Surface-engineered iron-nitrogen-selenium monatomic nano-enzyme loaded with all-transretinoic acid as well as preparation method and application of surface-engineered iron-nitrogen-selenium monatomic nano-enzyme

Abstract

The invention discloses a surface-engineered iron-nitrogen-selenium monatomic nano-enzyme loaded with all-trans retinoic acid, which comprises an inner core and all-trans retinoic acid loaded on the surface of the inner core, and the inner core is a selenium-rich organic nano-enzyme inner core. The invention also discloses a corresponding preparation method and application thereof. The ATRA is loaded on the surface of the inner core, the inner core can regulate the active oxygen level in target cells so as to cooperate with all-transretinoic acid to induce acute myelogenous leukemia cell differentiation, the regulating effect of the nano-enzyme on ROS and the differentiation promoting effect of the ATRA generate a synergistic effect, the differentiation induction effect on AML cells is remarkably enhanced, and the differentiation induction effect on the AML cells is remarkably improved. The problem of drug resistance of ATRA single-drug treatment is solved. The nano-enzyme disclosed by the invention not only can effectively inhibit proliferation and infiltration of leukemia cells, but also can promote repair of a bone marrow hematopoietic microenvironment and recovery of a hematopoietic function, and provides a targeted redox differentiation enhancement treatment strategy with clinical transformation potential for acute myelogenous leukemia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of preparation and application of nanoenzymes, and particularly relates to a surface-engineered iron-nitrogen-selenium monatomic nanoenzyme loaded with all-trans retinoic acid and a preparation method and application thereof. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous and aggressive hematological malignancy. In the past few decades, the treatment of AML has made relatively limited progress, especially for non-APL subtypes, and the prognosis of patients is still poor. Differentiation induction therapy is an important strategy for treating leukemia. All-trans retinoic acid (ATRA) has achieved remarkable efficacy in the treatment of acute promyelocytic leukemia (APL) subtype, but its therapeutic effect in other AML subtypes is not ideal. This is mainly due to the existence of intrinsic or acquired resistance to ATRA in many AML subtypes. Existing studies have shown that the imbalance of reactive oxygen species (ROS) plays a key role in regulating the self-renewal, proliferation and differentiation of leukemia cells. The differentiation-promoting activity of ATRA depends on the moderate level of oxidative stress signal in cells. However, in many AML subtypes, the cell antioxidant defense system is enhanced or the redox pathway is damaged, resulting in weakened response of cells to ROS, thereby producing resistance to ATRA. Therefore, how to restore the redox homeostasis in leukemia cells and re-sensitize the cells to ATRA is a very promising strategy to overcome differentiation disorders and improve the therapeutic effect of AML.

[0003] Nanoenzymes are artificial nanomaterials with enzyme-like catalytic activity, and show great application prospects in the field of biomedicine due to their high stability, controllable catalytic activity and easy functionalization. However, there is still a lack of nanoenzyme systems that can effectively regulate the redox balance in leukemia cells and synergize with differentiation inducers to enhance the anti-leukemia effect. SUMMARY

[0004] The present application aims to provide a surface-engineered iron-nitrogen-selenium monatomic nanoenzyme loaded with all-trans retinoic acid, which can effectively regulate the redox state in acute myeloid leukemia cells and synergize with all-trans retinoic acid (ATRA) to enhance the differentiation induction treatment effect on AML, to solve the problem of poor therapeutic effect and drug resistance of ATRA in non-APL type AML in the prior art, and to provide a corresponding preparation method and application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid, comprising a core and all-trans retinoic acid loaded on the surface of the core, wherein the core is a selenium-rich organic nanozyme core.

[0006] The core of the selenium-enriched organic nanozyme is a nanostructure containing single-atom sites of iron, nitrogen, and selenium.

[0007] The all-trans retinoic acid is loaded onto the surface of the core through physical adsorption or chemical bonding; the selenium-enriched organic nanozyme core is generated by introducing a selenium source into the Fe-ZIF-8 framework to form a Se-Fe diatomic nanozyme core; the average particle size of the iron-nitrogen-selenium single-atom nanozyme is 20-200 nm.

[0008] A method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid includes the following steps: (1) Preparation of Fe-ZIF-8; (2) Preparation of FeSAzyme: After dispersing Fe-ZIF-8 in an organic solvent in step (1), it was poured into an inorganic solvent, and then an aqueous solution of surfactant and an aqueous solution of strong alkali were added in sequence. Tetraethyl orthosilicate (TEOS) solution was injected, and after stirring and reacting, the intermediate product was collected by centrifugation. The intermediate product was washed, dried, annealed, and then etched in a strong alkali etching solution to obtain FeSAzyme. (3) Preparation of Se-Fe diatomic nanozyme: FeSAzyme from step (2) was ground with a selenium source to obtain a precursor, which was then annealed to obtain Se-Fe diatomic nanozyme (DSAzyme). (4) Preparation of iron-nitrogen-selenium single-atom nanozyme: The Se-Fe diatomic nanozyme and all-trans retinoic acid (ATRA) from step (3) were dispersed in an inorganic solvent, stirred, centrifuged, and the precipitate was collected. The precipitate was washed and freeze-dried to obtain iron-nitrogen-selenium single-atom nanozyme (DSAzyme-ATRA).

[0009] In step (1), Fe-ZIF-8 is prepared by chemical precipitation.

[0010] In step (1), the preparation method of Fe-ZIF-8 is as follows: Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) is dissolved in an organic solvent, and under stirring conditions, a solution of 2-methylimidazole and acetylacetone iron is added to react. After centrifugation, the precipitate is collected, washed, and activated under vacuum to obtain Fe-ZIF-8.

[0011] In step (1), the organic solvent is methanol, and the mass ratio of zinc nitrate hexahydrate, 2-methylimidazole, and iron acetylacetone is (1.50-1.85):(6.60-8.10):(0.18-0.22). The reaction conditions are: time 22-26 h, and the vacuum activation conditions are: temperature 90-110°C, time 22-26 h. In step (2), the organic solvent is methanol, the inorganic solvent is water, the surfactant aqueous solution is CTAB aqueous solution, the strong alkali aqueous solution is NaOH aqueous solution, the strong alkali etching solution is sodium hydroxide aqueous solution, and the annealing method is as follows: under a nitrogen atmosphere, the temperature is raised to 280-320 °C at a rate of 5 °C / min and held for 1-3 h, then raised to 850-950 °C at a rate of 5 °C / min and held for 4-6 h, and then cooled; the tetraethyl orthosilicate solution is prepared by dissolving the tetraethyl orthosilicate stock solution in an organic solvent. In step 1), the ratio of Fe-ZIF-8 to the tetraethyl orthosilicate stock solution is (150-200) mg : (0.8-1.4) mL.

[0012] In step (3), the annealing conditions are as follows: under a nitrogen atmosphere, the temperature is heated to 550-650 °C at a rate of 5 °C / min, held for 1-3 h, and then cooled to obtain Se-Fe diatomic nanozyme. The selenium source is selenium powder, and the mass ratio of FeSAzyme to selenium powder is (0.5-1.5):(1-3). In step (4), the inorganic solvent is water, and the mass ratio of Se-Fe diatomic nanozyme to all-trans retinoic acid in step (3) is (0.5-5):1. During washing, dimethyl sulfoxide (DMSO) is used first, followed by water.

[0013] Application of surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid in the preparation of drugs for treating leukemia.

[0014] The leukemia is acute myeloid leukemia; the drug includes an iron-nitrogen-selenium single-atom nanozyme and a pharmaceutically acceptable carrier, and the dosage form of the drug is tablets, capsules, granules, powders, oral liquids or injections.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1) Targeted regulation of redox homeostasis: The iron-nitrogen-selenium single-atom nanozyme of the present invention contains a selenium-rich organic nanozyme core with glutathione peroxidase-like activity. The selenium-rich organic nanozyme core is a nanostructure containing iron-nitrogen-selenium (Fe-N3-Se) dual single-atom sites. This specific single-atom structure endows the nanozyme with efficient and stable glutathione peroxidase-like activity, which can reduce the excessively high ROS level in AML cells, restore redox balance, break the differentiation resistance of AML cells to ATRA, and clear the obstacles for ATRA to play its role. 2) Synergistic effect: By loading all-trans retinoic acid (ATRA) onto the surface of the core, the core can regulate the level of reactive oxygen species in the target cells to synergistically induce the differentiation of acute myeloid leukemia cells with all-trans retinoic acid, thus constructing an integrated drug delivery platform. The regulatory effect of nanozymes on ROS and the differentiation-promoting effect of ATRA produce a synergistic effect, which significantly enhances the differentiation-inducing effect on AML cells and overcomes the drug resistance problem of ATRA monotherapy. 3) Good biocompatibility: The nanozyme core constructed based on organic materials has good biocompatibility and biodegradability, reducing potential in vivo toxicity; 4) Multiple therapeutic effects: The iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid of the present invention can not only effectively inhibit the proliferation and infiltration of leukemia cells, but also promote the repair of the bone marrow hematopoietic microenvironment and the recovery of hematopoietic function, showing comprehensive therapeutic potential. It provides a novel, efficient and clinically translational targeted redox differentiation enhancement therapy strategy for the treatment of clinically challenging acute myeloid leukemia. 5) The method for preparing the composite nanozyme of the present invention has mild conditions and simple steps, and can achieve stable loading while ensuring the structural integrity and ATRA activity of the nanozyme. It has good repeatability and batch-to-batch consistency, is suitable for large-scale preparation, and significantly improves the practical application value of the composite nanozyme. Attached Figure Description

[0016] Figure 1Figures show the preparation process and structural and performance characterization results of DSAzyme-ATRA prepared in Example 1 of this invention, wherein: (a) is a schematic diagram of the synthesis route of DSAzyme-ATRA; (b)–(d) are transmission electron microscope (TEM) images of FeSAzyme, DSAzyme, and DSAzyme-ATRA, respectively; (e) is a hydration particle size distribution diagram of FeSAzyme, DSAzyme, and DSAzyme-ATRA; (f) is an aberration-corrected high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of DSAzyme-ATRA and the corresponding three-dimensional atomic resolution structure distribution diagram and two-atom site line scan intensity distribution diagram; (g) is the nitrogen adsorption-desorption isotherm of FeSAzyme, DSAzyme, and DSAzyme-ATRA; (h) is the X-ray photoelectron spectroscopy (XPS) full spectrum of DSAzyme-ATRA; (i) and (j) are Fe 2p and Se in DSAzyme-ATRA, respectively. 3d high-resolution XPS spectra; (k) is a schematic diagram of the DTNB analytical method used to detect glutathione peroxidase (GPx) catalytic activity; (l) and (m) are the GPx catalytic activity test results of DSAzyme and DSAzyme-ATRA, respectively; Figure 2 The image shows the X-ray absorption fine structure (XAFS) characterization results of DSAzyme-ATRA in Example 1 of this invention and a schematic diagram of its glutathione peroxidase (GPx)-like catalytic process, wherein: (a) is the normalized spectrum of Se K-edge X-ray absorption near-edge structure (XANES); (b) is the R-space spectrum of Se K-edge Fourier transform extended X-ray absorption fine structure (FT-EXAFS); (c) is the k-space spectrum of Se K-edge EXAFS; (d) is the normalized spectrum of Fe K-edge XANES; (e) is the R-space spectrum of Fe K-edge FT-EXAFS; (f) is the k-space spectrum of Fe K-edge EXAFS; (g)–(i) are the Se K-edge wavelet transform (WT) contour plots of DSAzyme-ATRA, Se foil, and SeO2, respectively; (j)–(l) are the Fe K-edge wavelet transform (WT) contour plots of DSAzyme-ATRA, Fe foil, and FePC, respectively. K-side WT contour plot; (m) is a schematic diagram of the GPx-like reaction pathway involving DSAzyme-ATRA; (n) is a schematic diagram of the free energy change of the GPx-like reaction under Fe-N4 and Fe-N3-Se configurations; (o) and (p) are the projected density of states (PDOS) analysis results for Fe-N4 and Fe-N3-Se configurations, respectively. Figure 3This invention relates to the differentiation of leukemia cells under the action of DSAzyme-ATRA, the maturation of myeloid cells in mouse bone marrow, and the results of Example 1 of this invention. Tg (drl:hoxa9) The experimental results of hematopoietic status in transgenic zebrafish are shown in the figures, where: (a) and (b) are flow cytometry results of DSAzyme-ATRA uptake in U937 cells and HL-60 cells, respectively; (c) and (d) are morphological observation results of U937 cells and HL-60 cells after DSAzyme-ATRA treatment, respectively; (e) and (f) are flow cytometry analysis results of CD11b expression in U937 cells and HL-60 cells, respectively; (g) is a schematic diagram of the treatment process of mouse femoral bone marrow cells; (h) and (i) are flow cytometry analysis results of CD33⁺ and CD11b⁺ cell populations in bone marrow, respectively; (j) is... Tg (drl:hoxa9) Schematic diagram of the experimental procedure for DSAzyme-ATRA administration in transgenic zebrafish embryos; (k) represents the process of DSAzyme-ATRA administration in zebrafish embryos. cmyb , mpx and mfap4 The overall in situ hybridization detection results for expression status are shown in the figure. Figure 4 Figure 1 shows the experimental analysis results of the relationship between CXCR5-related signaling pathways, thioredoxin reductase (TrxR) activation, and DSAzyme-ATRA-induced differentiation of acute myeloid leukemia cells in Example 1 of this invention. Specifically: (a) is a statistical result of differentially expressed genes in the transcriptome of U937 cells after treatment; (b) is a result of gene set enrichment analysis (GSEA) of differentially expressed genes; (c) is a heatmap analysis result of some differentially expressed genes; (d) is a result of sorting differentially expressed genes by log2 fold change; (e) is the RT-qPCR verification result of transcriptional levels of some genes; (f) is the detection result of Cxcr5 expression in transgenic mouse and wild-type mouse cells; (g) is the flow cytometry analysis result of CD11b expression in U937 cells under CXCR5 gene interference conditions; (h) and (i) are the detection results of TrxR activity in U937 cells and CXCR5-interfered cells under different treatment conditions, respectively. Figure 5 This invention relates to Example 1 of the study, which describes the application of DSAzyme-ATRA and all-trans retinoic acid (ATRA) in acute myeloid leukemia cells and transgenic cells. The results are as follows: (a) and (b) show the morphological observations of U937 cells and HL60 cells after different treatments, respectively; (c) and (d) show the flow cytometry results of the CD11b⁺ cell ratio in U937 cells and HL60 cells, respectively; (e) shows the CD11b⁺ cell ratio in U937 cells after different treatments; and (f) shows the results of the transgenic cells in zebrafish embryos. cmyb , mpxand mfap4 (g) Results of in situ hybridization detection of expression status; (h) Results of immunoblotting detection of PML–RARα protein level in NB4 cells; (h) Statistical results of differentially expressed genes in the transcriptome of U937 cells under different treatment conditions; (i) Graph of zebrafish embryo morphology observation results. Figure 6 Figure 1 shows the experimental results of the therapeutic effect of DSAzyme-ATRA in an in vivo model of acute myeloid leukemia in Example 1 of this invention. (a) is a schematic diagram of the animal model construction and drug administration process; (b) shows the statistical results of animal survival in different treatment groups; (c) shows the statistical results of changes in animal body weight; (d) and (e) are the flow cytometry analysis results of the proportions of CD11b⁺ and CD33⁺ cells in peripheral blood, respectively; (f) and (g) are the results of peripheral blood leukocyte and platelet counts, respectively; (h) and (i) are the results of CD33 immunohistochemical staining and TUNEL staining in spleen and liver tissues, respectively; (j)–(l) are the results of animal serum biochemical index detection, respectively; and (m) shows the statistical results of the survival of the subcultured transplanted animals. Figure 7 The figure shows the experimental results of DSAzyme-ATRA on the recovery of hematopoietic function after whole-body radiation in Example 1 of the present invention, wherein: (a) is a schematic diagram of the animal drug treatment process after whole-body radiation; (b) is the statistical result of the animal weight change; (c)-(e) are the white blood cell, red blood cell and platelet count results respectively; (f) is a schematic diagram of the sorting strategy of hematopoietic stem cell population flow cytometry analysis; (g)-(j) are the statistical analysis results of the number of different hematopoietic cell subsets respectively. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0018] Example 1 A surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid comprises a core and all-trans retinoic acid loaded on the surface of the core. The core is a selenium-enriched organic nanozyme core, which is a nanostructure containing iron-nitrogen-selenium single-atom sites. The all-trans retinoic acid is loaded onto the surface of the core through physical adsorption or chemical bonding. The selenium-enriched organic nanozyme core is generated by introducing a selenium source into the Fe-ZIF-8 framework to form a Se-Fe diatomic nanozyme core. The average particle size of the surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid is 20–200 nm.

[0019] A method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid includes the following steps: (1) Preparation of Fe-ZIF-8: 1.68 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was dissolved in 80 mL of methanol. At room temperature and with a stirring speed of 1000 rpm, a solution of 2-methylimidazole and ferric acetylacetone (prepared by dissolving 7.40 g of 2-methylimidazole and 0.2 g of ferric acetylacetone (Fe(acac)2) in 80 mL of methanol) was rapidly added. After reacting for 24 h, the mixture was centrifuged at 10000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with 40 mL of methanol each time. The mixture was then activated under vacuum at 100 °C for 24 h to obtain Fe-ZIF-8. (2) Preparation of FeSAzyme: Fe-ZIF-8 from step (1) was dispersed in 120 mL of methanol and sonicated for 10 min at an ultrasonic power of 100 W. Then, it was quickly poured into 120 mL of water, followed by 6 mL of 25 mg / mL CTAB aqueous solution and 10 mL of 6 mg / mL NaOH aqueous solution. 7.2 mL of tetraethyl orthosilicate (TEOS) solution (1.2 mL of TEOS stock solution dissolved in 6 mL of methanol) was injected. After stirring for 2 h, the mixture was centrifuged at 10000 rpm for 10 min. The intermediate product was collected, washed with 40 mL of ethanol, and dried under vacuum at 100 °C. The dried powder was placed in a tube furnace and heated to 300 °C at a rate of 5 °C / min under a nitrogen atmosphere, held for 2 h, then heated to 900 °C at a rate of 5 °C / min, held for 5 h. After cooling, the material was etched in 6 mol / L NaOH aqueous solution at 60 °C for 2 hours. h is used to remove the silica coating layer to obtain FeSAzyme; (3) Preparation of Se-Fe diatomic nanozyme: FeSAzyme from step (2) and selenium powder were mechanically ground at a mass ratio of 1:2 to obtain a precursor. The precursor was evenly spread in a ceramic boat and heated to 600 °C at a rate of 5 °C / min under a nitrogen atmosphere. The temperature was maintained for 2 h and then cooled to room temperature to obtain Se-Fe diatomic nanozyme (DSAzyme). (4) Preparation of surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid: The Se-Fe diatomic nanozyme and all-trans retinoic acid (ATRA) from step (3) were co-dispersed in water at a mass ratio of 2:1. After ultrasonic treatment for 10 min at an ultrasonic power of 100 W, the mixture was stirred continuously for 8 h at room temperature to promote coordination. The mixture was centrifuged at 10000 rpm for 10 min, and the precipitate was collected. The precipitate was washed with 20 mL of dimethyl sulfoxide (DMSO) to remove free ATRA, and then washed twice with water. After freeze-drying, the iron-nitrogen-selenium single-atom nanozyme (DSAzyme-ATRA) was obtained. The synthetic route is as follows: Figure 1 As shown in (a).

[0020] Application of surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid in the preparation of drugs for the treatment of acute myeloid leukemia.

[0021] The drug includes iron-nitrogen-selenium single-atom nanozymes and pharmaceutically acceptable carriers, and the dosage form of the drug is tablets, capsules, granules, powders, oral liquids or injections.

[0022] Example 2 A surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid comprises a core and all-trans retinoic acid loaded on the surface of the core. The core is a selenium-enriched organic nanozyme core, which is a nanostructure containing iron-nitrogen-selenium single-atom sites. The all-trans retinoic acid is loaded onto the surface of the core through physical adsorption or chemical bonding. The selenium-enriched organic nanozyme core is generated by introducing a selenium source into the Fe-ZIF-8 framework to form a Se-Fe diatomic nanozyme core. The average particle size of the surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid is 20–200 nm.

[0023] A method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid includes the following steps: (1) Preparation of Fe-ZIF-8: 1.50 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was dissolved in 80 mL of methanol. At room temperature and with a stirring speed of 800 rpm, a solution of 2-methylimidazole and ferric acetylacetone (prepared by dissolving 7.00 g of 2-methylimidazole and 0.18 g of ferric acetylacetone (Fe(acac)2) in 80 mL of methanol) was rapidly added. After reacting for 22 h, the mixture was centrifuged at 10000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with 40 mL of methanol each time. The mixture was then activated under vacuum at 90 °C for 22 h to obtain Fe-ZIF-8. (2) Preparation of FeSAzyme: Fe-ZIF-8 from step (1) was dispersed in 120 mL of methanol and sonicated for 10 min at an ultrasonic power of 100 W. Then, it was quickly poured into 120 mL of water, followed by 6 mL of 25 mg / mL CTAB aqueous solution and 10 mL of 6 mg / mL NaOH aqueous solution. 7.2 mL of tetraethyl orthosilicate (TEOS) solution (0.8 mL of TEOS stock solution dissolved in 6 mL of methanol) was injected. After stirring for 2 h, the mixture was centrifuged at 10000 rpm for 10 min. The intermediate product was collected, washed with 40 mL of ethanol, and dried under vacuum at 100 °C. The dried powder was placed in a tube furnace and heated to 320 °C at a rate of 5 °C / min under a nitrogen atmosphere. The temperature was maintained for 1 h, then increased to 950 °C at a rate of 5 °C / min and maintained for 4 h. After cooling, the material was etched in a 6 mol / L NaOH aqueous solution at 60 °C. h is used to remove the silica coating layer to obtain FeSAzyme; (3) Preparation of Se-Fe diatomic nanozyme: FeSAzyme from step (2) and selenium powder were mechanically ground at a mass ratio of 1:3 to obtain a precursor. The precursor was evenly spread in a ceramic boat and heated to 600 °C at a rate of 5 °C / min under a nitrogen atmosphere. The temperature was maintained for 2 h and then cooled to room temperature to obtain Se-Fe diatomic nanozyme (DSAzyme). (4) Preparation of surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid: The Se-Fe diatomic nanozyme and all-trans retinoic acid (ATRA) in step (3) were co-dispersed in water at a mass ratio of 3:1. After ultrasonic treatment for 10 min at an ultrasonic power of 100 W, the mixture was stirred continuously for 8 h at room temperature to promote coordination. The mixture was centrifuged at a speed of 10000 rpm for 10 min, and the precipitate was collected. The precipitate was washed with 20 mL of dimethyl sulfoxide (DMSO) to remove free ATRA. The mixture was then washed twice with water and freeze-dried to obtain iron-nitrogen-selenium single-atom nanozymes (DSAzyme-ATRA).

[0024] Application of surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid in the preparation of drugs for the treatment of acute myeloid leukemia.

[0025] The drug includes iron-nitrogen-selenium single-atom nanozymes and pharmaceutically acceptable carriers, and the dosage form of the drug is tablets, capsules, granules, powders, oral liquids or injections.

[0026] Example 3 A surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid comprises a core and all-trans retinoic acid loaded on the surface of the core. The core is a selenium-enriched organic nanozyme core, which is a nanostructure containing iron-nitrogen-selenium single-atom sites. The all-trans retinoic acid is loaded onto the surface of the core through physical adsorption or chemical bonding. The selenium-enriched organic nanozyme core is generated by introducing a selenium source into the Fe-ZIF-8 framework to form a Se-Fe diatomic nanozyme core. The average particle size of the iron-nitrogen-selenium single-atom nanozyme is 20–200 nm.

[0027] A method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid includes the following steps: (1) Preparation of Fe-ZIF-8: 1.85 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was dissolved in 80 mL of methanol. At room temperature and with a stirring speed of 1200 rpm, a solution of 2-methylimidazole and ferric acetylacetone (prepared by dissolving 8.00 g of 2-methylimidazole and 0.22 g of ferric acetylacetone (Fe(acac)2) in 80 mL of methanol) was rapidly added. After reacting for 26 h, the mixture was centrifuged at 10000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with 40 mL of methanol each time. The mixture was then activated under vacuum at 100 °C for 24 h to obtain Fe-ZIF-8. (2) Preparation of FeSAzyme: Fe-ZIF-8 from step (1) was dispersed in 120 mL of methanol and sonicated for 10 min at an ultrasonic power of 100 W. Then, it was quickly poured into 120 mL of water, followed by 6 mL of 25 mg / mL CTAB aqueous solution and 10 mL of 6 mg / mL NaOH aqueous solution. 7.2 mL of tetraethyl orthosilicate (TEOS) solution (1.4 mL of TEOS stock solution dissolved in 6 mL of methanol) was injected. After stirring for 2 h, the mixture was centrifuged at 10000 rpm for 10 min. The intermediate product was collected, washed with 40 mL of ethanol, and dried under vacuum at 100 °C. The dried powder was placed in a tube furnace and heated to 320 °C at a rate of 5 °C / min under a nitrogen atmosphere, held for 2 h, then heated to 850 °C at a rate of 5 °C / min, held for 6 h. After cooling, the material was etched in a 6 mol / L NaOH aqueous solution at 60 °C. h is used to remove the silica coating layer to obtain FeSAzyme; (3) Preparation of Se-Fe diatomic nanozyme: FeSAzyme from step (2) and selenium powder were mechanically ground at a mass ratio of 1:2 to obtain a precursor. The precursor was evenly spread in a ceramic boat and heated to 630 °C at a rate of 5 °C / min under a nitrogen atmosphere. The temperature was maintained for 2 h and then cooled to room temperature to obtain Se-Fe diatomic nanozyme (DSAzyme). (4) Preparation of iron-nitrogen-selenium single-atom nanozyme: The Se-Fe diatomic nanozyme and all-trans retinoic acid (ATRA) in step (3) were co-dispersed in water at a mass ratio of 4:1. After ultrasonic treatment for 10 min at an ultrasonic power of 100 W, the mixture was stirred continuously for 8 h at room temperature to promote coordination. The mixture was centrifuged at 10000 rpm for 10 min, and the precipitate was collected. The precipitate was washed with 20 mL of dimethyl sulfoxide (DMSO) to remove free ATRA. The mixture was then washed twice with water and freeze-dried to obtain iron-nitrogen-selenium single-atom nanozyme (DSAzyme-ATRA).

[0028] Application of surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid in the preparation of drugs for the treatment of acute myeloid leukemia.

[0029] The drug includes iron-nitrogen-selenium single-atom nanozymes and pharmaceutically acceptable carriers, and the dosage form of the drug is tablets, capsules, granules, powders, oral liquids or injections.

[0030] Example 4 Performance Testing To systematically characterize the structure and physicochemical properties of the FeSAzyme, DSAzyme, and DSAzyme-ATRA nanomaterials prepared in Example 1, the following testing methods were used: 4.1 Structural Characterization of DSAzyme-ATRA The morphology and particle size distribution of FeSAzyme, DSAzyme, and DSAzyme-ATRA nanomaterials were observed and analyzed using transmission electron microscopy (TEM). Figure 1 As shown in (bd), FeSAzyme, DSAzyme, and DSAzyme-ATRA all maintained a clear ZIF-like polyhedral morphology, indicating that the introduction of diatomic sites and subsequent ATRA modification did not destroy the overall structure.

[0031] Dynamic light scattering (DLS) was used to determine the hydrated particle size distribution of nanomaterials in an aqueous system to evaluate dispersion stability and surface charge characteristics. Figure 1As shown in (e), the hydrodynamic dimensions of FeSAzyme, DSAzyme, and DSAzyme-ATRA are 72.48±17.63 nm, 75.32±19.78 nm, and 87.61±22.07 nm, respectively, which are similar in size.

[0032] High-resolution imaging of DSAzyme-ATRA was performed using aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), such as... Figure 1 As shown in (f), bright and uniformly dispersed diatomic signals were observed in DSAzyme-ATRA, and the three-dimensional atomic resolution reconstruction and line scan profile clearly confirmed the existence of Fe-Se diatomic centers. The specific surface area and pore size distribution of FeSAzyme, DSAzyme, and DSAzyme-ATRA were measured using the Brunauer-Emmett-Teller (BET) method, as shown... Figure 1 (g) As shown in the nitrogen adsorption-desorption isotherms, all three nanozymes exhibit typical type IV behavior, indicating that they possess a mesoporous structure. X-ray photoelectron spectroscopy (XPS) analysis further verified their elemental composition and chemical state. Figure 1 As shown in (h), in addition to C, O, and N, characteristic peaks for Fe and Se are clearly observed. The high-resolution Fe 2p spectrum shows peaks corresponding to Fe at 711.1 and 724.8 eV. 3+ The peak, and a satellite peak at 717.9 eV ( Figure 1 i). The Se 3d spectrum shows Se at 54.7 and 55.5 eV. 2- The signal showed Se at 56.1 and 58.0 eV. 4+ signals ( Figure 1 (j)).

[0033] 4.2 Analysis of the valence state and coordination environment of selenium (Se) and iron (Fe) atoms To clarify the existence state and local atomic coordination structure of selenium (Se) and iron (Fe) in the DSAzyme-ATRA nanomaterials prepared in Example 1, synchrotron X-ray absorption spectroscopy (XAS) was used to systematically characterize the samples, including X-ray absorption near-edge structure spectroscopy (XANES) and extended X-ray absorption fine structure spectroscopy (EXAFS). The valence state of selenium was analyzed using Se K-edge XANES spectra, such as... Figure 2 As shown in (a), the absorption edge of DSAzyme-ATRA lies between the Se foil and SeO2, indicating that Se exists in a partially oxidized, positively charged state. Combined with the k³-weighted EXAFS (FT-EXAFS) spectrum after Fourier transform, the local coordination structure of selenium is analyzed, as shown below. Figure 2As shown in (b), DSAzyme-ATRA exhibits two characteristic peaks in R space: a main peak at 1.76 Å corresponds to Se-C coordination, and a secondary peak at 2.57 Å originates from Se-Fe coordination.

[0034] like Figure 2 As shown in (d), the Fe K-edge XANES spectrum indicates that the absorption edge of DSAzyme-ATRA lies between the Fe foil and Fe₂O₃, suggesting that Fe exists in a partially oxidized state (0 < δ < +3). Figure 2 As shown in (e), the prominent peak at 1.64 Å and the shoulder peak at 2.44 Å in the FT-EXAFS spectrum are attributed to Fe-N and Fe-Se interactions, respectively. These results collectively confirm the coexistence of Fe-N, Se-C, and Se-Fe bonds in the nanozyme. The least squares method was used to fit the Se K-edge and Fe K-edge EXAFS data, as shown in... Figure 2 As shown in (cf), each Se atom is coordinated to two C atoms (1.91 Å) and one Fe atom (2.58 Å), while each Fe atom is coordinated to an average of 2.8 N atoms (1.96 Å) and 1.4 Se atoms (2.51 Å). Figure 2 As shown in (gl), wavelet transform analysis of the EXAFS data of Se K-edge and Fe K-edge confirms the atomic dispersion of Fe and Se and the formation of asymmetric diatomic sites.

[0035] 4.3 Theoretical Calculation To further analyze the effect of selenium introduction on the catalytic performance of the material as a glutathione peroxidase (GPx-like) catalytic agent, an active site model was constructed using density functional theory (DFT) calculations. Figure 2 As shown in (m), using the Fe-N3-Se diatomic structure model as the target model and the Fe-N4 single-atom structure model as the control model, the energy barrier changes of the two models in the key reaction steps of the GPx-type reaction pathway are calculated and compared. Figure 2 As shown in (n), H2O2 is first adsorbed and then deprotonated to form the *OOH intermediate. Subsequently, CH3SHH donates a proton to *OOH to generate the first H2O molecule, followed by the adsorption of another CH3SH to form an SS interaction. Finally, desorption produces the second H2O molecule. The energy barrier of this rate-determining step (RDS) is only 0.95 eV, while the corresponding energy barrier of the Fe-N4 model is as high as 3.55 eV. Figure 2 (n) indicates that selenium doping significantly reduces the energy barrier of the GPx-like catalytic pathway. Simultaneously, density of states (DOS) analysis was performed on the above model to assess its electronic structure characteristics. Figure 2As shown in (op), Fe-N4 has a relatively high electron density near the Fermi level, making it an electron-rich site that is difficult to stably adsorb electron-donating species such as H2O2 or *OOH, resulting in increased intermediate energy and suppressed reaction kinetics. Conversely, the introduction of Se reduces the electron density of the Fe-N3-Se center, enabling it to more strongly adsorb H2O2 and stabilize peroxide intermediates, thereby lowering the energy barrier and significantly enhancing GPx-like activity.

[0036] Example 5: In vitro activity and functional evaluation of the sample from Example 1 5.1 Assay for Class GPx Activity The glutathione peroxidase-like catalytic activities of FeSAzyme, DSAzyme, and DSAzyme-ATRA from the examples were evaluated using a glutathione peroxidase assay kit (Solepro, BC1195). Results are as follows: Figure 1 As shown in (km).

[0037] Reduced glutathione (GSH) reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to generate a chromogenic product with a characteristic absorbance at 412 nm. Glutathione peroxidase (GPx) catalyzes the reduction of hydrogen peroxide (H₂O₂), during which GSH is oxidized to glutathione disulfide (GSSG), leading to a decrease in absorbance at 412 nm. Therefore, the decrease in absorbance at 412 nm reflects the glutathione peroxidase-like activity of the nanozyme. Figure 1 (km) indicates that the activity of DSAzyme is significantly enhanced after the introduction of Se, suggesting that the introduction of Se is crucial for endowing GPx-like catalytic function.

[0038] 5.2 Synergistic Induction of Differentiation To evaluate the uptake capacity of DSAzyme-ATRA by acute myeloid leukemia (AML) cells, human AML cell lines U937 and HL-60 were selected as test subjects. DSAzyme-ATRA was co-incubated with these cells, and cell samples were collected at different time points. Flow cytometry was used to detect intracellular material signals to assess its uptake rate and efficiency. Figure 3 As shown in (ab), DSAzyme-ATRA was rapidly internalized, with uptake detectable in U937 cells within 30 minutes and in HL-60 cells as early as 5 minutes. To verify the induction of leukemia cell differentiation by DSAzyme-ATRA, U937 and HL-60 cells were co-cultured with DSAzyme-ATRA for 3 days, and cell morphological changes were observed under a microscope, focusing on analyzing typical differentiation-related characteristics such as nuclear morphology and nucleocytoplasmic ratio. Figure 3As shown in (cd), characteristic morphological changes in myeloid differentiation were observed, such as a decrease in the nucleus and nucleocytoplasmic ratio of kidney-shaped cells, confirming its role in inducing differentiation.

[0039] Under the same treatment conditions, flow cytometry was used to detect the expression level of CD11b, a myeloid differentiation-related marker, in U937 and HL-60 cells to evaluate the degree of cell differentiation. Figure 3 As shown in (ef), the myeloid maturation marker CD11b was significantly upregulated in both U937 and HL-60 cells after treatment. Additionally, bone marrow-derived cells from C57BL / 6J mice were co-cultured with DSAzyme-ATRA (5 μg / mL) for 3 or 5 days in vitro. After culture, flow cytometry was used to detect changes in the expression of hematopoietic progenitor cell markers CD33 and CD11b to assess their impact on the differentiation of normal hematopoietic progenitor cells. Figure 3 As shown in (gi), bone marrow cells from C57BL / 6J mice treated with DSAzyme-ATRA (5 μg / mL) for 3 or 5 days exhibited decreased CD33 expression and increased CD11b expression, indicating promotion of normal hematopoietic progenitor cell differentiation. (Selected) Tg ( drl:hoxa9 A transgenic zebrafish model in which... hoxa9 High specific expression in hematopoietic cells leads to myeloid developmental arrest. Green fluorescent protein (GFP) was used to label hematopoietic-related tissues at 24 hours post-fertilization (hpf). Embryos were treated with different concentrations of DSAzyme-ATRA (5, 10, and 20 μg / mL), and hematopoietic development was assessed at 3 days post-fertilization (dpf). Myeloid progenitor cell markers were detected. cmyb Macrophage markers mfap4 and neutrophil markers mpx Expression levels were assessed to evaluate the effect of DSAzyme-ATRA on improving hematopoietic differentiation arrest. For example... Figure 3 As shown in (jk), DSAzyme-ATRA treatment significantly salvaged hematopoietic development, manifested as follows: Tg (drl:hoxa9) In the embryo cmyb + Myeloid progenitor cells, mfap4 + macrophages and mpx + The increase in the number of neutrophils indicates that DSAzyme-ATRA can effectively restore myeloid differentiation despite the presence of hereditary hematopoietic block.

[0040] 5.3 Revealing the synergistic regulation of differentiation and hematopoietic repair mediated by the CXCR5–TrxR axis To analyze the molecular mechanism by which DSAzyme-ATRA induced acute myeloid leukemia (AML) cell differentiation obtained in Example 1, the human AML cell line U937 was selected as the test subject. U937 cells were co-incubated with DSAzyme-ATRA for 3 hours, cell samples were collected, total RNA was extracted, and whole transcriptome sequencing analysis was performed to capture the early transcriptional response after material treatment. Figure 4 As shown in (a), 343 genes were upregulated and 780 genes were downregulated in differentially expressed genes. Based on the differentially expressed gene data, KEGG pathway enrichment analysis was used to evaluate the biological pathways significantly regulated by DSAzyme-ATRA treatment, such as... Figure 4 As shown in (b), cells treated with DSAzyme-ATRA showed significant enrichment of chemokine signaling pathways, hematopoietic cell lineages, PI3K-Akt signaling pathways, and TNF-related pathways, including significant upregulation of 10 differentiation-related hematopoietic genes, such as CD38, CD22, ITGA6, CD5, IL1B, CD1C, CD34, CD55, HLA-DMB, and ITGB3. Figure 4 (c) indicates enhanced differentiation of leukemia cells. Statistical analysis was performed on representative genes closely related to hematopoietic differentiation screened from transcriptome analysis, and the expression changes of key genes were verified using real-time quantitative PCR (RT-qPCR). Figure 4 As shown in (de), CXCR5 (a chemokine receptor) was the most significantly upregulated transcript. To verify the role of CXCR5 in DSAzyme-ATRA-induced differentiation, CXCR5 in U937 cells was specifically silenced using a liposome-mediated small interfering RNA (siRNA) method. After confirming the silencing efficiency of CXCR5 by RT-qPCR, the expression changes of the differentiation marker CD11b in cells under DSAzyme-ATRA treatment were detected. Figure 4 As shown in (g), compared with cells treated with control siRNA, CD11b expression was significantly reduced in CXCR5-deficient cells after DSAzyme-ATRA treatment, indicating that CXCR5 is essential for differentiation induced by DSAzyme-ATRA. Given that DSAzyme-ATRA is rich in selenium, its effect on thioredoxin reductase (TrxR) activity was further investigated. Changes in TrxR enzyme activity before and after DSAzyme-ATRA treatment were measured in normal U937 cells and CXCR5-silenced U937 cells, respectively. Figure 4 As shown in (hi), DSAzyme-ATRA significantly increased the activity of TrxR, while this effect disappeared after knocking down CXCR5, suggesting the existence of a CXCR5-dependent regulatory mechanism.

[0041] Example 6: Comparison of the anti-leukemia effects of DSAzyme-ATRA and free ATRA in Example 1 6.1 Mouse AML Model Given that the mass fraction of ATRA in DSAzyme-ATRA is approximately one-twentieth that of free ATRA, the differentiation-inducing effects of DSAzyme-ATRA and free ATRA were compared using an equimolar dosage principle, applied to human acute myeloid leukemia cell lines U937 and HL60, respectively. Figure 5 As shown in (ae), the differentiation phenotype induced by DSAzyme-ATRA was more pronounced. The differentiation-inducing effect of DSAzyme-ATRA was not only superior to ATRA alone, but also superior to the clinically established ATRA combined with ATO regimen. Figure 5 (e) NB4 cells carrying the PML::RARA fusion gene were used as a model to compare the effects of DSAzyme-ATRA and free ATRA on the level of PML::RARA fusion protein, in order to assess the difference in their ability to clear pathogenic proteins. Figure 5 As shown in (g), compared with ATRA, DSAzyme-ATRA enhanced the degradation of fusion oncoprotein in NB4 cells carrying the PML::RARA fusion protein. The effects of the two treatments on embryonic development were also assessed in a zebrafish model, focusing on the occurrence of toxicity-related phenotypes such as pericardial edema, yolk sac retention, and tail deformities. Transcriptome sequencing analysis was used to compare the changes in the number of differentially expressed genes (DEGs) after DSAzyme-ATRA and free ATRA treatment. Figure 5 As shown in (h), fewer differentially expressed genes were observed after DSAzyme-ATRA treatment compared to the ATRA treatment group (1,123 vs. 1,309), indicating reduced cytotoxicity. Figure 5 As shown in (i), free ATRA (rather than DSAzyme-ATRA) can induce pericardial edema, yolk sac retention and tail deformity. The findings indicate that DSAzyme-ATRA achieves better therapeutic effects while reducing ATRA-related toxicity.

[0042] Example 7: Evaluation of the in vivo anti-leukemia efficacy of DSAzyme-ATRA in Example 1 7.1 Mouse AML Model A systemic AML model was established by tail vein injection of AML-carrying cells (e.g., U937 cells) into NOD / SCID mice irradiated with 250 cGy. After successful model establishment, mice were randomly assigned to groups and administered tail vein injections of saline (control group), ATRA, DSAzyme, and ATRA-loaded DSAzyme, respectively. The leukemia burden in the mice was monitored. Efficacy data are as follows: Figure 6As shown in (ac).

[0043] Depend on Figure 6 (dg) indicates that, compared with the control group and other treatment groups, the DSAzyme-ATRA treatment group mice had hematological parameters closest to those of healthy mice, significantly inhibited the progression of leukemia, and prolonged the survival of the mice. After treatment, bone marrow and spleen organs of the mice were collected for histopathological analysis (such as H&E staining) and immunohistochemical analysis. Figure 6 (hi) The results showed that the infiltration of leukemia cells in the bone marrow cavity of mice treated with DSAzyme-ATRA was significantly reduced, the proportion of normal hematopoietic cells increased, and the bone marrow structure was improved, indicating that this iron-nitrogen-selenium single-atom nanozyme helps repair the bone marrow hematopoietic microenvironment damage caused by leukemia. Creatinine levels in mice treated with DSAzyme-ATRA ( Figure 6 (j)) and alanine aminotransferase ( Figure 6 (k) levels remained within the normal range, comparable to the healthy control group. Due to liver dysfunction, AML mice had significantly reduced blood urea nitrogen (BUN) levels, but after DSAzyme-ATRA treatment, BUN levels recovered to near-normal levels. Figure 6 (l) indicates that the drug has a liver-protective effect.

[0044] Bone marrow cells from AML donors treated with PBS, ATRA, or DSAzyme-ATRA were transplanted into healthy recipient mice irradiated with a sublethal dose to construct a secondary transplantation experiment. The inhibitory effect of DSAzyme-ATRA on the relapse potential of leukemia was evaluated by monitoring mouse survival rates and other indicators. Healthy C57BL / 6J mice were selected and given a single 7.5 Gy whole-body irradiation to induce hematopoietic system damage, establishing a radiation injury model. Subsequently, mice were randomly assigned to receive DSAzyme-ATRA, free ATRA, or a control group. During treatment, changes in mouse body weight and general condition were continuously monitored. Body weight change was used as an important indicator of overall tolerability and systemic toxicity to assess the protective effect of DSAzyme-ATRA on overall homeostasis under hematopoietic stress. Femoral bone marrow cells were harvested at key early post-irradiation time points, and hematopoietic stem / progenitor cell subsets were finely analyzed by flow cytometry. Lineage was used... - Sca-1 + c-Kit + LSK (low-lying stem cells) serves as a marker for hematopoietic stem / progenitor cell populations, further distinguishing between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and multipotent progenitor cells (MPPs). By comparing changes in the number and proportion of each subpopulation in different treatment groups, the role of DSAzyme-ATRA in promoting hematopoietic stem cell survival, expansion, and functional recovery after radiation injury was evaluated. Figure 6(m) The results showed that mice that received PBS-treated bone marrow died within 30 days, while 16.7% and 33.3% of mice in the ATRA group and DSAzyme-ATRA group, respectively, survived for more than 42 days, indicating that DSAzyme-ATRA can not only reduce leukemia burden, but also delay relapse and improve long-term survival.

[0045] 7.2 Evaluation of the Zebrafish Model A chemically induced zebrafish model of myeloid differentiation arrest was established to rapidly evaluate the effect of DSAzyme-ATRA from Example 1 on hematopoietic recovery. Zebrafish were exposed to water containing different formulations. The degree of hematopoietic recovery was assessed by observing the regeneration of myeloid cells in the hematopoietic tissue region of the zebrafish tail (e.g., whole embryonic in situ hybridization staining). The results are as follows: Figure 5 As shown in (f).

[0046] Depend on Figure 5 (f) shows that the zebrafish treated with DSAzyme-ATRA recovered their hematopoietic function much faster than the control group, further confirming its potential to promote hematopoietic recovery.

[0047] 7.3 Repair of the bone marrow hematopoietic microenvironment A systemic radiotherapy injury model was established in C57BL / 6N mice irradiated with 7.5 Gy to evaluate the therapeutic potential of DSAzyme-ATRA in maintaining hematopoietic function under stress. Results are as follows: Figure 7 As shown in (aj).

[0048] Depend on Figure 7 (aj) indicates that the body weight of mice in the DSAzyme-ATRA group remained stable or increased slightly throughout the treatment period, and the long-term hematopoietic stem cells in the mice increased significantly.

[0049] In summary, the iron-nitrogen-selenium single-atom nanozyme of the present invention comprises a selenium-enriched organic nanozyme core with glutathione peroxidase-like activity. The selenium-enriched organic nanozyme core is a nanostructure containing iron-nitrogen-selenium (Fe-N3-Se) dual single-atom sites. This specific single-atom structure endows the nanozyme with highly efficient and stable glutathione peroxidase-like activity, which can reduce the excessively high ROS level in AML cells, restore redox balance, break the differentiation resistance of AML cells to ATRA, and clear obstacles for ATRA to play its role.

[0050] By loading all-trans retinoic acid (ATRA) onto the surface of the core, the core can regulate the level of reactive oxygen species (ROS) within target cells, synergistically inducing differentiation of acute myeloid leukemia (AML) cells with ATRA. This creates an integrated drug delivery platform. The regulatory effect of nanozymes on ROS and the differentiation-promoting effect of ATRA produce a synergistic effect, significantly enhancing the differentiation-inducing effect on AML cells and overcoming the drug resistance problem of ATRA monotherapy. The iron-nitrogen-selenium single-atom nanozyme of this invention not only effectively inhibits the proliferation and infiltration of leukemia cells but also promotes the repair of the bone marrow hematopoietic microenvironment and the recovery of hematopoietic function, demonstrating comprehensive therapeutic potential. It provides a novel, efficient, and clinically translational targeted redox differentiation-enhancing therapeutic strategy for the treatment of clinically challenging acute myeloid leukemia.

Claims

1. A surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid, characterized in that, It includes a core and all-trans retinoic acid loaded on the surface of the core, wherein the core is a selenium-enriched organic nanozyme core.

2. The surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid as described in claim 1, characterized in that, The core of the selenium-enriched organic nanozyme is a nanostructure containing single-atom sites of iron, nitrogen, and selenium.

3. The surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid as described in claim 2, characterized in that, The all-trans retinoic acid is loaded onto the surface of the core through physical adsorption or chemical bonding; the selenium-enriched organic nanozyme core is generated by introducing a selenium source into the Fe-ZIF-8 framework to form a Se-Fe diatomic nanozyme core; the average particle size of the iron-nitrogen-selenium single-atom nanozyme is 20-200 nm.

4. A method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of Fe-ZIF-8; (2) Preparation of FeSAzyme: After dispersing Fe-ZIF-8 in an organic solvent in step (1), pour it into an inorganic solvent, add an aqueous solution of surfactant and an aqueous solution of strong alkali in sequence, inject tetraethyl orthosilicate solution, stir the reaction, centrifuge to separate, collect the intermediate product, wash, dry and anneal the intermediate product, place it in a strong alkali etching solution for etching, and obtain FeSAzyme. (3) Preparation of Se-Fe diatomic nanozyme: FeSAzyme from step (2) was ground with a selenium source to obtain a precursor, which was then annealed to obtain Se-Fe diatomic nanozyme; (4) Preparation of iron-nitrogen-selenium single-atom nanozyme: The Se-Fe diatomic nanozyme and all-trans retinoic acid from step (3) were dispersed in an inorganic solvent, stirred, centrifuged, and the precipitate was collected. The precipitate was washed and freeze-dried to obtain iron-nitrogen-selenium single-atom nanozyme.

5. The method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid as described in claim 4, characterized in that, In step (1), Fe-ZIF-8 is prepared by chemical precipitation.

6. The method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid as described in claim 5, characterized in that, In step (1), the preparation method of Fe-ZIF-8 is as follows: zinc nitrate hexahydrate is dissolved in an organic solvent, and under stirring conditions, a solution of 2-methylimidazole and acetylacetone iron is added to react. After centrifugation, the precipitate is collected, washed, and vacuum activated to obtain Fe-ZIF-8.

7. The method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid as described in claim 6, characterized in that, In step (1), the organic solvent is methanol, and the mass ratio of zinc nitrate hexahydrate, 2-methylimidazole, and iron acetylacetone is (1.50-1.85):(6.60-8.10):(0.18-0.22). The reaction conditions are: time 22-26 h, and the vacuum activation conditions are: temperature 90-110°C, time 22-26 h. In step (2), the organic solvent is methanol, the inorganic solvent is water, the surfactant aqueous solution is CTAB aqueous solution, the strong base aqueous solution is NaOH aqueous solution, the strong base etching solution is sodium hydroxide aqueous solution, and the annealing method is as follows: under a nitrogen atmosphere, the temperature is raised to 280-320 °C at a rate of 5 °C / min and held for 1-3 h, then raised to 850-950 °C at a rate of 5 °C / min and held for 4-6 h, and then cooled; the tetraethyl orthosilicate solution is prepared by dissolving the tetraethyl orthosilicate stock solution in an organic solvent. In step 1), the ratio of Fe-ZIF-8 to the tetraethyl orthosilicate stock solution is (150-200) mg: (0.8-1.4) mL.

8. The method for preparing surface-engineered iron-nitrogen-selenium single-atom nanozymes loaded with all-trans retinoic acid as described in claim 7, characterized in that, In step (3), the annealing conditions are as follows: under a nitrogen atmosphere, the temperature is heated to 550-650 °C at a rate of 5 °C / min, held for 1-3 h, and then cooled to obtain Se-Fe diatomic nanozyme. The selenium source is selenium powder, and the mass ratio of FeSAzyme to selenium powder is (0.5-1.5):(1-3). In step (4), the inorganic solvent is water, and the mass ratio of Se-Fe diatomic nanozyme to all-trans retinoic acid in step (3) is (0.5-5):

1. During washing, dimethyl sulfoxide is used first, followed by water.

9. The use of the surface-engineered iron-nitrogen-selenium single-atom nanozyme loaded with all-trans retinoic acid as described in any one of claims 1-3 in the preparation of a drug for treating leukemia.

10. The application as described in claim 9, characterized in that, The leukemia is acute myeloid leukemia; the drug includes an iron-nitrogen-selenium single-atom nanozyme and a pharmaceutically acceptable carrier, and the dosage form of the drug is tablets, capsules, granules, powders, oral liquids or injections.

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