or the drug-loaded nanozyme prepared by any one of the preparation methods of claims 3-6, in the preparation of drugs to alleviate cognitive impairment after traumatic brain injury.

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