Magnesium monatomic nano-enzyme with multi-enzyme activity as well as preparation method and application of magnesium monatomic nano-enzyme

Abstract

The invention discloses a magnesium monatomic nano-enzyme with multienzyme activity and a preparation method and application thereof, the magnesium monatomic nano-enzyme takes a nitrogen-doped carbon material loaded magnesium monatomic as a core, and the surface of the magnesium monatomic nano-enzyme is connected with a brain-targeted ligand through polyethylene glycol as a connector to obtain the magnesium monatomic nano-enzyme modified by the brain-targeted ligand. According to the invention, monatomic magnesium is used as a catalytic activity center, and an oxidative stress treatment mechanism of multienzyme activity induction tumor microenvironment response, PEG-mediated long circulation and ligand-guided active targeting are integrated, so that the drug can accurately reach and be enriched in the most stubborn glioma stem cell area; and the glioblastoma can be efficiently removed through specific catalytic action, so that the problems of high recurrence rate and short lifetime in the current treatment of glioblastoma are expected to be solved.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to a magnesium single-atom nanozyme with multi-enzyme activity, its preparation method, and its application. Background Technology

[0002] Gliomas (GBM) are the most common and aggressive primary malignant brain tumors (WHO grade 4). They are characterized by rapid growth, diffuse infiltration, and are associated with extremely poor prognosis; the 2-year survival rate remains below 30%, the 5-year survival rate below 10%, and the median survival is only 12–15 months. Current standard treatment regimens include maximal safe surgical resection, supplemented with concurrent chemoradiotherapy with temozolomide and adjuvant chemotherapy, but the effects are unsatisfactory, and the tumors almost invariably recur within a short period.

[0003] GBM treatment faces multiple severe challenges. First, the presence of the blood-brain barrier severely limits the effective therapeutic concentrations of most therapeutic drugs (especially macromolecular and hydrophilic drugs) at the tumor site. Second, GBM exhibits high heterogeneity, with glioma stem cells, which are highly resistant to radiotherapy and chemotherapy, considered a fundamental cause of tumor recurrence, invasion, and treatment failure. Therefore, developing novel therapeutic strategies that can efficiently cross the blood-brain barrier and specifically target GSCs (glioma stem cells) is urgently needed.

[0004] In recent years, nanozymes, as a novel type of artificial enzyme that combines the properties of nanomaterials with enzyme-like catalytic activity, have shown great application potential in the biomedical field. In particular, single-atom nanozymes, by dispersing metal active centers in single-atom form on a support, achieve maximum atomic utilization and uniform catalytic active sites, often exhibiting catalytic activity and selectivity surpassing that of traditional nanozymes. However, the application of single-atom nanozymes in brain tumor treatment is still in the exploratory stage and faces several key bottlenecks: (1) Lack of targeting: Most reported single-atom nanozymes lack the ability to actively target GBM and GSCs, resulting in limited enrichment efficiency at tumor sites.

[0005] (2) Biocompatibility and safety of materials: The long-term biocompatibility and in vivo metabolic behavior of emerging main group metal (such as magnesium) nanozymes are still unclear and require systematic evaluation.

[0006] Although studies have reported single-atom nanozymes based on transition metals and some alkaline earth metals, as well as nanocarriers using target heads for brain targeting, there is currently no solution that can integrate efficient brain targeting, GSC killing, and multi-enzyme catalytic diagnostic and therapeutic functions, based on magnesium single-atom nanozymes with excellent biocompatibility. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a novel nanomedicine that can effectively overcome the blood-brain barrier, accurately identify and accumulate in glioblastoma and its stem cell regions, and possess excellent catalytic therapeutic function and real-time imaging capabilities, thereby providing new possibilities for the efficient diagnosis and treatment of GBM.

[0008] To achieve this objective, the present invention adopts the following technical solution: The first aspect of the present invention is to provide a magnesium single-atom nanozyme with multi-enzyme activity, wherein the magnesium single-atom nanozyme uses a magnesium single atom loaded on a nitrogen-doped carbon material as the core, and a brain-targeting ligand is connected to its surface through polyethylene glycol as a linker, thereby obtaining a brain-targeting ligand modified magnesium single-atom nanozyme.

[0009] In one alternative implementation, the brain-targeting ligand is an antibody, antibody fragment, or peptide.

[0010] In one alternative embodiment, the antibody or peptide is a molecule capable of specifically binding to the LRP1 receptor.

[0011] In one alternative embodiment, the peptide molecule is an Angiopep-2 peptide or a derivative thereof; or the antibody molecule is an Angiopep-2 monoclonal antibody.

[0012] A second aspect of the present invention is to provide a method for preparing a magnesium single-atom nanozyme with multi-enzyme activity, comprising the following steps: S1: The zinc source solution and the imidazole organic ligand solution were stirred and mixed. The resulting mixed solution was centrifuged, precipitated, and dried to obtain the zeolite imidazole ester framework structure material. S2: The zeolite imidazole ester framework material was placed in a tube furnace and pyrolyzed at high temperature in an inert atmosphere to obtain NC-doped material. After activation, centrifugal washing and drying, nanoporous nitrogen-doped carbon material was obtained. S3: Using nitrogen-doped carbon material as a carrier, magnesium and nitrogen sources are introduced, and after self-assembly mixing, centrifugal drying, and high-temperature pyrolysis in an inert gas, magnesium single-atom nanozymes are obtained. S4: Phospholipid-polyethylene glycol with maleimide active groups at the end is reacted with a brain-targeting ligand containing thiol groups under suitable conditions for forming thioether bonds to obtain a phospholipid-polyethylene glycol-brain-targeting ligand conjugate. S5: Dissolve the phospholipid-polyethylene glycol-brain-targeting ligand conjugate in an organic solvent to obtain solution A. Dissolve the magnesium single-atom nanozyme and add it to solution A and stir to mix. After dialysis and drying, the resulting mixed solution yields the magnesium single-atom nanozyme linked to the brain-targeting ligand.

[0013] In an optional embodiment, in step S1, the centrifugation conditions are: rotation speed 6000-10000 rpm, time 8-12 min; drying temperature 45-75 ℃; in steps S2 and S3, the high-temperature pyrolysis conditions are: temperature 900-12000 ℃, heating rate 5 ℃ / min, pyrolysis time 2-5 h.

[0014] In one optional embodiment, in step S3, the mass ratio of nitrogen-doped carbon material to magnesium source and nitrogen source is 1:(1-2):(1-2).

[0015] In an optional embodiment, in step S4, the molar ratio of phospholipid-polyethylene glycol to brain-targeting ligand is 1:(1-6).

[0016] In an optional embodiment, the mixed solution in step S5 further includes a phospholipid-polyethylene glycol-fluorescent label, wherein the mass ratio of the phospholipid-polyethylene glycol-brain-targeting ligand to the magnesium single-atom nanozyme is (3-7):(1-4).

[0017] A third aspect of the present invention is to provide the use of the magnesium single-atom nanozyme with multi-enzyme activity described above in the preparation of a drug for treating glioma.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The magnesium single-atom nanozyme prepared in this invention simulates the activities of various natural enzymes such as peroxidase and oxidase. Under the stimulation of the tumor microenvironment (such as weak acidity and high hydrogen peroxide), it can catalyze the generation of a large number of highly active free radicals and selectively induce tumor cell apoptosis. More importantly, its unique catalytic mechanism may directly interfere with the key survival pathways of tumor stem cells, providing a new pathway for fundamentally overcoming tumor recurrence and drug resistance.

[0019] (2) By covalently modifying the surface of the nanoenzyme with polyethylene glycol (PEG) chains, the present invention can effectively improve the hydrophilicity and stability of magnesium single-atom nanoenzymes, while giving them "invisibility" properties, effectively avoiding plasma protein adsorption and immune system clearance, thereby significantly prolonging the in vivo circulation time and creating conditions for drug accumulation in the brain.

[0020] (3) By connecting brain-targeting ligands such as Angiopep-2, the present invention can actively recognize the LRP1 receptor highly expressed on endothelial cells and cross the blood-brain barrier (BBB) ​​to achieve efficient intrabrain delivery of drugs. The targeting system can also specifically bind to LRP1 on the surface of glioma cells and GSCs to achieve dual-level targeting and enrichment of the lesion area, which greatly improves the treatment efficiency and reduces the dosage.

[0021] (4) This invention integrates a safe single-atom magnesium catalytic core, an oxidative stress treatment mechanism that induces tumor microenvironment response through multi-enzyme activity, PEG-mediated long circulation, and ligand-guided active targeting. This synergistic effect overcomes the three major bottlenecks in GBM treatment: "blood-brain barrier hindering drug entry into the brain," "tumor heterogeneity and GSC leading to relapse and drug resistance," and "poor biocompatibility of nanomaterials." It not only ensures that the drug can accurately reach and accumulate in the most refractory glioma stem cell region, but also efficiently eliminates it through its unique catalytic action. This is expected to overcome the current problems of high relapse rate and short survival in glioblastoma treatment and has significant clinical translational value. Attached Figure Description

[0022] Figure 1 The image shows a transmission electron microscope (TEM) image of ZIF-8, NC, and MgSA synthesized in Example 1 of this invention.

[0023] Figure 2 Chemical characterization of the MgSA synthesized in Example 1 of this invention; Figure 3 Enzyme activity characterization of MgSA synthesized in Example 1 of this invention; Figure 4 The results are the stability test results of pMgSA synthesized in Example 1 of this invention; Figure 5 The effect of pMgSA synthesized in Example 1 of this invention on intracellular ROS and mitochondrial MitoSox in glioma cells; Figure 6 The results show the effects of pMgSA and Ang-pMgSA synthesized in Example 1 of this invention on glioma cell cytotoxicity and apoptosis. Figure 7 These are confocal fluorescence images of cellular uptake of pMgSA and Ang-pMgSA synthesized in Example 1 of this invention. Figure 8 The image shows the cellular uptake effect of pMgSA and Ang-pMgSA synthesized in Example 1 of this invention. Figure 9 The in vivo experimental results are for pMgSA and Ang-pMgSA synthesized in Example 1 of this invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0025] In the following, pMgSA refers to MgSA mixed with DSPE-PEG. 2000 -COOH linkage; Ang-pMgSA is MgSA and DSPE-PEG 2000 -Angiopep-2 linkage; pMgSA@Cy5 is MgSA with DSPE-PEG 2000 -Cy5 linkage; Ang-pMgSA@Cy5 is MgSA and DSPE-PEG 2000 -Cy5 and DSPE-PEG 2000 -Angiopep-2 connection.

[0026] I. Preparation and Characterization of Magnesium Single-Atom Nanozymes (I) Preparation of ZIF-8 At room temperature, 1.68 g of hydrated Zn(NO3)2 and 4 g of 2-methylimidazole were dissolved in 20 ml and 60 ml of methanol, respectively, and stirred to obtain clear solutions. The two solutions were then mixed and magnetically stirred at 500 rpm for 1 h at room temperature (the solution containing the metal was added to the solution containing the binder). The mixture was centrifuged at 6000 rpm for 10 minutes at room temperature, washed three times with methanol, and the resulting precipitate was dried at 55 °C overnight to obtain ZIF-8 powder.

[0027] (II) Preparation of nitrogen-doped carbon (NC) ZIF-8 powder was spread evenly on the bottom of a quartz boat and placed in a vacuum tube furnace. Argon gas was introduced as a protective gas, and the material was pyrolyzed at 900 °C for 3 h at a heating rate of 5 °C / min to obtain CN-doped material. The CN-doped material was activated in 3M HCl for 3 h (with stirring), centrifuged at 6000 rpm for 20 min, and repeatedly washed with deionized water until the pH was neutral to obtain nitrogen-doped porous carbon. The carbon was then dried in a forced-air drying oven to obtain black nanoporous nitrogen-doped carbon powder NC.

[0028] (III) Preparation of magnesium single-atom nanozyme MgSA 50 mg of nitrogen-doped carbon powder, 100 mg of MgCl2·6H2O and 100 mg of melamine were dispersed in an isopropanol-water solution (in a 1:1 ratio), ultrasonically dispersed for 2 h, stirred for 5 h, and centrifuged to obtain the product. The centrifuged product was dried in an oven at 60 °C for at least 12 h and then pyrolyzed in argon at 900 °C at a heating rate of 5 °C / min for 2 h to form magnesium single-atom nanozyme MgSA.

[0029] (iv) Transmission electron microscopy characterization of magnesium single-atom nanozymes 10 μL each of the synthesized ZIF-8, NC, and MgSA were dropped onto a copper grid. After drying, the grid containing the samples was placed inside a TEM machine, and its microstructure was observed. The results are as follows. Figure 1 a- Figure 1 As shown in g.

[0030] Depend on Figure 1 As can be seen from a, ZiF-8 exhibits good dispersibility, with a particle size of approximately 120 ± 30 nm. It possesses a regular dodecahedral structure, uniform particle size, and complete morphology, indicating that the precursor has good crystallinity and a controllable nanostructure, making it suitable as a template for subsequent pyrolysis. Figure 1 As shown in b, NC is a porous carbon-based material transformed from ZIF-8 after pyrolysis. It retains the original dodecahedral framework structure, but the surface is denser and micropores appear, confirming that the structure is preserved and nitrogen doping is introduced during carbonization, which is beneficial for metal anchoring and electronic control. Figure 1 c indicates that MgSA exhibits good dispersion properties, with spherical particles of approximately 77 ± 10 nm in diameter. While retaining its original geometric morphology, MgSA displays a more clearly defined nanoscale rough structure on its surface, suggesting that magnesium atoms have successfully embedded themselves into the carbon framework without significant agglomeration. Figure 1 The high-magnification TEM image of MgSA (inset) shows highly ordered lattice fringes in the magnified region. Combined with the single bright spot in the inset, this suggests the existence of isolated atomic-level dispersion sites, initially supporting the existence of a single-atom dispersed structure. Figure 1 The EDS elemental distribution map of e shows that C, N, O, and Mg are uniformly distributed in the material, and the Mg signal is distributed as discrete points, indicating that magnesium atoms are uniformly anchored on the carbon-based support at the atomic level, rather than forming nanoparticles; from Figure 1 As can be seen from the AC-HAADF-STEM image and elemental mapping in f, multiple isolated bright spots (marked with white circles) are visible in the high-resolution image, corresponding to a single Mg atom; the elemental overlay image on the right (indicated by the arrow) further confirms that these bright spots are Mg signals, clearly demonstrating that Mg is stably dispersed in the carbon substrate in the form of single atoms, exhibiting typical characteristics of a single-atom catalyst. Figure 1 The three-dimensional AFM images of g reveal the topological morphology of the MgSA surface, showing that it has a uniform nanoscale undulation structure with a consistent high degree of distribution, reflecting good surface smoothness of the material, which is beneficial to cell interaction and drug delivery performance.

[0031] Based on the above characterization results, this invention successfully constructed an enzyme-like nanomaterial (MgSA) based on magnesium single atoms, which possesses atomically dispersed Mg active sites, a stable carbon-based support structure, and uniform morphology. This material exhibits good structural integrity and uniform elemental distribution, providing a reliable foundation for subsequent realization of multi-enzyme catalytic activity and brain-targeted tumor therapy.

[0032] (V) FTIR characterization of magnesium single-atom nanozymes like Figure 2 As shown in figure a, it can be seen from the figure that the FTIR spectrum compared MgSA / NC and no 500 cm⁻¹ was detected. -1 No Mg-O peak was observed (indicated by the red arrow), confirming the absence of the typical Mg-O vibrations found in magnesium oxides or hydroxides.

[0033] (vi) X-ray diffraction (XRD) characterization of magnesium single-atom nanozymes like Figure 2 As shown in b, the XRD pattern shows that the material has an amorphous carbon structure and no characteristic diffraction peaks of crystalline magnesium were identified, further confirming the highly dispersed state of magnesium in the material.

[0034] (vii) Raman spectroscopy characterization like Figure 2 As shown in c, compared to NC, the I of the MgSA sample... D / I G The increase in the ratio indicates that the structural disorder of the carbon-nitrogen support increases and the defect density improves after the introduction of magnesium single atoms, indicating that Mg is successfully anchored on the support and may form more catalytic active sites.

[0035] (viii) X-ray photoelectron spectroscopy (XPS) of magnesium single-atom nanozymes like Figure 2 As shown in d, the sample contains magnesium (Mg), nitrogen (N), oxygen (O), and carbon (C). High-resolution XPS spectra further reveal: Figure 2 e's C1s spectrum and Figure 2 The O1s spectrum region of f shows the presence of C–N and C=O bonds, but no signal corresponding to lattice oxygen (~529.8 eV) was observed, ruling out the possibility of the presence of magnesium oxides such as magnesium oxide.

[0036] Figure 2 g's Mg1s spectrum and Figure 2 The N1s spectrum of h reveals that the material mainly contains Mg–N coordination structure and nitrogen configuration dominated by pyridine nitrogen.

[0037] Figure 2The Mg-K edge X-ray absorption near-edge structure spectrum of i indicates that magnesium exists in the material in an isolated Mg–N4 configuration, which is significantly different from the reference magnesium phthalocyanine (MgPc) and magnesium oxide (MgO).

[0038] Based on the above analysis, the MgSA synthesized in this invention is an amorphous carbon material containing highly dispersed magnesium atoms. Magnesium primarily binds to nitrogen atoms through the formation of a Mg–N4 coordination structure, exhibiting a unique chemical environment distinct from traditional magnesium-containing compounds. This structural characteristic may endow the material with unique physicochemical properties, such as enhanced graphitization and a specific electronic structure.

[0039] (ix) Enzyme activity characterization of NC and MgSA A schematic diagram of the enzyme-like catalytic mechanism of MgSA is shown below. Figure 3 As shown in i, from Figure 3 As shown in i, MgSA exhibits various enzyme activities, mainly including POD-like activity: catalyzing the reaction of H2O2 with TMB to generate a colorimetric product; CAT-like activity: decomposing H2O2 to generate O2 and H2O; and OXD-like activity: catalyzing the reduction of O2 to generate ·O2. - NADH-like oxidase activity: catalyzes the oxidation of NADH to NAD. + And it produces H2O2.

[0040] from Figure 3 As shown in section a, the reaction uses H2O2 as a substrate, indicating that MgSA has a higher maximum reactivity. Rate (V) max =9.208×10 -5 Ms -1 and a lower Michaelis constant (K) m =9.30 mM), indicating that it has a stronger affinity and catalytic efficiency for H2O2 compared to NC.

[0041] from Figure 3 As shown in b, MgSA exhibits a higher V max =4.137×10 -7 Ms -1 and smaller K m = 0.070mM, indicating that its catalytic efficiency for TMB is significantly better than that for NC.

[0042] from Figure 3 As shown in c, the reaction rate of MgSA with TMB as substrate was measured at different concentrations, yielding V. max =1.036×10 -7 Ms -1 K m=0.121 mM, indicating that MgSA can effectively catalyze the generation of superoxide anions.

[0043] from Figure 3 As shown in d, V is obtained by measuring the solubility of oxygen produced by the decomposition of H2O2. max = 10.123 mg / L·min, K m = 0.357 M, confirming the ability of MgSA to efficiently decompose H2O2.

[0044] from Figure 3 As shown in equation e, after the addition of MgSA, the characteristic absorption peak of NADH at 340 nm decreased significantly, while the characteristic absorption peak of NAD at 260 nm appeared. + The absorption peak indicates that MgSA can effectively catalyze the oxidation of NADH to NAD. + .

[0045] The degradation process of methylene blue (MB) dye was monitored by ultraviolet-visible (UV-vis) spectroscopy to investigate the ability of activated MgSA nanozymes to continuously generate reactive oxygen species (ROS). Results are as follows: Figure 3 As shown in f, the MgSA-catalyzed colorimetric reaction of MB exhibits a concentration-dependent effect. With increasing H₂O₂ concentration, the absorbance of MB significantly decreases in the presence of MgSA nanozymes. In this context, MB can serve as a reference for verifying the generation of ·OH radicals in the system. The ·OH radicals generated in the system degrade MB, thereby weakening its optical signal.

[0046] To further confirm the OXD-like activity of MgSA, electron spin resonance (ESR) technology, which exhibits excellent sensitivity and specificity, was used to directly detect O2•-. Using DMPO (5,5-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent, a characteristic DMPO / O2•- adduct was formed. The results are as follows: Figure 3 g and Figure 3 As shown in h, from Figure 3 As shown in the data, after 10 minutes of MgSA addition, the MgSA group exhibited a significant quadruple signal, while the NC and control groups showed no significant signal, confirming that MgSA possesses hydroxyl radical and superoxide radical production capabilities, thus demonstrating that MgSA exhibits oxidase-like activity. Figure 3 As can be seen from h, the DMPO-O2•- signal intensity gradually increased over time, indicating that O2•- was continuously generated, which verified that the continuous oxidase-like activity of MgSA increased with time (0-10 min).

[0047] II. Performance Testing of Magnesium Single-Atom Nanoenzymes After Polyethylene Glycol Treatment The MgSA (magnesium single-atom nanozyme) prepared above was modified by stirring in DMSO at a mass ratio of 1:1 in DMSO at room temperature for 12 hours. Unreacted PEG and residual DMSO were then removed by dialysis to purify the modified product. Finally, the target product was obtained by freeze-drying, and MgSA coated with DSPE-PEG-COOH was named pMgSA.

[0048] (a) Zeta potential analysis The obtained pMgSA was subjected to Zeta potential testing. The results are as follows: Figure 4 Figure a shows that the Zeta potential of MgSA is approximately +27.47 mV, indicating that its surface is positively charged; while pMgSA (MgSA surface coated with DSPE-PEG) 2000 The zeta potential of the modified DSPE-PEG decreased to approximately -30.8 mV, exhibiting significant electronegativity. This change confirms the presence of DSPE-PEG. 2000 -COOH was successfully coated onto the MgSA surface, and DSPE-PEG was then coated onto the MgSA surface. 2000 The addition of -COOH can effectively improve its hydrophilicity and stability, which is beneficial for its subsequent dispersion and recycling in biological systems.

[0049] (ii) Stability test The pMgSA was subjected to stability testing. MgSA and pMgSA solutions with a concentration of 1 mg / ml were prepared, allowed to stand for 48 hours, and the suspension states of MgSA (a) and pMgSA (b) were observed and imaged at different time points (0-48 h). The results are shown below. Figure 4 As shown in b, unmodified MgSA began to show obvious precipitation after 12 h and almost completely settled by 48 h; while pMgSA remained uniformly dispersed throughout the process, without obvious aggregation or sedimentation.

[0050] Based on the results in (I) and (II) above, the polyethylene glycol treatment of magnesium single-atom nanozymes can significantly improve the long-term stability of nanozymes in aqueous phase, which helps to enhance their circulation time and bioavailability when used in vivo.

[0051] (III) Effects of pMgSA on intracellular ROS levels and MitoSOX 1. Intracellular reactive oxygen species detection U87MG cells were spaced at 2 × 10⁻⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours to ensure complete adhesion. Cells were then treated with different methods and incubated for 0, 6, or 12 hours respectively. After treatment, cells were digested with trypsin, centrifuged, and transferred to flow cytometry tubes. The cell pellet was resuspended in serum-free medium containing DCFH-DA (10 μM) dye and incubated at 37 °C in the dark for 30 minutes, gently mixing every 3-5 minutes to ensure uniform staining. After incubation, cells were washed once with PBS and centrifuged.

[0052] Finally, cells were resuspended in 500 μL PBS for flow cytometry analysis. Data acquisition was performed using a NovoCyte 2060R flow cytometer equipped with a NovoSamplerProNS200 (Agilent Technologies), and analysis was performed using the instrument's built-in software. Results are as follows: Figure 5 As shown in Figure a, treatment with nanozymes MgSA and pMgSA can significantly increase the level of reactive oxygen species (ROS) in U87MG glioma cells.

[0053] Further analysis of ROS levels in cells treated with pMgSA at different time points (0, 6, and 12 h) yielded the following results: Figure 5 As shown in b, a time-dependent increase in ROS was observed at different time points, confirming its excellent ability to produce reactive oxygen species.

[0054] 2. Flow cytometry detection of MitoSOX U87MG cells were spaced at 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours to ensure complete adhesion. pMgSA was then added, and incubation continued for 0, 6, or 12 hours. After treatment, cells were digested with trypsin, collected by centrifugation, and the supernatant was discarded. The cell pellet was resuspended in 1 mL of MitoSOX™ working solution (1 μM in serum-free medium) and incubated at 37°C in the dark for 30 minutes, gently mixing every 3-5 minutes to ensure uniform staining. After incubation, cells were washed once with cold PBS, centrifuged, and resuspended in 500 μL of PBS for flow cytometry analysis. Data acquisition was performed using a NovoCyte 2060R flow cytometer (Agilent Technologies) equipped with a NovoSampler ProNS200 autosampler, and analysis was performed using the instrument's built-in software (excitation / emission wavelength = 488 / 510 nm). Results are as follows: Figure 5 As shown in Figure c, time-dependent increases in ROS were observed at different time points (0, 6, and 12 h), confirming its excellent mitochondrial ROS capacity.

[0055] The above processes (measurement of intracellular reactive oxygen species (ROS) levels and MitoSOX) were observed using laser confocal microscopy (CLSM) co-localization technology. The results are as follows: Figure 5 As shown in d, from Figure 5 As shown in d, the fluorescence signals of Total ROS-Green and MitoSOX-Red significantly increased with the extension of treatment time (6 h to 12 h), indicating that pMgSA continuously catalyzes the production of reactive oxygen species intracellularly, exhibiting a typical time-dependent oxidative stress effect.

[0056] This indicates that pMgSA can induce significant oxidative stress. Total ROS and MitoSOX assays showed that the total ROS and mitochondrial ROS in the pMgSA-treated group increased significantly over time. This is consistent with the peroxidase-like (POD-like) and oxidase-like (OXD-like) activities of MgSA, which can catalyze the generation of highly toxic •OH and promote O2•- generation. Nanozymes can continuously generate ROS intracellularly through enzyme-like catalytic reactions, causing oxidative damage. The above demonstrates that polyethylene glycol modification of magnesium single-atom nanozymes (pMgSA) does not affect the peroxidase-like (POD-like) and oxidase-like (OXD-like) activities of the nanozymes (MgSA).

[0057] Example 1 DSPE-PEG-Mal (phospholipid-polyethylene glycol-maleimide, with a molecular weight of Mw=2000 for polyethylene glycol) was linked to thiol-containing Angiepep-2 at a molar ratio of 1:3 and reacted for 12 h under inert gas protection. After dialysis and lyophilization, DSPE-PEG-Angipep-2 was obtained. 10 mg of DSPE-PEG-Angiopep-2 was dissolved in 15 mL of DMSO to obtain solution A. 2 mg of magnesium single-atom nanozyme MgSA powder was dissolved in 5 mL of DMSO and ultrasonically dispersed. This dispersion was then added dropwise to solution A and stirred for 24 h. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 12 hours to remove unreacted DSPE-PEG-Angiopep-2 and residual DMSO. The dialysate was changed every 2 hours. The resulting product was lyophilized to obtain magnesium single-atom nanozyme Ang-pMgSA linked with a targeting antibody.

[0058] Example 2 The difference between this embodiment and Embodiment 1 is the molar ratio of DSPE-PEG-Mal to Anpepp-2. In this embodiment, the molar ratio of DSPE-PEG-Mal to Anpepp-2 is 1:1, and the remaining steps are the same as in Embodiment 1.

[0059] Example 3 The difference between this embodiment and Embodiment 1 is the molar ratio of DSPE-PEG-Mal to Anpepp-2. In this embodiment, the molar ratio of DSPE-PEG-Mal to Anpepp-2 is 1:6, and the remaining steps are the same as in Embodiment 1.

[0060] Example 4 The difference between this embodiment and Embodiment 1 is the mass ratio of DSPE-PEG-Angiopep-2 to magnesium single-atom nanozyme MgSA. In this embodiment, the mass ratio of DSPE-PEG-Angiopep-2 to magnesium single-atom nanozyme is 3:1. The remaining steps are the same as in Embodiment 1.

[0061] Example 5 The difference between this embodiment and Embodiment 1 is the mass ratio of DSPE-PEG-Angiopep-2 to magnesium single-atom nanozyme. In this embodiment, the mass ratio of DSPE-PEG-Angiopep-2 to magnesium single-atom nanozyme MgSA is 7:4. The remaining steps are the same as in Embodiment 1.

[0062] Effect verification

[0063] I. Cellular Experiments with Magnesium Single-Atom Nanozymes (a) Cytotoxicity test The CCK-8 cytotoxicity assay was used. U87MG cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [insert density here] in 96-well plates and incubated for 24 hours to allow complete cell adhesion. The medium was then replaced with fresh complete medium containing different concentrations (0, 3.5, 6.5, 12.5, 25, 50, 100, and 200 μg / mL) of pMgSA and Ang-pMgSA, and incubated for another 24 hours. After treatment, the cells were gently washed three times with PBS. Then, 100 μL of fresh complete medium containing 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 °C in the dark for 30 minutes. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula:

[0064] Where A S A represents the absorbance of the sample processing well. b A represents the absorbance of the blank control (containing only culture medium, without cells or nanoparticles). c The absorbance corresponding to the negative control (cells cultured in a medium without nanoparticles).

[0065] The results are as follows Figure 6As shown in Figure a, after 24 hours of treatment with different concentrations of pMgSA and Ang-pMgSA, the viability of U87MG glioma cells decreased with increasing concentration. Both exhibited dose-dependent cytotoxicity, but Ang-pMgSA was more cytotoxic than MgSA. This indicates that linking antibodies to the surface of the polyethylene glycol-modified magnesium single-atom nanozyme enhanced its inhibitory effect on cell activity.

[0066] (II) Apoptosis test Apoptosis was quantitatively detected using the Annexin V-FITC / PI apoptosis detection kit (catalog number C1062M, Beyotime Biotechnology Co., Ltd., China) according to the manufacturer's instructions. U87MG cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at high density in 6-well plates and incubated at 37°C for 24 hours to ensure complete adhesion. After adhesion, cells were treated with different nanomaterials and co-cultured for 24 hours. After treatment, the culture supernatant was collected into centrifuge tubes, and the cells were washed twice with ice-cold PBS to remove any remaining free nanomaterials. The remaining free nanomaterials were first dissociated using EDTA-free trypsin, followed by the addition of pre-collected serum-containing culture medium to terminate the digestion reaction. The resulting cell suspension was transferred to flow cytometry tubes and centrifuged to collect the cell pellet. The cell pellet was resuspended in 195 µL of 1× binding buffer, and 5 µL of Annexin V-FITC and 10 µL of propidium iodide (PI) were added. The mixture was gently mixed and incubated on ice in the dark for 20 minutes. Samples were analyzed directly by flow cytometry without fixation. Data were acquired using an Agilent Technologies NovoCyte 2060R flow cytometer system, and analysis was performed using the instrument's built-in software.

[0067] Flow cytometry Annexin V–FITC / PI double staining results are as follows Figure 6 As shown in Figure b, treatment with Ang-pMgSA nanozyme can significantly induce the apoptosis process.

[0068] (III) Cell uptake experiment Preparation of Ang-pMgSA@Cy5: First, mix DSPE-PEG-Cy5 and DSPE-PEG-Angiopep-2 at a mass ratio of 1:5 as solution A, and then mix it with solution B (MgSA) (the specific process is the same as in "III. Targeting antibody linked to the surface of magnesium single-atom nanoenzyme via phospholipid polyethylene glycol") to prepare Ang-pMgSA@Cy5.

[0069] Preparation of pMgSA@Cy5: MgSA and DSPE-PEG-Cy5 were mixed at a mass ratio of 1:1 and prepared according to the same scheme (the specific process is the same as in "III. Targeting antibody linked to the surface of magnesium single-atom nanoenzyme via phospholipid polyethylene glycol") to obtain pMgSA@Cy5.

[0070] Cell culture: U-87 MG-Luc cell lines were cultured in high-glucose DMEM supplemented with 1% penicillin-streptomycin, non-essential amino acids, and 10% fetal bovine serum (FBS). Cells were cultured in a humidified incubator at 37°C, 95% relative humidity, and 5% CO2. When cell confluence reached approximately 90%, cells were cultured using TrypLE. TM Digestion was performed using Express digestion solution (Gibco). This was followed by addition of complete medium containing FBS (volume ratio 1:2, i.e., TrypLE). TM Neutralize the enzymatic digests with culture medium. After collecting the cell suspension, centrifuge at 3000 rpm for 3 minutes. Resuspend the resulting cell pellet in fresh complete culture medium for subsequent passage or experimental use.

[0071] Cell uptake: U87MG cells were introduced at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per confocal culture dish and cultured overnight to allow them to adhere. Cells were then treated with free Cy5, pMgSA@Cy5, and Ang-pMgSA@Cy5 (100 μg / mL each) for 4 hours. After incubation, cells were washed three times with 1×PBS and fixed with ice-cold 4% paraformaldehyde for 30 minutes. After fixation, cells were rinsed 2-3 times with PBS (10 minutes each time) and then permeabilized with 0.5% Triton X-100 for 15 minutes. Finally, cells were incubated with Actin-Tracker Green-488 for 10 minutes at room temperature in the dark. Finally, the internalization of cells with different formulations was observed using a confocal laser scanning microscope (CLSM). Results are as follows: Figure 7 As shown in the figure. Figure 7 In the images, cell nuclei were stained with DAPI (blue), materials were labeled with Cy5 (red), and the cytoskeleton was stained with FITC-phalloidin (green). From... Figure 7 As can be seen, Ang-pMgSA@Cy5 has a significantly enhanced ability to target cell uptake. Compared with unmodified Ang (Angiopep-2) pMgSA@Cy5, the vascular-targeting peptide-modified Ang-pMgSA@Cy5 showed a stronger fluorescence signal in U87 glioma cells.

[0072] The cell uptake of pMgSA@Cy5 at different concentrations (12.5, 25, 50, 100 μg / ml) was investigated. U87MG cells were cultured at 2 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells per confocal culture dish and cultured overnight to allow adhesion. Cells were then treated with different concentrations of pMgSA@Cy5 (12.5, 25, 50, 100 μg / ml) and incubated for 4 hours. After incubation, cells were washed three times with 1×PBS and fixed with ice-cold 4% paraformaldehyde for 30 minutes. After fixation, cells were rinsed 2-3 times with PBS (10 minutes each time) and then permeabilized with 0.5% Triton X-100 for 15 minutes. Finally, cells were incubated with Actin-Tracker Green-488 for 10 minutes at room temperature in the dark, followed by counterstaining with DAPI for 10 minutes. Finally, cell uptake at different concentrations was observed using a confocal laser scanning microscope (CLSM). Results are as follows: Figure 8 As shown in Figure a, pMgSA@Cy5 can be effectively taken up by glioma cells, and the cellular uptake of pMgSA@Cy5 is concentration-dependent.

[0073] To investigate the cellular uptake of pMgSA@Cy5 at different time points, cells were co-incubated with pMgSA@Cy5 at a concentration of 100 μg / ml, and then the cellular uptake of the formulation at different time points was observed using a confocal laser scanning microscope (CLSM) (experimental procedure as above). The results are as follows. Figure 8 As shown in b, pMgSA@Cy5 can be effectively taken up by glioma cells, and the cellular uptake of pMgSA@Cy5 is time-dependent.

[0074] The cellular uptake capacity of pMgSA@Cy5 and Ang-pMgSA@Cy5 was investigated. U87MG cells were loaded at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per confocal culture dish and cultured overnight to promote cell adhesion. Cells were then treated with free Cy5, pMgSA@Cy5, and Ang-pMgSA@Cy5 (100 μg / mL each) and incubated for 4 hours. After incubation, cells were washed three times with 1×PBS and fixed with ice-cold 4% paraformaldehyde for 30 minutes. After fixation, cells were rinsed 2-3 times with PBS (10 minutes each time) and permeabilized with 0.5% Triton X-100 for 15 minutes. Cells were then incubated with Actin-TrackerGreen-488 at room temperature in the dark for 30 minutes, followed by counterstaining with DAPI for 10 minutes. Finally, cell uptake of the material was observed using a confocal laser scanning microscope (CLSM). Results are as follows: Figure 8As shown in Figure c, Ang-pMgSA@Cy5 exhibits stronger cellular uptake capacity compared to pMgSA@Cy5.

[0075] The cellular uptake efficiency of pMgSA@Cy5 and Ang-pMgSA@Cy5 was further analyzed by flow cytometry. The results are as follows: Figure 8 As shown in d, it can be seen that pMgSA@Cy5, after being modified with Ang peptide (Angipep-2), can effectively improve the uptake efficiency of nanozymes by cells.

[0076] II. In vivo experiments of magnesium single-atom nanozymes (i) Permeability test of nanozymes in three-dimensional tumor spherical models Three-dimensional tumor spheroids were prepared by seeding U87MG cells at a density of 3 × 10³ cells per well in 96-well ultra-low adhesion round-bottom plates. The culture plates were incubated at 37°C and 5% CO2 humidity for 48 hours, during which the cells aggregated to form dense, spherical tumor spheroids. Before treatment, the original culture medium was removed, and the spheroids were gently rinsed once with PBS. Fresh culture medium containing free Cy5, pMgSA@Cy5, and Ang-pMgSA@Cy5 (100 µg / mL) was then added, and incubation was continued for 4 hours. After incubation, the spheroids were gently rinsed three times with PBS to remove free nanoparticles. Fluorescence imaging was performed using a confocal laser scanning microscope (CLSM, Zeiss LSM980) with a 10x objective. The penetration efficiency of different Cy5-labeled nanocarriers into the tumor spheroids was evaluated by analyzing the intensity and distribution of Cy5 fluorescence within the spheroid structure.

[0077] The results are as follows Figure 9 3D cell model confocal images of b and Figure 9 The Cy5 fluorescence intensity at different depths of c is shown, combined with Figure 9 b and Figure 9 c indicates that pMgSA@Cy5 is only enriched on the surface, while free Cy5 has almost no permeability, suggesting that modification with Ang peptide (Angipep-2) can significantly enhance the permeability of nanozyme (Ang-pMgSA) in dense tumor tissue.

[0078] like Figure 9 As shown in figure a, confocal depth scanning (0-70 μm) shows that Ang-pMgSA@Cy5 can uniformly penetrate the entire U87 tumor organoid with a diameter of approximately 70 μm, and still maintains the strongest fluorescence signal at a depth of 50 μm.

[0079] (II) Plasma Pharmacokinetics Free Cy5, pMgSA@Cy5, and Ang-pMgSA@Cy5 were each prepared to a concentration of 1 mg / mL and dissolved in sterile physiological saline. 200 μL of each formulation was administered intravenously via tail vein to healthy BALB / c mice (n=3 per group). Blood samples (approximately 50 μL) were collected from the retroorbital sinus at predetermined time points after injection (0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours). Immediately after collection, each blood sample was mixed with 100 μL of lysis buffer containing 1% (v / v) Triton X-100 (Sigma, USA) to lyse red blood cells and release the fluorescent agent. Subsequently, 0.5 mL of dimethyl sulfoxide (DMSO) was added to each sample, and the mixture was incubated overnight to ensure complete dissolution and stabilize the fluorescence signal. After incubation, cell debris was removed by centrifugation at 15,000 rpm for 10 minutes. The supernatant was transferred to black 96-well plates, and fluorescence intensity was measured using a microplate reader (excitation / emission wavelength = 630 / 670 nm). The fluorescence signals were converted to concentrations of each Cy5-labeled compound in whole blood according to a pre-defined standard curve. All formulations were processed using the same method. Plasma concentration-time curves were generated for each formulation. Nonlinear regression analysis was performed using OriginPro software (OriginLab, USA) to determine the concentrations including elimination half-life (t0). 1 / 2 Pharmacokinetic parameters, including those mentioned above. The best fit of the data is a phase exponential decay model described by the following equation:

[0080] The results are as follows Figure 9 As shown in d, Ang-pMgSA@Cy5 has the longest circulating half-life (t). 1 / 2 = 70.33 min), much higher than pMgSA@Cy5 (t 1 / 2 = 55.0 min) and free Cy5 (t 1 / 2 = 18.4 min), indicating that it has excellent plasma stability and prolonged in vivo circulation time.

[0081] (III) In vivo fluorescence imaging in mice Free Cy5, pMgSA@Cy5, and Ang-pMgSA@Cy5 (10 mg / kg dissolved in 200 µL 1×PBS) were administered via tail vein injection to BALB / c nude mice orally inoculated with U87MG-Luc glioblastoma. Whole-body fluorescence imaging was performed at predetermined time points (0, 1, 2, 4, 6, 8, 12, and 24 hours post-injection) using a small animal in vivo imaging system with excitation wavelength of 605 nm and emission wavelength of 670 nm to assess the biodistribution and tumor accumulation of the Cy5-labeled formulation. Results are as follows: Figure 9As shown in f, in vivo fluorescence imaging in mice (0-24 h) showed that Ang-pMgSA@Cy5 was continuously and efficiently enriched at the intracranial tumor site and maintained a strong signal after 24 h; while the signals of pMgSA@Cy5 and free Cy5 decayed rapidly, indicating that Ang modification can endow nanozymes with long-term and specific brain tumor targeting ability.

[0082] Further investigation into the mechanism of action of Ang-pMgSA@Cy5 in targeting gliomas yielded the following results: Figure 9 As shown in e, This indicates that Ang-pMgSA@Cy5 crosses the blood-brain barrier (BBB) ​​efficiently by binding to the LRP1 receptor on the surface of brain vascular endothelial cells and then undergoing receptor-mediated transcytosis, thereby targeting gliomas.

[0083] (iv) Organ distribution and quantitative analysis Free Cy5, pMgSA@Cy5, and Ang-pMgSA@Cy5 (10 mg / kg dissolved in 200 µL 1×PBS) were administered via tail vein injection to BALB / c nude mice bearing orthotopic U87MG-Luc glioblastoma. Four hours after injection, the mice were sacrificed, and major organs such as the heart, liver, spleen, lungs, kidneys, and tumor-bearing brain were rapidly collected and rinsed with cold 1×PBS to remove residual blood. In vivo imaging systems (such as IVIS Spectrum or similar devices) were used with a Cy5 detection filter set (λEx = 630 nm, λEm = 670 nm). The fluorescence imaging results of major organs (ex vivo) are as follows: Figure 9 As shown in g. To further quantify the biodistribution of Cy5, each tissue sample was homogenized using a high-throughput tissue homogenizer (60 Hz, 10 min) in 0.6 mL of lysis buffer containing 1% Triton X-100. Subsequently, 0.8 mL of dimethyl sulfoxide (DMSO) was added to each homogenate, and the samples were incubated overnight at room temperature in the dark to ensure complete extraction of the Cy5 labeling material. The next day, the homogenates were centrifuged (e.g., 12000 × g, 10 min, 4 °C), and the supernatant was collected for fluorescence detection. The concentration of Cy5 in each tissue was determined by fluorescence spectroscopy according to a standard calibration curve, and expressed as a percentage of the injection dose per gram of tissue (% ID g). -1 Biodistribution is expressed as a percentage of the injection dose per gram of tissue (% ID g). -1 The formula is expressed as: [(fluorescence-derived CD content per gram of tissue) / (total injection dose)] × 100%. The result is as follows: Figure 9 Quantitative analysis of Cy5 content in various organs of h is shown. (Ex vivo organ imaging) Figure 9 g) and quantitative (% ID / g) analysis ( Figure 9h) Further confirmation showed that Ang-pMgSA@Cy5 accumulated significantly more in the brain than other groups (p < 0.0001), and was significantly better than free Cy5 (which could hardly enter the brain); at the same time, it was distributed in the liver, but the renal uptake was low, suggesting good biodistribution characteristics and potential safety.

[0084] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0085] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0086] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A magnesium single-atom nanozyme with multi-enzyme activity, characterized in that, The magnesium single-atom nanozyme uses a nitrogen-doped carbon material loaded with magnesium single atoms as the core, and connects brain-targeting ligands to its surface through polyethylene glycol as a linker, thus obtaining a brain-targeting ligand-modified magnesium single-atom nanozyme.

2. The magnesium single-atom nanozyme with multi-enzyme activity according to claim 1, characterized in that, The brain-targeting ligand is an antibody, antibody fragment, or peptide.

3. The magnesium single-atom nanozyme with multi-enzyme activity according to claim 2, characterized in that, The antibody or peptide is a molecule that can specifically bind to the LRP1 receptor.

4. The magnesium single-atom nanozyme with multi-enzyme activity according to claim 3, characterized in that, The peptide molecule is Angiopep-2 peptide or a derivative thereof; or the antibody molecule is Angiopep-2 monoclonal antibody.

5. A method for preparing a magnesium single-atom nanozyme with multi-enzyme activity as described in claim 1, characterized in that, Includes the following steps: S1: The zinc source solution and the imidazole organic ligand solution were stirred and mixed. The resulting mixed solution was centrifuged, precipitated, and dried to obtain the zeolite imidazole ester framework structure material. S2: The zeolite imidazole ester framework material was placed in a tube furnace and pyrolyzed at high temperature in an inert atmosphere to obtain NC-doped material. After activation, centrifugal washing and drying, nanoporous nitrogen-doped carbon material was obtained. S3: Using nitrogen-doped carbon material as a carrier, magnesium and nitrogen sources are introduced, and after self-assembly mixing, centrifugal drying, and high-temperature pyrolysis in an inert gas, magnesium single-atom nanozymes are obtained. S4: Phospholipid-polyethylene glycol with maleimide active groups at the end is reacted with a brain-targeting ligand containing thiol groups under suitable conditions for forming thioether bonds to obtain a phospholipid-polyethylene glycol-brain-targeting ligand conjugate. S5: Dissolve the phospholipid-polyethylene glycol-brain-targeting ligand conjugate in an organic solvent to obtain solution A. Dissolve the magnesium single-atom nanozyme and add it to solution A and stir to mix. After dialysis and drying, the resulting mixed solution yields the magnesium single-atom nanozyme linked to the brain-targeting ligand.

6. The method for preparing magnesium single-atom nanozymes with multi-enzyme activity according to claim 5, characterized in that, In step S1, the centrifugation conditions are: rotation speed 6000-10000 rpm, time 8-12 min; drying temperature 45-75℃; in steps S2 and S3, the high-temperature pyrolysis conditions are: temperature 900-12000 ℃, heating rate 5 ℃ / min, pyrolysis time 2-5 h.

7. The method for preparing magnesium single-atom nanozymes with multi-enzyme activity according to claim 5, characterized in that, In step S3, the mass ratio of nitrogen-doped carbon material to magnesium source and nitrogen source is 1:(1-2):(1-2).

8. The method for preparing magnesium single-atom nanozymes with multi-enzyme activity according to claim 5, characterized in that, In step S4, the molar ratio of phospholipid-polyethylene glycol to brain-targeting ligand is 1:(1-6).

9. The method for preparing magnesium single-atom nanozymes with multi-enzyme activity according to claim 5, characterized in that, The mixed solution in step S5 also includes a phospholipid-polyethylene glycol-fluorescent label, wherein the mass ratio of the phospholipid-polyethylene glycol-brain-targeting ligand to the magnesium single-atom nanozyme is (3-7):(1-4).

10. The use of a magnesium single-atom nanozyme with multi-enzyme activity according to any one of claims 1-4 in the preparation of a drug for treating glioma.

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