Lanthanide / transition metal ion coordination nano-enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antitumor drugs, and particularly relates to a lanthanide / transition metal ion coordination nano-enzyme as well as a preparation method and application thereof. The lanthanum ion / copper ion nano enzyme is formed by coordination of lanthanum ions, copper ions and inhibitors Lap and IWR-1, has SOD (superoxide dismutase), POD (peroxidase) and GSH-Px triple mimic enzyme activity, can respond to tumor microenvironment degradation and release active ingredients, synergistically reverses copper death resistance and induces comprehensive death of tumor cells, and is simple and efficient in preparation method, uniform in nano enzyme size, good in biological safety and suitable for industrial production. Wide application prospects are realized in the field of anti-tumor medicines.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a lanthanide / transition metal ion coordination nanozyme, its preparation method and application. This lanthanide / transition metal ion coordination nanozyme can be used as a nanomedicine to eliminate resistance to copper death in order to achieve comprehensive copper death therapy in tumors. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Copper death is a novel programmed cell death mechanism distinct from traditional apoptosis and pyroptosis. It induces mitochondrial-dependent oxidative damage by causing intracellular copper overload, providing a new strategy for cancer treatment. However, tumor cells easily develop resistance through mechanisms such as upregulating metallothionein expression, activating copper ion efflux pathways, or remodeling mitochondrial metabolism, leading to a gradual weakening of the efficacy of single copper death inducers.

[0004] Nanozymes combine the catalytic activity of enzymes with the properties of nanomaterials, but their clinical application still faces two major challenges: (1) The non-degradability of the noble metal-based active center hinders its metabolic clearance in the body; (2) Copper ion delivery efficiency is low and resistance-related signaling pathway regulation ability is limited.

[0005] Therefore, developing a coordination nanozyme system that combines multiple enzyme catalytic activities, biodegradability, and copper death resistance reversal function is key to breaking through the bottleneck of tumor copper death therapy. Summary of the Invention

[0006] To address the needs of existing technologies, the purpose of this invention is to provide a lanthanide / transition metal ion coordination nanozyme, its preparation method, and its applications. This invention involves the coordination of lanthanum ions, copper ions, and the inhibitors Lap and IWR-1 to form a nanozyme exhibiting triple enzyme-mimicking activities of SOD, POD, and GSH-Px. It can respond to degradation in the tumor microenvironment and release active ingredients, synergistically reversing copper death resistance and inducing comprehensive tumor cell death. The preparation method is simple and efficient, producing nanozymes with uniform size and good biosafety, showing broad application prospects in the field of anti-tumor drugs.

[0007] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides a lanthanide / transition metal ion coordination nanozyme, wherein the lanthanide / transition metal ion coordination nanozyme has a hierarchical network structure, including a first hierarchical network and a second hierarchical network; the first hierarchical network is a ternary coordination complex formed by lanthanide ions as central metal nodes, deprotonated tyrosine kinase inhibitor lapatinib (Lap), and Wnt pathway inhibitor IWR-1; the second hierarchical network is a quaternary coordination structure formed by copper ions as secondary metal nodes embedded in the first hierarchical network; wherein the molar ratio of lanthanide ions, lapatinib, IWR-1, and copper ions is 1:(1~3):(1~3):2.

[0008] Preferably, the molar ratio of lanthanum ions, Lap, IWR-1 to copper ions is 1:2:2:2.

[0009] Preferably, the ternary coordination complex is connected by lanthanum ions to the sulfonyl oxygen atom of lapatinib and the carbonyl oxygen atom of IWR-1; the quaternary coordination structure is based on the ternary complex and is connected by copper ions to the pyrimidine ring nitrogen atom of lapatinib and the pyridine ring / amide nitrogen atom of IWR-1.

[0010] Preferably, the ternary coordination complex is spherical with a particle size of 77-101 nm; the lanthanide / transition metal ion coordination nanozyme is clustered with a particle size of 61-83 nm.

[0011] Preferably, the lanthanide / transition metal ion coordination nanozyme has triple enzyme activity, including superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GSH-Px).

[0012] Preferably, the lanthanide / transition metal ion coordination nanozyme degrades under high concentrations of GSH, H2O2, and slightly acidic conditions, releasing therapeutically active lanthanum ions, copper ions, Lap, and IWR-1.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned lanthanide / transition metal ion coordination nanozyme, comprising the following steps: S1. Add methanol solution of Wnt pathway inhibitor IWR-1 and sodium hydroxide aqueous solution to a mixed solution of tyrosine kinase inhibitor lapatinib and soluble lanthanum salt, and perform ultrasonic and stirring reaction. After post-treatment, lanthanum-Lap / IWR-1 nanoparticles are obtained. S2. Lanthanum-Lap / IWR-1 nanoparticles were added to a soluble copper salt aqueous solution, and after ultrasonic treatment, sodium hydroxide solution was added. The reaction was continuously stirred, and the lanthanum-copper@Lap / IWR-1 nanozyme was obtained after post-treatment.

[0014] Preferably, in step S1, the mixed solution comprises a methanol solution containing the tyrosine kinase inhibitor lapatinib and an aqueous solution containing a soluble lanthanum salt; the ratio of the sum of the volumes of the methanol solutions containing the tyrosine kinase inhibitor lapatinib and the methanol solution containing the Wnt pathway inhibitor IWR-1 to the volume of the aqueous solution containing the soluble lanthanum salt is 3.5~4.5:5~5.5, more preferably 4:5.

[0015] Preferably, in step S1, the molar ratio of the tyrosine kinase inhibitor lapatinib, the soluble lanthanum salt, and the Wnt pathway inhibitor IWR-1 is 1:(1~3):(1~3), and more preferably 1:2:2.

[0016] Preferably, in step S1, the soluble lanthanum salt is selected from one or more of lanthanum chloride hexahydrate (LaCl3·6H2O) and lanthanum nitrate hexahydrate (La(NO3)3·6H2O).

[0017] Preferably, in step S1, the concentration of the Wnt pathway inhibitor IWR-1 is 0.45~0.55 mg / mL, the concentration of the sodium hydroxide aqueous solution is 380~420 mM, and the volume ratio of the methanol solution of the Wnt pathway inhibitor IWR-1 to the sodium hydroxide aqueous solution is 8:0.1~0.2.

[0018] In step S1, sodium hydroxide is used to provide an alkaline environment. Only sodium hydroxide can be used here. If other bases are used, other metal ions or ammonium ions will be introduced, which will interfere with the coordination of lanthanum ions with ligands.

[0019] Preferably, in step S1, the ultrasonic power is 15~250 W and the time is 2~10 min; the stirring rate of the stirring reaction is 300~800 rpm and the time is 22~26 h; the post-processing includes centrifugation, washing and drying, the centrifugation speed is 10000~14000 rpm and the centrifugation time is 5~15 min; the washing is performed 2~3 times.

[0020] Sonication for 2–10 min effectively breaks up the agglomeration of components in the reaction system, enabling uniform mixing of Lap, IWR-1 inhibitors, and lanthanum ions at the molecular level. This process not only accelerates the deprotonation of ligands but also significantly promotes the initial coordination between lanthanum ions and ligands, laying a homogeneous reaction foundation for subsequent continuous stirring.

[0021] Preferably, in step S2, the ratio of the soluble copper salt to the sodium hydroxide aqueous solution is (1.7~1.71) mg:(80~120) μL. Sodium hydroxide is added to provide an alkaline environment.

[0022] Preferably, in step S2, the soluble copper salt is selected from one or more of copper chloride dihydrate (CuCl2·2H2O) and copper nitrate trihydrate (Cu(NO3)2·3H2O).

[0023] Preferably, in step S2, the ultrasonic treatment power is 15~250 W, and the time is 5~15 min; the stirring rate of the continuous stirring reaction is 500~1000 rpm, and the time is 10~14 h; the post-treatment includes centrifugation, washing, and drying, the centrifugation speed is 10000~14000 rpm, and the centrifugation time is 5~15 min; the washing is performed 2~3 times.

[0024] A third aspect of the present invention provides the application of the lanthanide / transition metal ion coordination nanozyme described in the first aspect in the preparation of tumor microenvironment improvers or products that induce copper cell death.

[0025] A fourth aspect of the present invention provides a pharmaceutical composition comprising the lanthanide / transition metal ion coordination nanozyme and pharmaceutical carrier described in the first aspect.

[0026] Preferably, the pharmaceutical carrier is selected from one or more of hyaluronic acid, polyethylene glycol, polysaccharides, and glycolic acid.

[0027] Preferably, the pharmaceutical composition further includes other active ingredients selected from one or more of antitumor small molecule drugs, natural enzymes, and sound-sensitive agents.

[0028] The beneficial effects achieved by one or more of the above technical solutions of the present invention are as follows: (1) The lanthanide / transition metal ion coordination nanozyme prepared in this invention has triple mimicry activities of SOD, POD, and GSH-Px. This unique combination of enzyme activities enables it to precisely regulate the intracellular redox balance, thereby effectively remodeling the tumor microenvironment. It is worth noting that, due to the low concentrations of hydrogen peroxide (H2O2) and glutathione (GSH) in normal tissues, this nanozyme exhibits almost no catalytic activity in healthy tissues, thus demonstrating excellent tumor targeting specificity and good biosafety.

[0029] Specifically: 1) It has SOD-like activity and can catalyze superoxide anion (O2) ·- ) is converted into H2O2; 2) It has POD-like activity and can catalyze the generation of hydroxyl radicals (·OH) from H2O2; 3) It possesses GSH-Px activity, which involves oxidizing reduced GSH to oxidized GSHSG. This multi-enzyme synergy enables it to effectively regulate reactive oxygen species metabolism.

[0030] (2) The lanthanum-copper@Lap / IWR-1 nanozyme prepared in this invention can amplify the oxidative stress of copper ions and eliminate copper death resistance in the tumor microenvironment through an enzyme-driven cascade reaction, thereby inducing comprehensive copper death in tumor cells, breaking the immunosuppressive microenvironment, and achieving long-term effective immunotherapy and metastasis inhibition.

[0031] (3) The lanthanide / transition metal ion coordination nanozyme prepared by the present invention can be specifically degraded by GSH, H2O2 and microacids, releasing lanthanum ions, copper ions, Lap and IWR-1 components, effectively solving the problems of long retention time and slow metabolism of traditional nanozymes in the body, realizing rapid clearance after treatment, and significantly improving the system safety of nanozymes.

[0032] (4) The lanthanide / transition metal ion coordination nanozymes prepared by this invention have the characteristics of uniform size and good dispersibility. The unique non-smooth morphology of its surface can not only significantly improve the internalization efficiency and uptake rate of the nanozymes by tumor cells, but also facilitate functional modification on the surface, linking or loading small molecule drugs, thereby enhancing its therapeutic effect as an anti-tumor active ingredient.

[0033] (5) The preparation method of the present invention is simple. It can be synthesized by stirring at room temperature. It is practical and easy to promote. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 The images show scanning electron microscope (SEM) images (a) and particle size distribution diagram (b) of the lanthanum-Lap / IWR-1 nanomaterials prepared in Example 1 of this invention. Figure 2 The images show transmission electron microscopy (TEM) images (a) and particle size distribution (b) of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention. Figure 3 The images show TEM images (a) and (b) of the lanthanum-Lap / IWR-1 nanoparticles prepared in Comparative Example 1 of the present invention, and TEM images (c) and (d) of the lanthanum-copper@Lap / IWR-1 nanoparticles prepared in Comparative Example 1. Figure 4The images show TEM images (a) of the lanthanum-Lap / IWR-1 nanoparticles prepared in Comparative Example 2 of the present invention and their tumor cell endocytosis (b), and TEM images (c) of the lanthanum-copper@Lap / IWR-1 nanoparticles prepared in Comparative Example 2 and their dispersibility index (d). Figure 5 The images show TEM images (a) of the lanthanum-Lap / IWR-1 nanoparticles prepared in Comparative Example 3 of the present invention, including their tumor cell endocytosis map (b) and dispersibility index map (c), and TEM images (d) of the lanthanum-copper@Lap / IWR-1 nanoparticles prepared in Comparative Example 3, including their tumor cell endocytosis map (e). Figure 6 TEM images (a) of lanthanum-Lap / IWR-1 nanoparticles prepared in Comparative Example 4 of the present invention, and TEM images (b) of lanthanum-copper@Lap / IWR-1 nanoparticles prepared in Comparative Example 4 and their hydrated particle size distribution (c). Figure 7 The images show TEM images (a) and (b) of the lanthanum-Lap / IWR-1 nanoparticles prepared in Comparative Example 5 of the present invention, and TEM images (c) and (d) of the lanthanum-copper@Lap / IWR-1 nanoparticles prepared in Comparative Example 5. Figure 8 The images show TEM images (a) and (b) of the lanthanum-Lap / IWR-1 nanoparticles prepared in Comparative Example 6 of the present invention, and TEM images (c) and (d) of the lanthanum-copper@Lap / IWR-1 nanoparticles prepared in Comparative Example 6. Figure 9 The image shows a simulated SOD activity diagram (a) of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of the present invention, and a comparison diagram (b) of the simulated SOD activity of the nanozyme with that of the lanthanum-copper@Lap / IWR-1 nanozymes of Comparative Examples 1 to 6. Figure 10 The image shows the simulated POD activity of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention (a), and the comparison image shows the simulated POD activity of the nanozyme with that of the lanthanum-copper@Lap / IWR-1 nanozymes of Comparative Examples 1 to 6 (b). Figure 11 The image shows the simulated GSH-Px activity of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention (a), and the comparison image shows the simulated GSH-Px activity of the nanozyme with that of the lanthanum-copper@Lap / IWR-1 nanozymes of Comparative Examples 1 to 6 (b). Figure 12 The image shows the degradation TEM image of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention in a simulated tumor microenvironment. Figure 13 The TEM image shows the degradation of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 1 of this invention in a simulated tumor microenvironment. Figure 14 The TEM image shows the degradation of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 2 of this invention in a simulated tumor microenvironment. Figure 15 The TEM image shows the degradation of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 3 of this invention in a simulated tumor microenvironment. Figure 16 This is a TEM image showing the degradation of the lanthanum-copper@Lap / IWR-1 nanozyme described in Comparative Example 4 of the present invention in a simulated tumor microenvironment; Figure 17 The TEM image shows the degradation of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 5 of this invention in a simulated tumor microenvironment. Figure 18 The TEM image shows the degradation of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 6 of this invention in a simulated tumor microenvironment. Figure 19 The image shows the cytotoxicity (MTT) curve (a) of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention, and the MTT curve (b) comparing the nanozyme with the lanthanum-copper@Lap / IWR-1 nanozymes prepared in Comparative Examples 1-6. Figure 20 This is a diagram showing the protein expression of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention, which eliminates the resistance of tumor cells to copper death. Figure 21 This is a comparison of the expression of tumor cell copper death resistance proteins and representative copper death proteins between the lanthanum-copper@Lap / IWR-1 nanozyme described in Example 1 of the present invention and the lanthanum-copper@Lap / IWR-1 nanozymes prepared in Comparative Examples 1 to 6. Figure 22 This is a diagram showing the expression of representative proteins in tumor cells induced by the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 of this invention; Figure 23 Distribution of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 in tumor-bearing mice in vivo; Figure 24 Hematological and biochemical parameters of tumor-bearing mice after injection of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1 This embodiment provides a lanthanide / transition metal ion coordination nanozyme, the preparation method of which includes the following steps: In this embodiment, the molar ratio of lanthanum ions, Lap, and IWR-1 is 1:2:2; the molar ratio of lanthanum ions, Lap, IWR-1, and copper ions is 1:2:2:2; and the volume ratio of the sum of the methanol solutions of Lap and IWR-1 to the aqueous solution of LaCl3·6H2O is 4:5.

[0039] (1) Disperse 5.81 mg Lap in 8 mL of methanol solution, add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 8 mL of 0.51 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0040] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme. The obtained lanthanum-copper@Lap / IWR-1 nanozyme has a concentration of 3.8 mg / mL and can be diluted to different concentrations according to the needs of subsequent experiments.

[0041] Comparative Example 1 This comparative example provides a lanthanide / transition metal ion coordination nanozyme and its preparation method. The difference between this comparative example and Example 1 is that the volume ratio of the sum of the methanol solutions of Lap and IWR-1 to the aqueous solution of LaCl3·6H2O is 2:5; the other preparation methods are the same as in Example 1.

[0042] The specific preparation method is as follows: (1) Disperse 5.81 mg Lap in 4 mL of methanol solution, and add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 4 mL of 1.02 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0043] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme.

[0044] Comparative Example 2 This comparative example provides a lanthanide / transition metal ion coordination nanozyme and its preparation method. The difference between this comparative example and Example 1 is that the volume ratio of the sum of the methanol solutions of Lap and IWR-1 to the aqueous solution of LaCl3·6H2O is 1:10; the other preparation methods are the same as in Example 1.

[0045] The specific preparation method is as follows: (1) Disperse 5.81 mg Lap in 1 mL of methanol solution, and add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 1 mL of 4.09 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0046] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme.

[0047] Comparative Example 3 This comparative example provides a lanthanide / transition metal ion coordination nanozyme and its preparation method. The difference between this comparative example and Example 1 is that the molar ratio of lanthanum ions, Lap, and IWR-1 is 1:1:1, and the molar ratio of lanthanum ions, Lap, IWR-1, and copper ions is 1:1:1:2; all other preparation methods are the same as in Example 1.

[0048] The specific preparation method is as follows: (1) Disperse 2.905 mg Lap in 8 mL of methanol solution, add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 8 mL of 0.51 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0049] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme.

[0050] Comparative Example 4 This comparative example provides a lanthanide / transition metal ion coordination nanozyme and its preparation method. The difference between this comparative example and Example 1 is that the molar ratio of lanthanum ions, Lap, and IWR-1 is 1:3:3, and the molar ratio of lanthanum ions, Lap, IWR-1, and copper ions is 1:3:3:2; the other preparation methods are the same as in Example 1.

[0051] The specific preparation method is as follows: (1) Disperse 8.715 mg Lap in 8 mL of methanol solution, add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 8 mL of 1.53 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0052] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme.

[0053] Comparative Example 5 This comparative example provides a lanthanide / transition metal ion coordination nanozyme and its preparation method. The difference between this comparative example and Example 1 is that the molar ratio of lanthanum ions, Lap, and IWR-1 is 1:1:2, and the molar ratio of lanthanum ions, Lap, IWR-1, and copper ions is 1:1:2:2; all other preparation methods are the same as in Example 1.

[0054] The specific preparation method is as follows: (1) Disperse 2.905 mg Lap in 8 mL of methanol solution, add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 8 mL of 1.02 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0055] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme.

[0056] Comparative Example 6 This comparative example provides a lanthanide / transition metal ion coordination nanozyme and its preparation method. The difference between this comparative example and Example 1 is that the molar ratio of lanthanum ions, Lap, and IWR-1 is 1:2:1, and the molar ratio of lanthanum ions, Lap, IWR-1, and copper ions is 1:2:1:2; all other preparation methods are the same as in Example 1.

[0057] The specific preparation method is as follows: (1) Disperse 5.81 mg Lap in 8 mL of methanol solution, add 20 mL of deionized water containing 1.77 mg LaCl3·6H2O while stirring at 800 rpm to obtain mixed solution A; (2) After vigorously stirring the mixed solution A for 5 min, add 8 mL of 0.51 mg / mL IWR-1 methanol solution and 100 μL of 400 mM sodium hydroxide aqueous solution to obtain mixed solution B; (3) After sonicating the mixed solution B at 200 W power for 5 min, it was stirred continuously at 500 rpm for 24 h to obtain a uniformly dispersed mixed solution C; (4) The mixed solution C was centrifuged at 12000 rpm for 10 min to collect the precipitate. The precipitate was then ultrasonically dispersed to obtain lanthanum-Lap / IWR-1 nanomaterials.

[0058] (5) 1.705 mg CuCl2·2H2O was dispersed in 20 mL of deionized water, followed by the addition of lanthanum-Lap / IWR-1 solution. After ultrasonic treatment at 200 W for 10 min, 100 μL of 400 mM sodium hydroxide aqueous solution was added, and the mixture was stirred continuously at 700 rpm for 12 h to obtain mixed solution D. (6) After centrifuging the mixed solution D at 12000 rpm for 10 min, the precipitate was collected and ultrasonically dispersed to obtain lanthanum-copper@Lap / IWR-1 nanozyme.

[0059] Experimental Example 1 This experiment characterizes the structure of the lanthanide / transition metal ion coordination nanozymes prepared in Example 1 and Comparative Examples 1-6. like Figure 1 As shown in Figure a, the SEM image of the lanthanum-Lap / IWR-1 nanomaterials prepared in Example 1 shows that the lanthanum-Lap / IWR-1 nanomaterials are spherical; as Figure 1 As shown in Figure b, the particle size distribution map was obtained by recording and analyzing the size of lanthanum-Lap / IWR-1 nanomaterials. The data shows that the particle size of lanthanum-Lap / IWR-1 nanomaterials is 77~101 nm.

[0060] like Figure 2 As shown in Figure a, the TEM image of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 shows that the lanthanum-copper@Lap / IWR-1 nanozyme is in a clustered state; as Figure 2As shown in Figure b, the particle size distribution map was obtained by recording and analyzing the size of the lanthanum-copper@Lap / IWR-1 nanozyme. The data shows that the particle size of the lanthanum-copper@Lap / IWR-1 nanozyme is 61~83 nm.

[0061] like Figure 3 As shown in Figure a, the TEM image of the lanthanum-Lap / IWR-1 nanomaterial prepared in Comparative Example 1 shows a tendency towards a spherical morphology, but compared with Example 1, the morphology formation of this material is less than ideal; Figure 3 As shown in Figure b, the lanthanum-Lap / IWR-1 nanomaterial prepared in Example 1 maintained a stable hydrated particle size for 7 consecutive days; however, the hydrated particle size of the lanthanum-Lap / IWR-1 nanomaterial in Comparative Example 1 gradually decreased over time, indicating that the nanomaterial prepared in Comparative Example 1 had poor stability. Figure 3 As shown in Figure c, the TEM image (scale bar 1 μm) of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 1 shows that it failed to form nanoparticles with a uniform and regular morphology; Figure 3 As shown in Figure d, the lanthanum-copper@Lap / IWR-1 nanomaterial of Comparative Example 1 has poor stability, and its hydrated particle size decreases rapidly over time, which does not meet the product requirements.

[0062] like Figure 4 As shown in Figure a, the lanthanum-Lap / IWR-1 nanomaterials prepared in Comparative Example 2 exhibit a chain-like aggregate morphology. Due to its large aspect ratio, this structure is difficult to be completely encapsulated by the cell membrane during tumor cell endocytosis, leading to a decrease in endocytosis efficiency. Figure 4 As can be seen from b, compared to the lanthanum-Lap / IWR-1 nanomaterials prepared in Example 1, this chain-like morphology weakens the uptake ability of tumor cells. Figure 4 As shown in Figure c, the TEM image (scale bar 500 nm) of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 2 shows that its morphology does not exhibit a chain-like structure, but there is a problem of morphological inhomogeneity, and the degree of aggregation is also aggravated; as shown in Figure c. Figure 4 As shown in Figure d, the dispersibility of the lanthanum-copper@Lap / IWR-1 nanozyme in Comparative Example 2 is significantly worse than that of the lanthanum-copper@Lap / IWR-1 nanozyme in Example 1, and its dispersibility index is significantly higher than that of Example 1. Therefore, this product does not meet the requirements.

[0063] like Figure 5 As shown in Figure a, the morphology of the lanthanum-Lap / IWR-1 nanomaterials prepared in Comparative Example 3 underwent significant changes, forming large-sized sheet-like structures with obvious stacking. On the one hand, the excessively large size significantly reduces the endocytosis efficiency of the material by cells, thus affecting the results of subsequent cell and in vivo experiments. Figure 5As shown in Figure b, with the extension of incubation time, the uptake efficiency of lanthanum-Lap / IWR-1 nanoparticles by tumor cells in Example 1 was significantly higher than that in Comparative Example 3; on the other hand, the severe stacking of materials led to a significant decrease in their dispersibility, which not only affected the stability of the nanomaterials but also had an adverse effect on the in vivo drug delivery process, such as... Figure 5 As shown in Figure c, the dispersibility index of the lanthanum-Lap / IWR-1 prepared in Example 1 is significantly lower than that of Comparative Example 3. Figure 5 As shown in Figure d, the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 3 exhibits a sheet-like morphology, but its size is larger than that of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1. This difference will affect the endocytosis efficiency of the nanozyme by cells; Figure 5 As shown in Figure e, the uptake efficiency of tumor cells on the lanthanum-copper@Lap / IWR-1 nanozyme of Comparative Example 3 was significantly lower than that on the nanozyme of Example 1, and the product did not meet the requirements.

[0064] like Figure 6 As shown in Figure a, the morphology of the lanthanum-Lap / IWR-1 nanomaterials prepared in Comparative Example 4 became finer and exhibited agglomeration. This small-particle-size and agglomerated structure will adversely affect subsequent surface modification and binding with other drugs. According to the data in Table 1, after the lanthanum-Lap / IWR-1 nanomaterials prepared in Example 1 were bound to the chemotherapy drug DOX, the DOX loading rate reached 13.6% and the encapsulation efficiency was 47.4%; in contrast, the DOX loading rate in Comparative Example 4 was only 3.51% and the encapsulation efficiency was only 10.9%. Figure 6 As shown in Figure b (scale bar 100 nm), the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 4 exhibits a spherical morphology, but suffers from an unclean backing and the formation of a small number of large particles; for example... Figure 6 As shown in Figure c, compared with the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1, the lanthanum-copper@Lap / IWR-1 nanozyme in Comparative Example 4 exhibits a multi-size distribution of hydrated particle size, indicating that its particle size uniformity has significantly decreased, and this product does not meet the requirements.

[0065] Table 1

[0066] like Figure 7 As shown in Figure a, a TEM image (scale bar 500 nm) of the lanthanum-Lap / IWR-1 nanomaterial prepared in Comparative Example 5 shows that no nanoparticles were formed; Figure 7 As shown in Figure b (scale bar 500 nm), the lanthanum-Lap / IWR-1 nanomaterials in Comparative Example 5 exhibit poor stability, with their hydrated particle size decreasing rapidly over time. Figure 7As shown in Figure c, the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 5 did not form nanoparticles; as Figure 7 As shown in Figure d, the lanthanum-copper@Lap / IWR-1 nanozyme of Comparative Example 5 has extremely poor stability, and the hydrated particle size fluctuates over time. This product does not meet the requirements.

[0067] like Figure 8 As shown in Figure a (scale bar 100 nm), the TEM image of the lanthanum-Lap / IWR-1 nanomaterial prepared in Comparative Example 6 shows that the nanoparticles exhibit significant aggregation, indicating a serious problem of agglomeration. Figure 8 As shown in b (scale bar 200 nm), the dispersibility index of this nanomaterial is higher than that of Example 1, indicating that its dispersibility is worse than that of Example 1; Figure 8 As shown in Figure c, although the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Comparative Example 6 exhibits a tendency towards spherical morphology, it still shows aggregation; as Figure 8 As shown in Figure d, the dispersibility of this nanozyme is also lower than that of Example 1, and the product does not meet the requirements.

[0068] Application Example 1 This application example provides an investigation into the enzyme-mimicking properties of the lanthanide / transition metal ion coordination nanozymes prepared in Example 1 and Comparative Examples 1-6. (1) Study on simulated superoxide dismutase activity Nitrotetrazolium chloride was used as O2. ·- The detection probe, which is in O2 ·- Under certain conditions, it can be reduced to a formazan product with a characteristic absorption peak. O2 is generated through a riboflavin / methionine photochemical system. ·- As a control, the simulated SOD activity of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 was tested.

[0069] like Figure 9 As shown in Figure a, with the increase of the concentration gradient of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 (0, 1, 5, 10, 20 μg / mL), the intensity of the characteristic absorption peak of formazan decreased in a concentration-dependent manner, indicating that the nanozyme has significant concentration-dependent SOD mimicry activity.

[0070] like Figure 9 As shown in Figure b, compared with other comparatively prepared lanthanum-copper@Lap / IWR-1 nanozymes, the lanthanum-copper@Lap / IWR-1 nanozyme treatment group prepared in Example 1 had the lowest formazan characteristic absorption peak intensity. This result indicates that among all the nanozymes tested, the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 exhibits excellent SOD mimicry activity.

[0071] (2) Study on simulated peroxidase activity 3,3',5,5'-Tetramethylbenzidine was used as the chromogenic substrate for POD activity. It can be oxidized by ·OH to generate a blue oxTMB product, which exhibits a characteristic absorption peak at 652 nm. The simulated POD activity of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 was detected using an H2O2-TMB reaction system, yielding the following results: Figure 10 Figure a shows the simulated POD activity of the lanthanum-copper@Lap / IWR-1 nanozyme at different concentrations.

[0072] like Figure 10 As shown in Figure a, the concentrations of lanthanum-copper@Lap / IWR-1 nanozymes were 0, 5, 10, 20, and 40 μg / mL, respectively, and the absorbance at 652 nm showed a significant concentration-dependent increase, indicating that the lanthanum-copper@Lap / IWR-1 nanozyme has POD-like activity.

[0073] like Figure 10 As shown in Figure b, compared with other comparatively prepared lanthanum-copper@Lap / IWR-1 nanozymes, the oxTMB product exhibited the highest characteristic absorption peak intensity in the lanthanum-copper@Lap / IWR-1 nanozyme treatment group prepared in Example 1. This test result indicates that, among all the nanozymes tested, the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 possesses excellent POD-mimicking activity.

[0074] (3) Study on simulated glutathione peroxidase activity 5,5-Dithio-bis-(2-nitrobenzoic acid) was used as an indicator for GSH-Px enzyme activity, producing a yellow product with a characteristic absorption peak at 412 nm under GSH. The lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 was diluted to different concentrations and reacted with GSH for the same time before DTNB was added to detect simulated GSH-Px activity, yielding the following results: Figure 11 The simulated GSH-Px activity diagram of the lanthanum-copper@Lap / IWR-1 nanozyme is shown in Figure a.

[0075] like Figure 11 As shown in Figure a, after reacting with GSH for the same amount of time, the absorption peak at 412 nm of different concentrations of lanthanum-copper@Lap / IWR-1 nanozyme (0, 5, 10, 20, 40 μg / mL) showed a concentration-dependent decreasing trend, indicating that the lanthanum-copper@Lap / IWR-1 nanozyme has GSH-Px mimicking activity.

[0076] like Figure 11As shown in Figure b, compared with other comparative examples of lanthanum-copper@Lap / IWR-1 nanozymes, the nanozyme treatment group prepared in Example 1 exhibited the lowest characteristic absorption peak intensity at 412 nm, indicating the lowest GSH content. Therefore, among all tested nanozymes, the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 demonstrated excellent GSH-Px mimicry activity.

[0077] Application Example 2 This application example provides a study on the degradation behavior of lanthanide / transition metal ion coordination nanozymes prepared in Example 1 and Comparative Examples 1-6, simulating the tumor microenvironment. To investigate the biodegradability of the lanthanum-copper@Lap / IWR-1 nanozymes prepared in Example 1 and Comparative Examples 1-6, environmental systems with pH (5.4), GSH (10 mM), and H2O2 (0.1 mM) were established to simulate tumor microenvironment conditions in vitro. Morphological changes of the lanthanum-copper@Lap / IWR-1 nanozymes were observed using TEM, such as... Figures 12-18 As shown.

[0078] The results showed that the nanozyme cluster structure of Example 1 exhibited significant dissociation under acidic, GSH, and H2O2 conditions, respectively, and significant degradation characteristics were observed. These findings confirm that the lanthanum-copper@Lap / IWR-1 nanozyme has good biodegradability under TME conditions. This characteristic not only facilitates the in vivo metabolic clearance of nanomaterials but also effectively meets the safety requirements of biomedical materials. Although the lanthanum-copper@Lap / IWR-1 nanozymes of Comparative Examples 1-6 showed some degree of structural damage under acidic conditions, GSH, and H2O2, this phenomenon was partly due to the instability of their own morphological structure, and their degradation degree was less than that of Example 1.

[0079] Application Example 3 This application example provides an in vitro antitumor activity study of the lanthanide / transition metal ion coordination nanozymes prepared in Example 1 and Comparative Examples 1-6. (1) Cytotoxicity test: 4T1 cells were seeded at a density of 8000 cells / well in 96-well plates and incubated for 24 h. After incubation, the cells were co-incubated for 12 h with 0, 25, 50, 100, 200, and 400 μg / mL of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1. Cytotoxicity was assessed using the MTT assay, and absorbance at 490 nm was read using a microplate reader. Figure 19 As shown in Figure a.

[0080] like Figure 19As shown in Figure a, the killing effect on tumor cells is enhanced with the increase of the concentration of lanthanum-copper@Lap / IWR-1 nanozyme, demonstrating its tumor-killing effect.

[0081] like Figure 19 As shown in Figure b, the lanthanum-copper@Lap / IWR-1 nanozymes prepared in Comparative Examples 1-6 all exhibited significantly weaker tumor cell-killing effects compared to the nanozymes of the same type prepared in Example 1. This result confirms the superior functional performance of the lanthanum-copper@Lap / IWR-1 nanozyme of Example 1, demonstrating its greater potential in tumor therapy-related applications.

[0082] (2) Copper death resistance elimination test: 4T1 cells were loaded at 5.0 × 10⁻⁶. 6 The nanozymes were seeded at a density of 100 cells / well in culture dishes. After 24 h of culture, 200 μg / mL of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 was added, and the cells were co-cultured for another 12 h. The expression levels of copper death-related resistance proteins in the cells were detected by Western blotting, and the results were as follows: Figure 20 As shown.

[0083] Experimental results showed that after treatment with lanthanum-copper@Lap / IWR-1 nanozyme, the expression of multiple copper death resistance proteins in 4T1 cells was significantly downregulated, confirming that lanthanum-copper@Lap / IWR-1 nanozyme can effectively inhibit the resistance mechanism of tumor cells to copper death.

[0084] The elimination of copper death resistance in tumor cells by the lanthanum-copper@Lap / IWR-1 nanozymes prepared in Example 1 and Comparative Examples 1-6 was compared, and the results were as follows: Figure 21 As shown.

[0085] Experimental results showed that the lanthanum-copper@Lap / IWR-1 nanozymes prepared in Comparative Examples 1-6 were significantly less effective than the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 in eliminating copper death resistance proteins (HIF-1α, MT2A, PDK1, β-catenin, WNT3A). This indicates that the nanozyme of Example 1 can more effectively reduce the resistance of tumor cells to copper death, thereby further enhancing its effect in inducing copper death in tumor cells.

[0086] (3) Copper death test: 4T1 cells were fed at 5.0 × 10⁻⁶ 6 The nanozymes were seeded at a density of 100 cells / well in culture dishes. After 24 h of culture, 200 μg / mL of the lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 was added, and the cells were co-cultured for another 12 h. The expression levels of proteins related to copper death in the cells were detected by Western blotting, and the results were as follows: Figure 22 As shown.

[0087] The levels of lipoic acid synthase (LIAS) and ferroreductase 1 (FDX1) in 4T1 cells treated with lanthanum-copper@Lap / IWR-1 nanozyme were reduced, and dihydrolipoamide S-acetyltransferase (DLAT) protein aggregated. These results indicate that tumor cells undergo significant copper death under the treatment of lanthanum-copper@Lap / IWR-1 nanozyme.

[0088] like Figure 21 As shown, in tumor cells treated with the lanthanum-copper@Lap / IWR-1 nanozymes in Comparative Examples 1-6, the expression levels of LIAS and FDX1 proteins, as well as the aggregation level of DLAT, were lower than those in the lanthanum-copper@Lap / IWR-1 nanozyme treatment group of Example 1. This phenomenon indicates that the nanozyme of Example 1 can induce copper death in tumor cells more efficiently.

[0089] Application Example 4 This application example provides a study on the biosafety of the lanthanide / transition metal ion coordination nanozyme prepared in Example 1. To ensure the biosafety of the lanthanum-copper@Lap / IWR-1 nanozyme, the in vivo distribution characteristics of the material and its effects on the blood system were evaluated using a mouse tumor model.

[0090] The lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 at a concentration of 15 mg / kg was injected subcutaneously into Balb / c mice carrying 4T1 cells via tail vein injection. The distribution of the material in the mice was detected at 0, 6, 12, 24, and 48 h. Figure 23 .

[0091] like Figure 23 As shown, within 24 hours after injection of lanthanum-copper@Lap / IWR-1 nanozyme, the copper content in tumor tissue increased in a time-dependent manner, significantly higher than that in other normal tissues. Notably, the copper content in the tumor site began to decrease after 48 hours, while the copper content in the kidneys continued to increase. These results indicate that lanthanum-copper@Lap / IWR-1 nanozyme can selectively accumulate in tumor tissue through the enhanced osmotic retention (EPR) effect, then undergo responsive degradation in the slightly acidic environment of the tumor, and finally, the metabolites are mainly cleared through the kidneys.

[0092] Healthy Balb / c mice and Balb / c mice treated with lanthanum-copper@Lap / IWR-1 nanozyme prepared in Example 1 at a concentration of 15 mg / kg were subjected to blood biochemistry and routine blood tests to obtain... Figure 24 .

[0093] like Figure 24As shown, compared with healthy mice, the mice treated with the drug showed no significant differences in any of their indicators, all of which were within the normal range, indicating good biocompatibility.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lanthanide / transition metal ion coordination nanozyme, characterized in that, The lanthanide / transition metal ion coordination nanozyme has a hierarchical network structure, including a first-level network and a second-level network. The first-level network is a ternary coordination complex formed by lanthanum ions as the central metal node, and deprotonated tyrosine kinase inhibitor lapatinib and Wnt pathway inhibitor IWR-1. The second-level network is a quaternary coordination structure formed by copper ions as secondary metal nodes embedded in the first-level network. The molar ratio of lanthanum ions, lapatinib, IWR-1 and copper ions is 1:(1~3):(1~3):

2.

2. The lanthanide / transition metal ion coordination nanozyme as described in claim 1, characterized in that, The molar ratio of lanthanum ions, Lap, IWR-1 to copper ions is 1:2:2:2; Preferably, the ternary coordination complex is connected by lanthanum ions to the sulfonyl oxygen atom of lapatinib and the carbonyl oxygen atom of IWR-1; the quaternary coordination structure is based on the ternary complex and is connected by copper ions to the pyrimidine ring nitrogen atom of lapatinib and the pyridine ring / amide nitrogen atom of IWR-1. Preferably, the ternary coordination complex is spherical with a particle size of 77-101 nm; the lanthanide / transition metal ion coordination nanozyme is clustered with a particle size of 61-83 nm.

3. The lanthanide / transition metal ion coordination nanozyme as described in claim 1, characterized in that, The lanthanide / transition metal ion coordination nanozyme has triple enzyme activity, including superoxide dismutase, peroxidase and glutathione peroxidase. Preferably, the lanthanide / transition metal ion coordination nanozyme degrades under high concentrations of GSH, H2O2, and slightly acidic conditions, releasing therapeutically active lanthanum ions, copper ions, Lap, and IWR-1.

4. A method for preparing a lanthanide / transition metal ion coordination nanozyme according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Add methanol solution of Wnt pathway inhibitor IWR-1 and sodium hydroxide aqueous solution to a mixed solution of tyrosine kinase inhibitor lapatinib and soluble lanthanum salt, and perform ultrasonic and stirring reaction. After post-treatment, lanthanum-Lap / IWR-1 nanoparticles are obtained. S2. Lanthanum-Lap / IWR-1 nanoparticles were added to a soluble copper salt aqueous solution, and after ultrasonic treatment, sodium hydroxide solution was added. The reaction was continuously stirred, and the lanthanum-copper@Lap / IWR-1 nanozyme was obtained after post-treatment.

5. The preparation method according to claim 4, characterized in that, In step S1, the mixed solution comprises a methanol solution containing the tyrosine kinase inhibitor lapatinib and an aqueous solution containing a soluble lanthanum salt; the ratio of the sum of the volumes of the methanol solutions containing the tyrosine kinase inhibitor lapatinib and the methanol solution containing the Wnt pathway inhibitor IWR-1 to the volume of the aqueous solution containing the soluble lanthanum salt is 3.5~4.5:5~5.5, preferably 4:

5.

6. The preparation method according to claim 4, characterized in that, In step S1, the molar ratio of the tyrosine kinase inhibitor lapatinib, the soluble lanthanum salt, and the Wnt pathway inhibitor IWR-1 is 1:(1~3):(1~3); Preferably, the soluble lanthanum salt is selected from one or more of lanthanum chloride hexahydrate and lanthanum nitrate hexahydrate; Preferably, the concentration of the Wnt pathway inhibitor IWR-1 is 0.45~0.55 mg / mL, the concentration of the sodium hydroxide aqueous solution is 380~420 mM, and the volume ratio of the methanol solution of the Wnt pathway inhibitor IWR-1 to the sodium hydroxide aqueous solution is 8:0.1~0.

2. Preferably, the ultrasonic power is 15~250 W, and the time is 2~10 min; the stirring rate of the stirring reaction is 300~800 rpm, and the time is 22~26 h; the post-treatment includes centrifugation, washing, and drying, the centrifugation speed is 10000~14000 rpm, and the centrifugation time is 5~15 min; the washing is performed 2~3 times.

7. The preparation method according to claim 4, characterized in that, In step S2, the ratio of the soluble copper salt to the sodium hydroxide aqueous solution is (1.7~1.71) mg:(80~120) μL; Preferably, the soluble copper salt is selected from one or more of copper chloride dihydrate and copper nitrate trihydrate; Preferably, the ultrasonic treatment power is 15~250 W, and the time is 5~15 min; the stirring rate of the continuous stirring reaction is 500~1000 rpm, and the time is 10~14 h; the post-treatment includes centrifugation, washing, and drying, the centrifugation speed is 10000~14000 rpm, and the centrifugation time is 5~15 min; the washing is performed 2~3 times.

8. The use of the lanthanide / transition metal ion coordination nanozyme according to any one of claims 1 to 3 in the preparation of tumor microenvironment improvers or products that induce copper cell death.

9. A pharmaceutical composition, characterized in that, Includes the lanthanide / transition metal ion coordination nanozyme and pharmaceutical carrier as described in any one of claims 1 to 3.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical carrier is selected from one or more of hyaluronic acid, polyethylene glycol, polysaccharides, and glycolic acid; The pharmaceutical composition also includes other active ingredients, which are selected from one or more of antitumor small molecule drugs, natural enzymes, and sound-sensitive agents.

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