Preparation method of ternary metal single-atom nanomaterial with high-efficiency enzyme activity sensitized ferroptosis effect

Abstract

The application relates to a preparation method of a ternary metal single-atom nanomaterial with a high-efficiency enzyme activity sensitization iron death effect, and relates to a preparation method of a ternary metal single-atom nanomaterial.The application aims to solve the problems that a single catalytic path of an existing nanometer enzyme is limited by a tumor antioxidant defense system, resulting in low iron death sensitization efficiency, and that a multi-metal single-atom active site is prone to migration and agglomeration, and multi-component components are difficult to realize atomic-level uniform dispersion.Method: I. synthesizing MIL-101(Fe); II. synthesizing Ce,Mo@MIL(Fe)@PZS; III. synthesizing Ce,Mo,Fe MEO-SACs.The application is used for the preparation of a ternary metal single-atom nanomaterial with a high-efficiency enzyme activity sensitization iron death effect.

Description

Technical Field

[0001] This invention relates to a method for preparing ternary metal single-atom nanomaterials. Background Technology

[0002] Enzymatic catalysis induces cell death by generating reactive oxygen species (ROS) in situ within the tumor microenvironment, mimicking the activity of natural enzymes. However, natural enzymes suffer from limitations such as poor stability, high cost, and short in vivo half-life. Traditional nanozymes are often constrained by inefficient catalytic kinetics and single catalytic pathways, making it difficult to overcome the tumor's endogenous antioxidant defense system, such as the scavenging effect of highly expressed glutathione (GSH), leading to less than ideal therapeutic effects. Among the many ROS-mediated cell death modes, ferroptosis has attracted considerable attention due to its unique lipid peroxidation mechanism. Sensitizing ferroptosis through specific catalytic systems involves continuously supplying highly toxic ROS, such as hydroxyl radicals (·OH) and singlet oxygen (·OH), to an external catalyst. 1 O2 can trigger a lipid peroxidation chain reaction, bypassing the classic apoptosis resistance pathway and achieving efficient clearance of refractory tumors.

[0003] The single-atom structure of polymetallic materials can achieve extremely high atomic utilization. Their catalytic mechanism relies on atomic-level dispersion to expose homogeneous active sites, thereby optimizing intermediate adsorption energies through strong metal-support interactions. These interactions can modulate d-band centers and charge distribution. However, under reaction conditions, single atoms in polymetallic materials may still migrate, aggregate, or leach, threatening material stability. To address this issue, research focuses on constructing support-single-atom synergistic interfaces and developing dynamic stabilization strategies. Multi-element single-atom loading at interfaces with locally highly mixed configurations inhibits metal aggregation through a high-entropy-like stabilization effect. Leveraging multi-element synergistic effects and tunable d-band centers, these components have shown great potential for inducing tumor oxidative stress and sensitized ferroptosis in in vitro models. However, achieving atomically uniform dispersion of the multi-element components remains challenging. Summary of the Invention

[0004] This invention aims to address the problem of low ferroptosis sensitization efficiency caused by the single catalytic pathway of existing nanozymes being limited by the tumor antioxidant defense system, and to solve the problems of easy migration and aggregation of single-atom active sites of multi-metals and the difficulty in achieving atomic-level uniform dispersion of multi-components. Therefore, it provides a method for preparing ternary metal single-atom nanomaterials with highly efficient enzyme activity to sensitize ferroptosis.

[0005] A method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity and sensitized iron death effect, comprising the following steps:

[0006] I. Synthesis of MIL-101(Fe):

[0007] ① Add the iron source and the organic bridging ligand to an organic solvent and sonicate to obtain a mixed solution A;

[0008] ② Heat mixed solution A to 100℃~200℃ and react until the solution turns reddish-brown. After the reaction is complete, cool it to room temperature to obtain solution B.

[0009] ③ Centrifuge solution B, then wash and dry the product to obtain MIL-101(Fe) powder;

[0010] II. Synthesis of Ce,Mo@MIL(Fe)@PZS:

[0011] ① Add MIL-101(Fe) powder to methanol and sonicate until homogenized. Then add Ce source and Mo source to obtain solution C.

[0012] ② The polymer coordination shell component was added to methanol and ultrasonically homogenized to obtain solution D;

[0013] ③ Mix solution C with solution D, then add an acid-binding agent and disperse by ultrasonication to obtain solution E;

[0014] ④ Stir solution E at room temperature, then centrifuge and wash sequentially to obtain Ce,Mo@MIL(Fe)@PZS;

[0015] III. Synthesis of Ce, Mo, Fe MEO-SACs:

[0016] The method for preparing ternary metal single-atom nanomaterials with highly efficient enzyme activity sensitized by iron death effect is completed by drying and grinding Ce,Mo@MIL(Fe)@PZS and reacting it in an argon atmosphere at a temperature of 200℃~500℃.

[0017] The beneficial effects of this invention are:

[0018] This invention proposes a trimetallic nanozyme CMF MESACs with a single-atom-support structure. By precisely controlling the d-band center through heterometallic orbital hybridization, charge transfer is optimized within its narrow bandgap structure characterized by lattice distortion. A high configurational entropy-driven interface-driven thermodynamic stabilization effect enables multi-level catalytic cascades between the single-atom sites and the multi-component metal substrate through controllable electronic interactions. Mo's 4d orbital hybridization narrows the bandgap, while Ce suppresses electron-hole recombination and enhances charge transfer through band alignment and Fermi level pinning. Electronic modifications lead to rapid internal electron transfer and catalytic adsorption / desorption processes, maintaining high catalytic activity. In catalysis, the surface-confined single-atom structure utilizes electron enrichment provided by the internal matrix to regulate the reactant adsorption energy of ROS burst reactions at active sites and promotes rapid responses to GSH consumption and O2 generation. Under near-infrared light excitation, CMF MESACs achieve deep-structure excitation, electron-hole separation and recombination, resulting in thermally driven catalytic performance enhancement. Attached Figure Description

[0019] Figure 1 The images are TEM images and elemental scan images. a is the MIL prepared in step one of Example 1, b is the CM@MIL@PZS prepared in step two ④ of Comparative Experiment 2, and c is the CM@MIL@PZS prepared in step two ④ of Example 1.

[0020] Figure 2 The images show the TEM image, elemental scan image, HAADF-STEM image, and energy spectrum of the CMF MESACs prepared in step three of Example 1. a is the TEM image, b is the elemental scan image, c is the HAADF-STEM image, and d is the energy spectrum.

[0021] Figure 3 XPS spectra of CMF MESACs prepared in Example 1, CF LESACs prepared in Comparative Experiment 3, and MF LESACs prepared in Comparative Experiment 4 are shown. a is N 1s spectrum, b is C 1s spectrum, c is O 1s spectrum, d is Fe 2p spectrum, e is Ce 3d spectrum, and f is Mo 3d spectrum.

[0022] Figure 4 Solid-state diffuse reflectance spectra of MIL, CM@MIL@PZS and CMF MESACs prepared in Example 1;

[0023] Figure 5 XRD patterns of MIL, CM@MIL@PZS and CMF MESACs prepared in Example 1;

[0024] Figure 6 Zeta potential diagrams of MIL, CM@MIL@PZS and CMF MESACs prepared in Example 1;

[0025] Figure 7 The FT-IR infrared spectra of MIL, CM@MIL@PZS and CMF MESACs prepared in Example 1 are shown.

[0026] Figure 8 To investigate the formation of ·OH in CMF MESACs prepared in Example 1, a is the ESR spectrum of ·OH formation with DMPO as the capture agent, and b is the UV absorption spectrum of ·OH generated by combining TMB as the colorimetric agent with different concentrations of CMF MESACs.

[0027] Figure 9 The CMF MESACs prepared in Example 1 1 O2 generation investigation, 'a' represents TEMP as a capture agent. 1 O2 generation ESR spectrum, b is the O2 generation ESR spectrum catalyzed by DPBF as chromogenic agent combined with MESACs of different concentrations of CMF. 1 UV absorption spectrum after O2;

[0028] Figure 10 This is a diagram illustrating the GSH-catalyzed decomposition of CMF MESACs prepared in Example 1.

[0029] Figure 11 This is a graph illustrating the O2 release performance of the CMF MESACs prepared in Example 1.

[0030] Figure 12 This is a diagram illustrating the photothermal effect of CMF MESACs prepared in Example 1.

[0031] Figure 13 To investigate the effects of CM@MIL@PZS and CMF MESACs prepared in Example 1 on the ferroptosis pathway, (1) control group, (2) CM@MIL@PZS + laser irradiation group (808nm, 1.0W / cm) 2 (5 min), (3) CMF MESACs + laser irradiation group (808 nm, 1.0 W / cm 2 (5 min), (4) CMF MESACs + laser irradiation (808 nm, 1.0 W / cm 2 (5 min) + ferroptosis inhibitor + apoptosis inhibitor group;

[0032] Figure 14 To investigate the cellular-level therapeutic performance of CM@MIL@PZS and CMF MESACs prepared in Example 1, (1) control group, (2) laser irradiation treatment (808nm, 1.0W / cm) 2(3) CM@MIL@PZS treatment, (4) CMF MESACs treatment, (5) CM@MIL@PZS + laser treatment (808nm, 1.0W / cm) 2 (5 min), (6) CMF MESACs + laser treatment (808 nm, 1.0 W / cm 2 (5 min). Detailed Implementation

[0033] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0034] Specific Implementation Method 1: This implementation method is a preparation method of a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect, which is carried out according to the following steps:

[0035] I. Synthesis of MIL-101(Fe):

[0036] ① Add the iron source and the organic bridging ligand to an organic solvent and sonicate to obtain a mixed solution A;

[0037] ② Heat mixed solution A to 100℃~200℃ and react until the solution turns reddish-brown. After the reaction is complete, cool it to room temperature to obtain solution B.

[0038] ③ Centrifuge solution B, then wash and dry the product to obtain MIL-101(Fe) powder;

[0039] II. Synthesis of Ce,Mo@MIL(Fe)@PZS:

[0040] ① Add MIL-101(Fe) powder to methanol and sonicate until homogenized. Then add Ce source and Mo source to obtain solution C.

[0041] ② The polymer coordination shell component was added to methanol and ultrasonically homogenized to obtain solution D;

[0042] ③ Mix solution C with solution D, then add an acid-binding agent and disperse by ultrasonication to obtain solution E;

[0043] ④ Stir solution E at room temperature, then centrifuge and wash sequentially to obtain Ce,Mo@MIL(Fe)@PZS;

[0044] III. Synthesis of Ce, Mo, Fe MEO-SACs:

[0045] The method for preparing ternary metal single-atom nanomaterials with highly efficient enzyme activity sensitized by iron death effect is completed by drying and grinding Ce,Mo@MIL(Fe)@PZS and reacting it in an argon atmosphere at a temperature of 200℃~500℃.

[0046] In step one of this embodiment, the MIL-101(Fe) metal-organic framework synthesized by the heat of solution method exhibits good dispersibility and a distinct symmetrical octahedral structure.

[0047] In this embodiment, sonication and stirring are used to better disperse the reagents and achieve complete reaction and uniform heating.

[0048] In step 1② of this embodiment, the temperature should be preheated to 40°C~60°C, and the subsequent heating should be carried out at a heating rate of 1°C / min~10°C / min. This allows the reaction to proceed in a timely manner and facilitates observation and control.

[0049] In step 1③ of this embodiment, the product is washed in anhydrous ethanol, which can more effectively separate nanoparticles from organic solvents and stably obtain pure products.

[0050] The polymer coordination shell method used in step two of this embodiment has a higher doping amount compared to direct stirring doping or direct doping in solvothermal mode. The polymer coordination effectively disperses heterogeneous metals and avoids the formation of large-sized particles and clusters through pyrolysis.

[0051] The pyrolysis of materials under an argon atmosphere used in step three of this embodiment can effectively avoid the formation of a large number of oxides.

[0052] The key challenge in the material design of this specific embodiment lies in how to precisely balance the interface engineering of single-atom anchoring to avoid thermodynamically driven phase separation and single-atom aggregation traps, while simultaneously designing materials capable of providing multi-element enzyme-like catalysis for highly efficient sensitization of ferrode tumor therapy. This specific embodiment utilizes a dual heterometallic anchoring to an Fe-based metal-organic framework structure, followed by pyrolysis to form a ternary single-atom-support structure. By controlling the synergistic regulation of the d-band center by multiple elements and optimizing the adsorption energy of the active sites for reaction intermediates, a stable, tumor microenvironment-responsive nanocatalytic system is constructed.

[0053] This embodiment features a stable and mature process, employing a PZS-assisted interface confinement strategy. The reaction system is environmentally friendly, successfully constructing a ternary metal single-atom nanomaterial catalytic system with promising application prospects. The obtained Ce / Mo / Fe trimetallic sites are atomically dispersed and anchored to the support, effectively avoiding phase separation and maximizing atom utilization. The material exhibits excellent hydrophilicity, low toxicity, and good biocompatibility.

[0054] This implementation leverages the tumor microenvironment's responsiveness to induce apoptosis and ferroptosis synergistically by paralyzing tumor antioxidant defense mechanisms through GSH depletion and lipid peroxidation. The introduction of photothermal effects further optimizes the electronic structure and accelerates catalytic kinetics, constructing a multimodal synergistic therapeutic platform that significantly enhances antitumor efficacy, demonstrating significant clinical translational potential and innovative leadership value.

[0055] The beneficial effects of this embodiment are:

[0056] This embodiment proposes a trimetallic nanozyme CMF MESACs with a single-atom-support structure. By precisely controlling the d-band center through heterometallic orbital hybridization, charge transfer is optimized within its narrow bandgap structure characterized by lattice distortion. A high configurational entropy-driven interface-driven thermodynamic stabilization effect enables multi-level catalytic cascades between the single-atom sites and the multi-component metal substrate through controllable electronic interactions. Mo's 4d orbital hybridization narrows the bandgap, while Ce, through band alignment and Fermi level pinning, suppresses electron-hole recombination and enhances charge transfer. Electronic modifications lead to rapid internal electron transfer and catalytic adsorption / desorption processes, maintaining high catalytic activity. In catalysis, the surface-confined single-atom structure utilizes electron enrichment provided by the internal matrix to modulate the reactant adsorption energy of ROS burst reactions at active sites and promotes rapid responses to GSH consumption and O2 generation. Under near-infrared light excitation, CMF MESACs achieve deep-structure excitation, electron-hole separation and recombination, resulting in thermally driven catalytic performance enhancement.

[0057] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that: the iron source in step one ① is ferric chloride hexahydrate; the organic bridging ligand in step one ① is 2-aminoterephthalic acid; the organic solvent in step one ① is N,N-dimethylformamide; the mass ratio of the iron source to the organic bridging ligand in step one ① is 1:(0.3~0.5); and the mass ratio of the total mass of the iron source and the organic bridging ligand to the organic solvent in step one ① is 1:(20~100). Everything else is the same as in Specific Implementation Method One.

[0058] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that: the ultrasonic homogenization mentioned in step one ① is specifically performed under the condition of ultrasonic power of 50W~200W for 1min~60min. Everything else is the same as in Specific Implementation Method One or Two.

[0059] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that: in step one, solution A is first heated to 40℃~60℃, then heated to 120℃~160℃ at a rate of 1℃ / min~10℃ / min, and reacted for 12 h~24 h at 120℃~160℃ until the solution turns reddish-brown. After the reaction is complete, the solution is cooled to room temperature to obtain solution B. Everything else is the same as in Specific Implementation Methods One to Three.

[0060] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in the following ways: The centrifugation described in step one (③) is specifically performed at a centrifugation speed of 7000 r / min to 12000 r / min for 3 to 15 minutes; the washing described in step one (③) is performed as follows: first, wash with anhydrous ethanol, then centrifuge at a centrifugation speed of 7000 r / min to 12000 r / min for 3 to 15 minutes, and repeat the washing and centrifugation 2 to 3 times; the drying described in step one (③) is performed at a temperature of 60℃ to 80℃ for 2 to 24 hours. Everything else is the same as in Specific Implementation Methods One to Four.

[0061] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: the Ce source in step two① is cerium acetylacetone; the Mo source in step two① is molybdenum oxyacetylacetone; the mass ratio of MIL-101(Fe) powder to methanol in step two① is 1:(100~500); the mass ratio of Ce source to Mo source in step two① is 1:(0.5~2); and the total mass ratio of Ce source and Mo source in step two① to the mass ratio of MIL-101(Fe) powder is 1:(1~10). Everything else is the same as in Specific Implementation Methods One to Five.

[0062] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that: the polymer coordination shell component in step two ② is a combination of 4,4'-dihydroxydiphenyl sulfone and trichlorophosphine, and the mass ratio of 4,4'-dihydroxydiphenyl sulfone to trichlorophosphine is 1:(0.3~0.6); the mass ratio of the polymer coordination shell to methanol in step two ② is 1:(150~250). Everything else is the same as in Specific Implementation Methods One to Six.

[0063] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that: the mass ratio of solution C to solution D in step two ③ is 1:(1~1.5); the acid-binding agent in step two ③ is triethylamine; and the mass ratio of the total mass of solution C and solution D to the mass of the acid-binding agent in step two ③ is (100~200):1. Everything else is the same as in Specific Implementation Methods One to Seven.

[0064] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One through Eight in the following ways: In Step Two ① and ②, the ultrasonic homogenization is specifically performed under ultrasonic power of 50W~200W for 1min~60min; in Step Two ③, under stirring speed of 200rpm / min~800rpm / min, solution C and solution D are mixed for 1min~60min, then an acid-binding agent is added, and ultrasonic dispersion is performed under ultrasonic power of 50W~200W for 1min~60min; in Step Two ④, the room temperature... The stirring process is specifically carried out at room temperature and a stirring speed of 200 rpm / min to 800 rpm / min for 12 to 24 hours. The centrifugation described in step two (④) is specifically carried out at a centrifugation speed of 7000 rpm / min to 12000 rpm for 3 to 15 minutes. The washing process described in step two (④) involves first washing with ethanol, then centrifuging at a centrifugation speed of 7000 rpm / min to 12000 rpm for 3 to 15 minutes, repeating the washing and centrifugation process 2 to 3 times. Other procedures are the same as in specific embodiments one through eight.

[0065] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One to Nine in that: the drying and grinding described in step three is specifically carried out at a temperature of 40℃~90℃ for 2h~24h, followed by grinding to 80 mesh~200 mesh at room temperature; the reaction in an argon atmosphere at a temperature of 200℃~500℃ described in step three is carried out as follows: under an argon atmosphere, first heat to 40℃~60℃, then increase the temperature to 200℃~500℃ at a heating rate of 3℃ / min~10℃ / min, and react for 2h~4h under an argon atmosphere at a temperature of 200℃~500℃. Everything else is the same as in Specific Implementation Methods One to Nine.

[0066] The beneficial effects of the present invention are verified using the following embodiments:

[0067] Example 1:

[0068] A method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity and sensitized iron death effect, comprising the following steps:

[0069] I. Synthesis of MIL-101(Fe):

[0070] ① Under the condition of ultrasonic power of 100W, the iron source and the organic bridging ligand were added to the organic solvent and ultrasonicated for 5 minutes to obtain mixed solution A;

[0071] The iron source is ferric chloride hexahydrate; the organic bridging ligand is 2-aminoterephthalic acid; and the organic solvent is N,N-dimethylformamide.

[0072] The mass ratio of the iron source to the organic bridging ligand is 1:0.33; the mass ratio of the total mass of the iron source and the organic bridging ligand to the organic solvent is 1:39.38.

[0073] ② First heat the mixed solution A to 40℃, then raise the temperature to 150℃ at a rate of 5℃ / min, and react at 150℃ for 12 hours until the solution turns reddish-brown. After the reaction is completed, cool it to room temperature to obtain solution B.

[0074] ③ Centrifuge solution B, then wash the product, and finally dry it at 70℃ for 3 hours to obtain MIL-101(Fe) powder (abbreviated as MIL).

[0075] The centrifugation specifically refers to centrifugation at a speed of 10000 r / min for 5 min;

[0076] The washing process is carried out in the following steps: first, the product is washed with anhydrous ethanol, and then centrifuged at a speed of 10000 r / min for 10 min. The washing and centrifugation are repeated 3 times.

[0077] II. Synthesis of Ce,Mo@MIL(Fe)@PZS:

[0078] ① Under the condition of ultrasonic power of 100W, MIL-101(Fe) powder was added to methanol and ultrasonicated for 2 minutes. Then Ce source and Mo source were added to obtain solution C.

[0079] The Ce source is cerium acetylacetone; the Mo source is molybdenum oxyacetylacetone; the mass ratio of MIL-101(Fe) powder to methanol is 1:329.58; the mass ratio of Ce source to Mo source is 1:1; the total mass of Ce source and Mo source to the mass ratio of MIL-101(Fe) powder is 1:1.2.

[0080] ② Under the condition of ultrasonic power of 100W, the polymer coordination shell component was added to methanol and ultrasonicated for 2 minutes to obtain solution D;

[0081] The polymer coordination shell component is a combination of 4,4'-dihydroxydiphenyl sulfone and trichlorophosphazene, and the mass ratio of 4,4'-dihydroxydiphenyl sulfone to trichlorophosphazene is 1:0.47; the mass ratio of the polymer coordination shell to methanol is 1:165.83.

[0082] ③ Under the condition of stirring speed of 600 rpm / min, solution C and solution D are mixed for 1 hour, then an acid-binding agent is added, and under the condition of ultrasonic power of 100W, the mixture is ultrasonically dispersed for 2 minutes to obtain solution E;

[0083] The mass ratio of solution C to solution D is 1:1; the acid-binding agent is triethylamine; the total mass ratio of solution C and solution D to the mass ratio of the acid-binding agent is 140:1.

[0084] ④ Under the conditions of room temperature and stirring speed of 600 rpm / min, solution E was stirred for 18 h, and then centrifuged and washed sequentially to obtain Ce,Mo@MIL(Fe)@PZS (abbreviated as CM@MIL@PZS).

[0085] The centrifugation specifically refers to centrifugation at a speed of 10000 r / min for 10 min;

[0086] The washing process involves first washing with ethanol, then centrifuging at 10000 r / min for 10 min, and repeating the washing and centrifugation process three times.

[0087] III. Synthesis of Ce, Mo, Fe MEO-SACs:

[0088] Ce,Mo@MIL(Fe)@PZS was dried for 2 hours at 70°C and then ground to 100 mesh. Ce,Mo@MIL(Fe)@PZS was first heated to 40°C under an argon atmosphere, and then heated to 300°C at a heating rate of 3°C / min. The mixture was then reacted for 3 hours under an argon atmosphere and at 300°C to obtain CMF MESACs.

[0089] Comparative Experiment 1: This comparative experiment differs from Example 1 in that the Ce source in step 2① is cerium chloride; and the Mo source in step 2① is molybdenum chloride. Everything else is the same as in Example 1.

[0090] Comparative Experiment 2: This comparative experiment differs from Example 1 in that the total mass ratio of the Ce source and Mo source mentioned in step 2① to the mass ratio of MIL-101(Fe) powder is 1:0.6. Everything else is the same as in Example 1.

[0091] Comparative Experiment 3: This comparative experiment differs from Example 1 in the following ways: the addition of the Mo source is omitted in step 2①; the mass ratio of the Ce source to the MIL-101(Fe) powder in step 2① is 1:2.4; Ce@MIL@PZS is prepared in step 2④; and CF LESACs are prepared in step 3. Everything else is the same as in Example 1.

[0092] Comparative Experiment 4: This comparative experiment differs from Example 1 in the following ways: the addition of the Ce source is omitted in step 2①; the mass ratio of the Mo source to the MIL-101(Fe) powder in step 2① is 1:2.4; Mo@MIL(Fe)@PZS is prepared in step 2④; and MF LESACs are prepared in step 3. Everything else is the same as in Example 1.

[0093] Figure 1 Figures 1 and 2 show TEM images and elemental scans. a) shows the MIL prepared in step one of Example 1; b) shows the CM@MIL@PZS prepared in step two of Comparative Experiment 2; and c) shows the CM@MIL@PZS prepared in step two of Example 1. The TEM images show the octahedral MIL in a monodisperse state, and a uniform distribution of Fe elemental signals was observed in the EDS spectrum. Figures b and c show that the transmission electron microscope images reveal successful incorporation of heterometals. The core challenge in the successful construction of multi-metal single atoms lies in the differences in reduction potential and diffusion rate between different metal precursors, which easily leads to phase separation or local enrichment. Elemental distribution analysis shows that the incorporation of cerium is significantly more restricted than that of molybdenum. This difference can be attributed to the difference in ionic radius: cerium ions are significantly larger than iron and molybdenum ions, leading to increased lattice distortion during the substitution process. This indicates that the incorporation behavior of heterometals is strictly controlled by ionic radius and lattice matching. In Comparative Experiment 2, the lack of doping amount control led to excessive incorporation of heterometallic salts, manifested as a distinct virus-like surface morphology and aggregation in the TEM image. Example 1, through optimized doping kinetics, obtained a uniformly structured, non-aggregated CM@MIL@PZS precursor (Figure b), laying the structural foundation for subsequent uniform dispersion of single atoms.

[0094] Figure 2Figure 1 shows the TEM image, elemental scan image, HAADF-STEM image, and energy dispersive spectroscopy (EDS) spectrum of the CMF MESACs prepared in step 3 of Example 1. a is the TEM image, b is the elemental scan image, c is the HAADF-STEM image, and d is the EDS spectrum. In step 2 of Example 1, a polyphosphazene (PZS) coating layer was introduced into the outer layer of the precursor. After pyrolysis at 300℃ in step 3, the PZS layer was transformed into a nitrogen-phosphorus crosslinked network, tightly encapsulating the carbon matrix surface. As shown in the figure, the structure of CM@MIL@PZS disintegrates and reassembles during pyrolysis, causing partial corrosion of the originally flat surface and forming a rough surface (Figure a). The elemental distribution map reveals the uniform distribution of Ce, Mo, and Fe atoms in the structure, indicating that heterogeneous atomic sites are precisely embedded in the three-dimensional matrix. AC-STEM characterization of the CMF MESACs shows that the bright spots in the dark field correspond to single-atom structures, proving that the surface contains atomically dispersed metal centers, rather than clusters. EDS analysis (Figure d) simultaneously verifies that these sites contain Ce, Mo, and Fe elements. This demonstrates that PZS-derived anchor points can successfully stabilize heterogeneous metals, overcome the thermodynamic aggregation tendency of multi-metal systems, and achieve single-atom-level dispersion.

[0095] Figure 3 XPS spectra of CMF MESACs prepared in Example 1, CF LESACs prepared in Comparative Experiment 3, and MF LESACs prepared in Comparative Experiment 4 are shown. a is the N 1s spectrum, b is the C 1s spectrum, c is the O 1s spectrum, d is the Fe 2p spectrum, e is the Ce 3d spectrum, and f is the Mo 3d spectrum. The XPS spectra show that the two Fe 2p peaks at 710.9 eV and 724.7 eV correspond to Fe 2p3 / 2 and Fe 2p1 / 2, respectively. Compared to the almost identical binding energies of MF LESACs and CMF MESACs, Mo doping induces an oxidation state shift through Fe~3d / Mo~4d orbital hybridization. The relatively low electronegativity of Ce and Mo promotes electron transfer from the heterometal to Fe, increasing the Fe electron density and reducing the binding energy. The XPS of Ce shows that Ce... 3+ The dominant configuration. Similarly, Mo doping leads to a negative shift in its binding energy. In the 3d spectrum of Mo, the peaks at 232.7 eV and 236.0 eV correspond to Mo 3d5 / 2 and 3d3 / 2, indicating that Mo has multiple valence states. CMF MESACs simultaneously accommodate metal-nitrogen coordination and mixed metal-metal / metal-oxygen configurations. This heterogeneous atomic structure promotes defect formation and lattice distortion, synergistically enhancing the intrinsic charge transport properties of the semiconductor.

[0096] Figure 4The solid-state diffuse reflectance spectra of MIL, CM@MIL@PZS, and CMF MESACs prepared in Example 1 are shown. Diffuse reflectance spectroscopy analysis revealed the semiconductor properties of MIL, CM@MIL@PZS, and CMF MESACs, and the band gap energy was calculated through band structure analysis. As shown in the figure, MIL exhibits a relatively wide band gap (1.67 eV). After doping with a heterometallic material, the band gap of CM@MIL@PZS narrowed slightly to 1.63 eV, indicating modulation of the electronic structure at the interface. The band gap of the pyrolyzed CMF MESAC material decreased significantly (1.09 eV) due to the introduction of abundant defect states and the strong electron-metal-carrier interaction induced by the single-atom metal center, extending the light absorption range into the near-infrared region.

[0097] Figure 5 XRD patterns of MIL, CM@MIL@PZS, and CMF MESACs prepared in Example 1 are shown. The formation process of the crystalline phases in the MIL, CM@MIL@PZS, and CMF MESACs catalysts was investigated using XRD patterns. Significant broad diffraction characteristic peaks were observed in the samples before pyrolysis, located at 20–30° (9.1°, 10.6°, 12.5°, 17.5°, 18.2°, 19.1°, 25.3°, and 27.2°), and these peak shapes highly matched the typical diffraction peaks of MIL-101(Fe). With Ce and Mo doping, CM@MIL@PZS exhibits isomorphic characteristics with MIL-101(Fe), which has a highly crystalline structure. The disappearance of the original peaks after pyrolysis is attributed to the collapse of the MIL structure.

[0098] Figure 6 The Zeta potential diagrams for MIL, CM@MIL@PZS, and CMF MESACs prepared in Example 1 are shown. The synthesis process of the materials was determined through the Zeta potential diagrams. The CM@MIL@PZS catalyst exhibited a stronger positive charge (16.80 mV), while the MIL catalyst showed 13.83 mV, indicating that PZS functionalization was achieved. The positive charge (17.09 mV) of the CMF MESACs is beneficial for biological applications.

[0099] Figure 7 The FT-IR spectra of MIL, CM@MIL@PZS, and CMF MESACs prepared in Example 1 are shown below. The FT-IR spectra of MIL, CM@MIL@PZS, and CMF MESACs can be used to determine the synthesis of the materials and changes in surface groups. After the formation of PZS, the stretching vibration intensity of OH is significantly enhanced by approximately 3400 cm⁻¹. -1 CMF MESACs catalyst at 3460 cm⁻¹ -1 (NH), 2850cm -1(CH) and -COC- (approximately 1000~1300cm) -1 The disappearance of the peak indicates that calcination leads to the loss of surface groups.

[0100] Figure 8 To investigate the ·OH generation of CMF MESACs prepared in Example 1, a is the ESR spectrum of ·OH generation with DMPO as the scavenger, and b is the UV absorption spectrum of ·OH generated by combining TMB as the colorimetric agent with different concentrations of CMF MESACs. DMPO was used as the scavenger in the experiment to identify the type and signal intensity of reactive oxygen species. Figure 8 The results showed that after CMF MESACs+NIR (808nm, 1.0W / cm²), 2 After treatment (1 min), the typical signal intensity ratio of ·OH was 1:2:2:1, which was 1.8 times that of the CMF MESACs group, confirming the catalytic activity. The CMF MESACs were characterized by colorimetric reaction. After catalyzing the generation of ·OH from endogenous H₂O₂, they could oxidize TMB to ox TMB, producing a UV absorption peak at 652 nm. The absorbance at 652 nm increased with increasing CMF MESACs concentration, indicating the continuous accumulation of ·OH. Comparison of catalytic efficiency shows that the CMF MESACs have a strong ·OH generation ability.

[0101] Figure 9 The CMF MESACs prepared in Example 1 1 O2 generation investigation, 'a' represents TEMP as a capture agent. 1 O2 generation ESR spectrum, b is the O2 generation ESR spectrum catalyzed by DPBF as chromogenic agent combined with MESACs of different concentrations of CMF. 1 The UV absorption spectrum after O2; using 1 ESR spectra were collected using the O2 trapping agent TEMP. Figure 9 As shown, a typical 1:1:1 signal appeared after the addition of CMF MESACs catalyst and H2O2. After near-infrared irradiation (808 nm, 1.0 W / cm²), 2 The signal intensity was significantly enhanced (after 1 minute), and was 1.6 times that of the ROS signal intensity of the CMF MESACs group, indicating that the CMF MESACs catalyst has potential application value as a photosensitizer. 1 The generation process of O2 can be characterized by the decrease in fluorescence peak after binding to the DPBF chromogenic probe. The absorbance decreases with increasing CMF MESACs concentration, indicating... 1 The continuous accumulation of O2.

[0102] Figure 10The figure shows the GSH-catalyzed decomposition of CMF MESACs prepared in Example 1; the figure shows the absorbance of DTNB-GSH solutions catalyzed by different concentrations of CMF MESACs; GSH depletion can be determined by reducing the absorbance after the reaction of DTNB and GSH. The change in DTNB absorbance was measured by mixing CMF MESACs / GSH / DTNB. Figure 10 GSH was consumed more quickly with increasing CMF MESACs concentration, indicating that CMF MESACs have a good catalytic effect on GSH depletion.

[0103] Figure 11 The figure shows the O2 release performance of CMF MESACs prepared in Example 1; the figure shows the results of dissolved oxygen determination for 200 mg / mL CMF MESACs and 200 mg / mL CMF MESACs + NIR (808 nm, 1.0 W / cm²). 2 O2 generation capacity at different time points (1 min); Figure 11 The amount of O2 released after the decomposition of hydrogen peroxide was detected by dissolved oxygen concentration. The experimental group with added CMF MESACs showed an increase of 13.98 mg / mL in dissolved oxygen concentration after 30 min, indicating significant O2 generation. With the assistance of laser irradiation, the slope of the curve within 5 min proved that the O2 generation rate was accelerated, and the increase in dissolved oxygen concentration of about 19.97 mg / mL also proved that photothermal drive accelerated catalysis.

[0104] Figure 12 The figure shows the photothermal effect of CMF MESACs prepared in Example 1; the figure shows the temperature change of CMF MESACs at different concentrations with different laser irradiation times; CMF MESACs solutions of different concentrations were irradiated with an 808nm laser and the temperature changes were recorded. Figure 12 The study presented the concentration-dependent temperature rise behavior of different concentrations of CMF MESACs over 10 minutes. As the concentration increased, the temperature showed a stable upward trend; 1 mL of a 500 μg / mL aqueous solution of CMF MESACs achieved a temperature rise of 24.3 °C after 10 minutes of laser irradiation. The results demonstrated that the therapeutic temperature threshold required for mild hyperthermia can be reached at relatively low concentrations, achieving local hyperthermia within 10 minutes. This heating performance meets the clinical requirements for precision tumor treatment while minimizing non-specific thermal damage to healthy tissues.

[0105] Figure 13 To investigate the effects of CM@MIL@PZS and CMF MESACs prepared in Example 1 on the ferroptosis pathway, (1) control group, (2) CM@MIL@PZS + laser irradiation group (808nm, 1.0W / cm) 2(5 min), (3) CMF MESACs + laser irradiation group (808 nm, 1.0 W / cm 2 (5 min), (4) CMF MESACs + laser irradiation (808 nm, 1.0 W / cm 2 The study included two groups: a 5-minute interval between treatments, one with ferroptosis inhibitors and the other with apoptosis inhibitors. The figure shows the effect of Western blotting on the glutathione peroxidase 4 (GPX4) pathway. GPX4 is a core component of the ferroptosis defense system, its function being to reduce toxic lipid peroxides to non-toxic lipid alcohols in reliance on GSH, thereby maintaining cell membrane integrity. GPX4 dysfunction leads to the inability to reduce lipid hydroperoxides, ultimately stimulating lipid peroxidation and achieving ferroptosis sensitization. Figure 13 The results of Western blot analysis of protein expression profiles were presented. In group (3), the significant inhibition of GPX4 confirmed the depletion of glutathione and its subsequent inability to neutralize lipid peroxides, thereby triggering ferroptosis. This effect disappeared in group (4) after the combined use of ferroptosis inhibitors and apoptosis inhibitors, confirming the pathway specificity. This indicates that CMF MESACs, with their narrower band gap and excellent photothermal conversion ability, generated a stronger thermal effect and a large amount of ROS burst and GSH depletion. Since the activity of GPX4 is highly dependent on GSH as a cofactor, the depletion of GSH directly leads to the inactivation of GPX4, which is unable to clear lipid peroxides, thereby disrupting the redox balance of the cell and ultimately achieving highly efficient ferroptosis sensitization.

[0106] Figure 14 To investigate the cellular-level therapeutic performance of CM@MIL@PZS and CMF MESACs prepared in Example 1, (1) control group, (2) laser irradiation treatment (808nm, 1.0W / cm) 2 (3) CM@MIL@PZS treatment, (4) CMF MESACs treatment, (5) CM@MIL@PZS + laser treatment (808nm, 1.0W / cm) 2 (5 min), (6) CMF MESACs + laser treatment (808 nm, 1.0 W / cm 2 (5 min). The cell therapy capability of CT26 cells was evaluated using the thiazolyl bromide blue tetrazolium method. Figure 14 It can be seen that the cell survival rate under near-infrared light stimulation in group (2) is greater than 90%, maintaining a high level and effectively eliminating laser-induced cytotoxicity. At a concentration of 500 μg / mL, the mortality rate of group (3) treated with CM@MIL@PZS was 33.4%, which was lower than that of group (4) treated with CMFMESACs (35.1%). At a concentration of 500 μg / mL, group (6) treated with CMF MESACs + laser (808 nm, 1.0 W / cm 2The combined treatment of CMF MESACs and laser treatment (808nm, 1.0W / cm²) showed a significant inhibitory effect on CT26 cells (61.9%). 2 In the group with 5 min), the increased concentration of the material also led to a gradual decrease in cell survival rate, indicating that the abundance of active sites can generate concentration-dependent reactive oxygen species and regulate the tumor microenvironment.

Claims

1. A method for preparing ternary metal single-atom nanomaterials with highly efficient enzyme activity-sensitized iron death effect, characterized in that... It is done in the following steps: I. Synthesis of MIL-101(Fe): ① Add the iron source and the organic bridging ligand to an organic solvent and sonicate to obtain a mixed solution A; ② Heat mixed solution A to 100℃~200℃ and react until the solution turns reddish-brown. After the reaction is complete, cool it to room temperature to obtain solution B. ③ Centrifuge solution B, then wash and dry the product to obtain MIL-101(Fe) powder; II. Synthesis of Ce,Mo@MIL(Fe)@PZS: ① Add MIL-101(Fe) powder to methanol and sonicate until homogenized. Then add Ce source and Mo source to obtain solution C. ② The polymer coordination shell component was added to methanol and ultrasonically homogenized to obtain solution D; ③ Mix solution C with solution D, then add an acid-binding agent and disperse by ultrasonication to obtain solution E; ④ Stir solution E at room temperature, then centrifuge and wash sequentially to obtain Ce,Mo@MIL(Fe)@PZS; III. Synthesis of Ce, Mo, Fe MEO-SACs: The method for preparing ternary metal single-atom nanomaterials with highly efficient enzyme activity sensitized by iron death effect is completed by drying and grinding Ce,Mo@MIL(Fe)@PZS and reacting it in an argon atmosphere at a temperature of 200℃~500℃.

2. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The iron source mentioned in step 1① is ferric chloride hexahydrate; the organic bridging ligand mentioned in step 1① is 2-aminoterephthalic acid; the organic solvent mentioned in step 1① is N,N-dimethylformamide; the mass ratio of the iron source to the organic bridging ligand mentioned in step 1① is 1:(0.3~0.5); the mass ratio of the total mass of the iron source and the organic bridging ligand to the organic solvent mentioned in step 1① is 1:(20~100).

3. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The ultrasonic homogenization mentioned in step 1① specifically refers to ultrasonication for 1 to 60 minutes under the condition of ultrasonic power of 50W to 200W.

4. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... In step 1②, mixed solution A is first heated to 40℃~60℃, and then heated to 120℃~160℃ at a heating rate of 1℃ / min~10℃ / min. The reaction is carried out at 120℃~160℃ for 12 h~24 h until the solution turns reddish-brown. After the reaction is completed, the temperature is lowered to room temperature to obtain solution B.

5. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The centrifugation described in step 1③ specifically involves centrifuging at a speed of 7000 r / min to 12000 r / min for 3 min to 15 min; the washing described in step 1③ specifically involves the following steps: first washing with anhydrous ethanol, then centrifuging at a speed of 7000 r / min to 12000 r / min for 3 min to 15 min, and repeating the washing and centrifugation 2 to 3 times; the drying described in step 1③ specifically involves drying at a temperature of 60℃ to 80℃ for 2 h to 24 h.

6. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The Ce source mentioned in step 2① is cerium acetylacetone; the Mo source mentioned in step 2① is molybdenum oxyacetylacetone; the mass ratio of MIL-101(Fe) powder to methanol mentioned in step 2① is 1:(100~500); the mass ratio of Ce source to Mo source mentioned in step 2① is 1:(0.5~2); the total mass of Ce source and Mo source mentioned in step 2① to the mass ratio of MIL-101(Fe) powder is 1:(1~10).

7. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The polymer coordination shell component mentioned in step 2② is a combination of 4,4'-dihydroxydiphenyl sulfone and trichlorophosphazene, and the mass ratio of 4,4'-dihydroxydiphenyl sulfone to trichlorophosphazene is 1:(0.3~0.6); the mass ratio of the polymer coordination shell to methanol mentioned in step 2② is 1:(150~250).

8. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The mass ratio of solution C to solution D in step 2③ is 1:(1~1.5); the acid-binding agent in step 2③ is triethylamine; the total mass of solution C and solution D in step 2③ to the mass ratio of the acid-binding agent is (100~200):

1.

9. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized iron death effect according to claim 1, characterized in that... The ultrasonic homogenization described in steps 2① and 2 specifically involves ultrasonication for 1 to 60 minutes at an ultrasonic power of 50W to 200W; in step 2③, solution C and solution D are mixed for 1 to 60 minutes at a stirring speed of 200 rpm / min to 800 rpm / min, then an acid-binding agent is added, and ultrasonic dispersion is performed for 1 to 60 minutes at an ultrasonic power of 50W to 200W; the stirring at room temperature described in step 2④ specifically involves stirring at room temperature and a stirring speed of 200 rpm / min to 800 rpm / min for 12 to 24 hours; the centrifugation described in step 2④ specifically involves centrifugation for 3 to 15 minutes at a centrifugation speed of 7000 rpm / min to 12000 rpm; the washing described in step 2④ specifically involves washing with ethanol first, then centrifuging for 3 to 15 minutes at a centrifugation speed of 7000 rpm / min to 12000 rpm, and repeating the washing and centrifugation 2 to 3 times.

10. The method for preparing a ternary metal single-atom nanomaterial with highly efficient enzyme activity-sensitized ferrode death effect according to claim 1, characterized in that... The drying and grinding described in step three specifically involves drying at a temperature of 40℃~90℃ for 2h~24h, and then grinding to 80 mesh~200 mesh at room temperature. The reaction in an argon atmosphere at a temperature of 200℃~500℃ described in step three specifically involves the following steps: under an argon atmosphere, first heat to 40℃~60℃, then raise the temperature to 200℃~500℃ at a heating rate of 3℃ / min~10℃ / min, and react for 2h~4h under an argon atmosphere at a temperature of 200℃~500℃.

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