Two-dimensional metal-organic nanolayer multi-enzyme mimetic active nanoszymes for inducing ferroptosis and their anticancer applications

Abstract

The application discloses a two-dimensional metal-organic nanolayer multi-enzyme mimetic active nanozyme for inducing ferroptosis and an anticancer application thereof, and relates to the following steps: forming a monolayer metal-organic framework material by coordination of metal ions and organic ligands in a solution through a liquid-phase synthesis method, and then modifying hematin chloride in the framework. The preparation method is simple in process and mild in conditions, and the obtained nanozyme can be used for inducing ferroptosis for antitumor treatment, and has a good application prospect in the field of biological medicine.

Description

Technical Field

[0001] This invention belongs to the field of two-dimensional metal nanomaterial preparation and biomedicine, specifically relating to a method for preparing a two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme for inducing ferroptosis. Background Technology

[0002] Cancer is one of the most serious threats to human health worldwide, with its incidence and mortality rates rising annually. Therefore, cancer treatment research has become a major global issue. Currently, surgery is the primary treatment for cancer, especially suitable for patients with early-stage cancer. While surgery has certain effectiveness, it also has drawbacks. Surgery requires incisions, causing trauma to the patient's body and potentially leading to complications such as bleeding, infection, and tissue damage. Post-operative recovery requires time, which may disrupt the patient's normal life and work. Complications during surgery include anesthesia risks, bleeding, and infection, which can be fatal in severe cases. During surgery, tumor cells may spread to surrounding tissues and distant organs, leading to tumor recurrence or metastasis.

[0003] Ferroptosis is an atypical form of programmed cell death, distinct from traditional cell death mechanisms such as apoptosis, necrosis, and autophagy. It is characterized by its reliance on iron ions and reactive oxygen species (ROS) to induce cell death, particularly closely related to intracellular lipid peroxidation. During ferroptosis, intracellular oxidative stress significantly increases, leading to lipid peroxidation, which in turn damages the cell membrane, ultimately causing cell death. This process involves multi-level regulation, including iron metabolism-related proteins, antioxidant systems, and molecules involved in cellular energy metabolism. Morphological features of ferroptosis include mitochondrial shrinkage, increased membrane density, and a reduction or disappearance of mitochondrial cristae. In scientific research, ferroptosis has wide applications, including cancer research, neurodegenerative disease research, and immunology.

[0004] In view of the shortcomings of tumor treatment technology and inspired by the mechanism of ferroptosis, this invention has successfully developed a two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme for inducing ferroptosis, which significantly enhances the anti-tumor treatment effect and inhibits tumor cell growth, and has significant scientific research and clinical application value. Summary of the Invention

[0005] The purpose of this invention is to provide a two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme for inducing ferroptosis. This nanozyme treats tumors by inducing ferroptosis in tumor cells, thereby avoiding the risk factors associated with surgical treatment. When combined with traditional chemotherapy drugs, radiotherapy, immunotherapy, and other methods, this treatment strategy can exert anti-tumor effects and improve treatment efficacy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The preparation method of a two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme for inducing ferroptosis includes the following steps:

[0008] Step 1: Dissolve cerium ammonium nitrate in pure water and disperse it evenly by ultrasonication to obtain cerium ammonium nitrate solution; dissolve H3BTB in N,N-dimethylformamide and disperse it evenly by ultrasonication to obtain H3BTB solution;

[0009] Step 2: Mix the cerium ammonium nitrate solution from Step 1 with the H3BTB solution and react them. After centrifugation and washing, collect the precipitate to obtain two-dimensional metal-organic nanosheets, denoted as Ce-BTB.

[0010] Step 3: Disperse the two-dimensional metal-organic nanosheets obtained in Step 2 in ethanol, add heme chloride to react, centrifuge and wash the resulting reaction solution, and collect the precipitate to obtain the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme, denoted as He@Ce-BTB.

[0011] Preferably, in step 2, the mass ratio of cerium ammonium nitrate to H3BTB is (5.85–58.5 mg): (7.5–75 mg).

[0012] Preferably, in step 2, the H3BTB solution is slowly added dropwise to the cerium ammonium nitrate solution under ultrasonic conditions, which is conducive to the full reaction between the two.

[0013] Preferably, the reaction conditions in step 2 are as follows: first, ultrasonic heating at 70–90°C for 10–20 min, then a water bath at room temperature for 3–5 min, and finally, ultrasonic heating at 70–90°C for another 10–20 min. The ultrasonic frequency is 40 kHz, and the power is 150 W. The low-temperature water bath not only balances the heat generated during the ultrasonic process but also, to some extent, prevents the oxidation of the two-dimensional metal-organic nanosheets during ultrasonication.

[0014] Preferably, in step 3, the mass ratio of two-dimensional metal-organic nanosheets to heme chloride is (50-500 mg): (32-320 mg).

[0015] Preferably, the reaction conditions in step 3 are as follows: first, magnetic stirring in an oil bath at 50–70°C for 10–14 hours, followed by ultrasonication at 50–70°C for 2–4 hours. The ultrasonic frequency is 40 kHz, and the power is 150 W. During this process, the metal ions on the two-dimensional metal-organic nanosheets undergo coordination exchange with the iron ions in heme chloride. These reaction conditions allow the two-dimensional metal-organic nanosheets to fully react with heme chloride, forming a new metal-organic complex.

[0016] Preferably, the centrifugation speed in both steps 2 and 3 is 9000-9500 rpm, which is beneficial for collecting the precipitate.

[0017] Preferably, in steps 2 and 3, the organic solvent used for washing is N,N-dimethylformamide and dimethyl sulfoxide, which can wash away impurities in the reaction solution.

[0018] The two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme prepared according to the above method has a radial dimension of 190-230 nm and a thickness of 0.7-1.2 nm. It possesses multi-enzyme mimicking activity functions of catalase (CAT), peroxidase (POD), and glutathione peroxidase (GSH-OXD). This function can autocatalytically supply oxygen, generating oxygen to improve the hypoxic environment of tumors. The iron ions in this nanozyme catalyze the generation of hydrogen peroxide, producing lipid peroxides and a series of reactive oxygen species, and catalyze glutathione oxidation. Glutathione depletion leads to a decrease in glutathione peroxidase (GPX-4) activity. Lipid oxides cannot be metabolized by the GPX-4-catalyzed glutathione reductase reaction, which will lead to the peroxidation of phospholipids on the cell membrane, disrupting cell membrane integrity. Mitochondria are the main site of intracellular ROS production. The multi-enzyme mimicking activity function of this nanozyme leads to an imbalance between ROS production and clearance, resulting in impaired mitochondrial function. These effects ultimately promote ferroptosis in tumor cells. This nanozyme has promising applications in the pharmaceutical and biological fields of anti-tumor therapy.

[0019] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0020] 1. This invention successfully synthesizes two-dimensional metal-organic nanolayer multi-enzyme mimicking nanozymes with desired morphology and size using a liquid-phase synthesis method. In this method, metal ions and organic ligands form a single-layer metal-organic framework material through coordination in solution, which has the advantages of simple operation and mild reaction conditions.

[0021] 2. This invention uses a method of modifying heme chloride onto a single-layer metal-organic framework to prepare a two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme. By modifying it onto a single-layer metal-organic framework, the highly ordered and tunable structure of the single-layer metal-organic framework can be utilized to improve the dispersibility and catalytic performance of heme chloride, thus giving the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme highly efficient catalytic performance.

[0022] 3. The present invention uses a method of preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes by modifying heme chloride on a single-layer metal-organic framework. The single-layer metal-organic framework has excellent stability and dispersibility, which can protect heme chloride from the influence of environmental factors and avoid the low solubility aggregation of heme chloride, so that the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes have high stability and dispersibility.

[0023] 4. This invention uses a method of preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes by modifying heme chloride on a single-layer metal-organic framework. The single-layer metal-organic framework has a highly ordered pore structure, which is conducive to the transport of substances and can ensure effective contact between heme chloride and reactants, thereby improving catalytic efficiency. This results in the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme having excellent catalytic performance.

[0024] 5. The preparation method of the present invention is simple, the conditions are mild, the raw materials used are relatively inexpensive and easy to obtain, and the product size is controllable, the reproducibility is high, and the yield is high.

[0025] 6. The two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme prepared by this invention has the function of inducing tumor ferroptosis and anti-tumor function, and has good application prospects in the field of biomedicine. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the synthesis and application of the two-dimensional metal-organic nanolayer multi-enzyme simulated active nanozyme of the present invention.

[0027] Figure 2 Transmission electron microscopy image of the He@Ce-BTB nanozyme prepared in Example 1.

[0028] Figure 3 An atomic force microscopy image of the He@Ce-BTB nanozyme prepared in Example 1, wherein... Figure 3 (a) is the atomic force diagram of He@Ce-BTB nanozyme. Figure 3 (b) shows the thickness of the He@Ce-BTB nanozyme.

[0029] Figure 4 The graph shows the 24-hour stability of the He@Ce-BTB nanozyme prepared in Example 1. Figure 4 (a) is a graph showing the particle size distribution of He@Ce-BTB nanozymes after storage at room temperature under different pH conditions and 10% FBS for 0 h. Figure 4 (b) is a graph showing the particle size distribution of He@Ce-BTB nanozymes after 24 hours of storage at different pH levels and with 10% FBS.

[0030] Figure 5The image shows the CAT activity of the He@Ce-BTB nanozyme prepared in Example 1.

[0031] Figure 6 The image shows the POD activity of the He@Ce-BTB nanozyme prepared in Example 1.

[0032] Figure 7 The image shows the GSH-OXD activity of the He@Ce-BTB nanozyme prepared in Example 1.

[0033] Figure 8 The graph shows the mitochondrial membrane potential changes of the He@Ce-BTB nanozyme prepared in Example 1.

[0034] Figure 9 The image shows a hemolysis experiment of the He@Ce-BTB nanozyme prepared in Example 1.

[0035] Figure 10 The image shows a Western blot of the He@Ce-BTB nanozyme prepared in Example 1. Figure 10 (a) is a band diagram of the target protein GPX-4 and the internal control GADPH. Figure 10 (b) is a quantitative analysis chart of the gray values ​​of GPX-4.

[0036] Figure 11 Cell viability staining of the He@Ce-BTB nanozyme prepared in Example 1.

[0037] Figure 12 The image shows the cytotoxicity experiment of the He@Ce-BTB nanozyme prepared in Example 1. Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0039] Example 1: Preparation of two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis

[0040] In this embodiment, a two-dimensional metal-organic nanolayer multi-enzyme simulated active nanozyme was prepared according to the following method:

[0041] Step 1: Weigh 11.7 mg of cerium ammonium nitrate and dissolve it in 120 μL of pure water. After sonication for 5 seconds, a cerium ammonium nitrate solution of 97.5 mg / mL is obtained. Weigh 15 mg of H3BTB and dissolve it in 360 μL of N,N-dimethylformamide. After sonication for 5 seconds, a 41.7 mg / mL H3BTB solution is obtained.

[0042] Step 2: Slowly add the H3BTB solution from Step 1 to the cerium ammonium nitrate solution under ultrasonic conditions, and then carry out the reaction. The reaction conditions are: first, ultrasonically heat at 80℃ for 15 minutes, then in a water bath at room temperature for 5 minutes, and finally continue to ultrasonically heat at 80℃ for 15 minutes.

[0043] After the reaction was completed, the resulting reaction solution was subjected to alternating centrifugation and washing as follows: the centrifugation speed was 9000 rpm and the duration of each centrifugation was 10 min. The specific method was as follows: centrifugation, washing once with N,N-dimethylformamide, centrifugation, washing once with dimethyl sulfoxide, centrifugation, washing once with dimethyl sulfoxide, centrifugation, and collection of precipitate to obtain two-dimensional metal-organic nanosheets, namely Ce-BTB.

[0044] Step 3: Disperse the two-dimensional metal-organic nanosheets obtained in Step 2 in 20 mL of ethanol to obtain a dispersion with a mass concentration of 20 mg / mL. Add 64 mg of heme chloride to 5 mL of the two-dimensional metal-organic nanosheet dispersion for reaction. The reaction conditions are: first, magnetic stirring in an oil bath at 60 °C for 12 h, and then sonication at 60 °C for 3 h. The sonication frequency is 40 kHz and the power is 150 W.

[0045] After the reaction was complete, the resulting reaction solution was subjected to alternating centrifugation and washing in the same manner as in step 2. The precipitate was collected to obtain the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme, namely He@Ce-BTB.

[0046] Figure 2 The image shows a transmission electron microscope (TEM) image of the He@Ce-BTB nanozyme obtained in this embodiment. The material exhibits a sheet-like structure, and the nanozyme size is approximately 190-230 nm.

[0047] Figure 3 The image shows an atomic force microscope image of the He@Ce-BTB nanozyme obtained in this embodiment. It can be seen that the thickness of the nanozyme is less than 0.7-1.2 nm.

[0048] Figure 4 The 24-hour stability graph of the He@Ce-BTB nanozyme obtained in this embodiment is shown. The characterization method is as follows: 0.1 mL of He@Ce-BTB methanol dispersion (concentration of 5 mg / mL) was taken into a 2 mL EP tube and diluted to 2 mL with PBS at pH 7.4, pH 6.5, and pH 5 and 10% FBS, respectively. The data were then measured with a particle size analyzer and plotted. It can be seen from the graph that the particle size of the He@Ce-BTB nanozyme prepared in this invention did not change much after 24 hours of storage at room temperature under different pH conditions and 10% FBS, indicating that the He@Ce-BTB nanozyme has good environmental stability.

[0049] Figure 5The CAT activity map of the He@Ce-BTB nanozyme prepared in Example 1 was obtained by the following method: 1 mL of 20 mM H2O2 was added to a 24-well plate, and the dissolved oxygen meter probe was placed in the H2O2. After the dissolved oxygen meter data stabilized, the measurement was started. After reading the first data, 25 μL of He@Ce-BTB methanol dispersion (5 mg / mL) was slowly added. After gently mixing with a pipette, the instrument automatically read the data 15 times (the instrument automatically read the data every 5 seconds). The dissolved oxygen data of 25 μL of heme chloride methanol dispersion (5 mg / mL) and 25 μL of Ce-BTB methanol dispersion (5 mg / mL) were measured in the same way. As shown in the figure, the dissolved oxygen concentration of He@Ce-BTB is at its highest overall trend after the addition of H2O2, indicating that the material has CAT activity and can improve the hypoxic environment of tumors. The figure shows that He@Ce-BTB has a good catalytic effect on the decomposition of hydrogen peroxide. Hydrogen peroxide is decomposed into water and oxygen. The oxygen further reacts with other molecules in the cell to produce hydroxyl radicals with high cytotoxicity. These radicals can damage the cell membrane, leading to an imbalance in the intracellular environment and ultimately causing ferroptosis in tumor cells.

[0050] Figure 6 The image shows the POD activity of the He@Ce-BTB nanozyme prepared in Example 1. The characterization method was as follows: 1 mL of PBS solution at different pH values ​​was taken, and 50 μL of He@Ce-BTB methanol dispersion (0.6 mg / mL) and 51 μL of a dimethyl sulfoxide solution (0.6 mg / mL) of 3,3',5,5'-tetramethylbenzidine (TMB) were added. The UV absorbance was then measured. The TMB color reaction was used to detect the generation of hydroxyl radicals. The principle is that hydroxyl radicals can cause a color reaction in TMB (changing from colorless to blue or yellow). The image shows that He@Ce-BTB has POD activity and can catalyze the decomposition of hydrogen peroxide to generate hydroxyl radicals. The generation of hydroxyl radicals by He@Ce-BTB was most effective at pH 5.0. These radicals can attack cancer cell membranes, leading to lipid peroxidation and ferrodeogenesis. At pH 7.4, the generation of hydroxyl radicals was very weak, and He@Ce-BTB did not produce significant toxicity to normal cells, thus achieving a specific killing effect on cancer cells.

[0051] Figure 7The GSH-OXD activity diagram of the He@Ce-BTB nanozyme prepared in Example 1 is shown. The characterization method is as follows: 4 mM glutathione (GSH) was added to different concentrations of He@Ce-BTB methanol dispersions (0 mg / mL, 0.3 mg / mL, 0.5 mg / mL), and the mixture was separated using a 0.22 μm filter. 0.2 mM of probe 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to the separated solution, and the UV absorbance of the final solution was measured (1 min, 3 min, 5 min, 8 min, 10 min). The experiment needs to be carried out in the dark because glutathione is easily destroyed and oxidized under light. The figure shows that glutathione gradually decreases over time, indicating that He@Ce-BTB has GSH-OXD activity and catalyzes glutathione oxidation. As glutathione is depleted, the activity of glutathione peroxidase (GPX-4) decreases. Lipid oxides cannot be metabolized by glutathione reductase catalyzed by GPX-4, which will lead to the peroxidation of phospholipids on the cell membrane, destroy the cell membrane integrity, and promote ferroptosis in tumor cells.

[0052] Figure 8 The image shows the mitochondrial membrane potential changes of the He@Ce-BTB nanozyme obtained in this embodiment. The characterization method is as follows: Take an appropriate amount of TMRE (1000X) and dilute TMRE at a ratio of 1 mL of detection buffer per 1 μL of TMRE (1000X) to obtain TMRE working solution. After incubating tumor cells in a 6-well plate for 24 h, aspirate the supernatant, wash once with PBS, add fresh cell culture medium, and dilute the methanol dispersion of He@CeBTB to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. Incubate for 24 h, add 1 mL of TMRE staining working solution to each well, and incubate at 37°C for 20 min in a cell culture incubator. After incubation at 37°C, aspirate the supernatant, wash twice with preheated cell culture medium, and then observe and photograph under a fluorescence microscope. As shown in the figure, the mitochondrial membrane potential of cells incubated with He@Ce-BTB nanozymes changed significantly. The decrease in mitochondrial membrane potential promotes the release of iron ions from the mitochondria into the cytoplasm, thereby activating the ferroptosis pathway. Furthermore, changes in mitochondrial membrane potential also affect the redox balance within the mitochondria, leading to increased ROS production and further promoting ferroptosis, playing a crucial role in anti-tumor therapy.

[0053] Figure 9The image shows the hemolysis experiment of the He@Ce-BTB nanozyme obtained in this embodiment. The characterization method was as follows: The methanol dispersion of He@CeBTB was diluted to 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. 0.2 mL of each of these solutions was mixed with 0.2 mL of treated blood (500 μL of fresh blood was added to 4.5 mL of physiological saline and centrifuged 5-8 times at 3000 rpm for 10 min until the blood supernatant was clear and transparent; the supernatant was then discarded, and the volume was adjusted to 5 mL with physiological saline) and 0.6 mL of physiological saline, respectively. The mixture was incubated at 37°C for 4 h, followed by centrifugation at 3000 rpm for 10 min. The absorbance at OD541 nm was measured, and the hemolysis rate was calculated. In the figure, RBCs+ represents the blood group, and RBCs- represents the control group without blood (using the same concentration of material and physiological saline as the blood group). As can be seen from the figure, the hemolysis rate of He@CeBTB is less than 10%, indicating that the material has good biocompatibility.

[0054] Figure 10The image shows a Western blotting of the He@CeBTB nanozyme obtained in this embodiment. The characterization method was as follows: After the 4T1 tumor cells in the 6-well plate reached confluence, He@CeBTB (solvent dimethyl sulfoxide) and 1 mM H2O2 were added, and the plate was irradiated under a UV lamp for 30 min. The culture medium was aspirated, and 2 mL of fresh culture medium was added to each well. Three wells were grouped together, and each well in each group was incubated with He@CeBTB at equivalent concentrations of 0 μg / mL, 100 μg / mL, and 200 μg / mL. One group was further incubated with 125 μL of H2O2 in each well for 24 h. (1) Protein extraction and sample preparation, and determination of BCA protein concentration: Gently aspirate the culture medium from the 6-well culture plate; add 100 μL of lysis buffer (1:50, 4 mL cell lysis buffer + 80 μL proteinase inhibitor PMSF) to each well, prepare fresh before use, and lyse at 4℃ for 30 min; transfer the lysed solution to 6 1.5 mL EP tubes, centrifuge at 12000 rpm for 10 min at 4℃, discard the precipitate, and collect the supernatant; take 5 μL of supernatant from each of the 6 tubes and add 95 μL of lysis buffer (diluted 20 times), and determine the concentration of BCA protein in the 6 tubes according to the BCA protein concentration determination method; add 5x loading buffer (volume ratio of supernatant: 5x loading buffer = 4:1) to the remaining supernatant in the 6 tubes to ensure consistent protein concentration in each group of samples, seal with sealing film, boil for 10 min to denature the protein, cool to room temperature, and store at -20℃. The calculated loading amount is 20 μg, and 20 μL is taken. (2) Electrophoresis steps. ① Gel preparation: Mount the glass plates on the gel preparation rack. Prepare a separating gel of appropriate concentration according to the molecular size of the target protein. Slowly pour the separating gel along the gap between the two glass plates, taking care to avoid air bubbles. Stop when the gel is 2-3 cm from the top. Add a certain amount of isopropanol to the top of the separating gel for sealing. Place it on a horizontal platform for 30 minutes to allow it to solidify. Pour off the top layer of isopropanol and blot dry with filter paper. Prepare the stacking gel. Slowly add the stacking gel to the top of the glass plate and immediately insert the comb, taking care to avoid air bubbles. Let it stand on a horizontal platform at room temperature for 30 minutes. ② Electrophoresis: After the stacking gel has solidified, remove the glass plates and place them in the electrophoresis tank. Carefully pull out the comb parallel to the gel wells. Pour fresh electrophoresis buffer into the inner tank. After confirming there are no leaks, add a certain amount of electrophoresis buffer to the outer tank and remove any air bubbles from the bottom of the tank. Add 5 μL of protein marker to each well at both ends of the gel, and add the same volume of protein sample (20 μg) to each of the remaining wells. Connect the electrophoresis tank to the electrophoresis apparatus and maintain a constant voltage of 80V for about 30 minutes. When the loading buffer shows that the protein has reached the boundary between the separating gel and the stacking gel, increase the voltage to 120V and continue electrophoresis for 60 minutes. Stop electrophoresis when the target protein has reached about 1 cm from the bottom of the glass plate.③ Transfer: Pour the membrane into the transfer pan. Soak the required filter paper, sponge, and PVDF membrane in the transfer solution. The PVDF membrane needs to be activated in methanol for 10 seconds before being soaked in the transfer solution. Remove the glass plate that has already undergone electrophoresis and cut off the top of the stacking gel. When transferring the membrane, place the black plate down and add the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in sequence. Remove air bubbles after each layer. After securing the membrane, place it in the transfer tank. Place an ice box in the transfer tank and pour in the transfer solution. Place the transfer tank in a basin of ice water mixture and start the transfer. The transfer conditions are: constant current: 250mA, 120 minutes. The voltage during the transfer should not be less than 90V, otherwise the transfer effect will be affected. ④ Blocking: Prepare a skim milk blocking buffer (2.25g skim milk powder, 40mL 1xTBST solution) using 1xTBST solution. Shake on a shaker until fully dissolved. Remove the transferred PVDF membrane, mark the front and back sides, and place it in the milk blocking buffer. Then, place it on a horizontal shaker and shake slowly at room temperature for 1 hour. Wash 6 times with 1xTBST solution, 5 minutes each time, and cut the bands. ⑤ Primary antibody incubation: a. After blocking, compare the size of the protein marked on the PVDF membrane with that on the marker band to locate the region containing the target protein, and cut the membrane. b. Dilute the primary antibody according to the specified concentrations: GADPDH (mouse antibody) at 1:20000, take 0.25μL of GADPH and add 5mL of primary antibody diluent; GPX-4 (rabbit antibody) at 1:1000, take 5μL of GPX-4 and add 5mL of primary antibody diluent. Add the diluted primary antibody to the corresponding PVDF membrane surface and incubate overnight at 4°C. ⑥ Secondary antibody incubation: Dilute the secondary antibody with 1xTBST at the specified concentration. After incubating with the primary antibody overnight, recover the primary antibody, remove the band from the refrigerator, place it in a washing box containing 1xTBST, and gently wash it 6 times on a horizontal shaker for 5 minutes each time. Place the thoroughly washed band into a box containing the secondary antibody and incubate the box on a horizontal shaker at room temperature for 1 hour. After the secondary antibody incubation is complete, recover the secondary antibody and wash the membrane 6 times on a horizontal shaker for 5 minutes each time with 1xTBST. (Secondary antibody preparation concentration: 2 μL of secondary antibody (mouse antibody), 5 mL of 1xTBST; 2 μL of secondary antibody (rabbit antibody), 5 mL of 1xTBST). Finally, observe and photograph the band under a developing apparatus, and repeat the above experiment twice. As can be seen from the figure, as the concentration of the two-dimensional metal-organic nanolayer multi-enzyme mimicking nanozyme increases, the expression level of GPX-4 in cells decreases. The activity of GPX-4 is negatively correlated with the occurrence of ferroptosis, and ferroptosis is more pronounced in cells with lower GPX-4 activity.

[0055] Figure 11The image shows the cell viability staining of He@CeBTB obtained in this embodiment. The characterization method was as follows: To demonstrate that the He@CeBTB nanozyme can inhibit tumor cell survival by generating ROS, after incubating tumor cells for 24 hours, the supernatant was aspirated, and the cells were washed once with PBS. H2O2, He@CeBTB, and He@CeBTB+H2O2 were added sequentially to each well, followed by cell culture medium. The cells were incubated for 24 hours, the culture medium was removed, and the cells were washed once with PBS. 12 μL of Calcein AM (calcein acetoxymethyl ester) and 4 μL of PI (propidium iodide) were mixed together and added to 4 mL of PBS. The culture medium was removed, and the cells were washed once with 500 μL of PBS. 300 μL of dye was added, and the cells were incubated at 37°C for 20 minutes in the dark. The images were then taken using a fluorescence microscope. The images show that the red fluorescence of He@CeBTB and He@CeBTB treated with hydrogen peroxide increased, indicating that the He@CeBTB nanozyme has a tumor cell killing effect.

[0056] Figure 12 The image shows the cytotoxicity experiment of the He@CeBTB nanozyme obtained in this embodiment. The characterization method was as follows: 100 μL (1 × 10⁻⁶) was seeded into each well of two plates. 5 NCM460 cells and 100 μL (1×10⁻⁶) 5 4T1 cells were incubated for 24 hours. The supernatant was aspirated, and each well was processed according to... Figure 12 Different concentrations of He@CeBTB were added and incubated for 24 h. After incubation, 20 μL MTT (5 mg / mL) was added to each well, and the culture was incubated for 4 h. Finally, the supernatant was aspirated, and 150 μL of dimethyl sulfoxide was added to each well to terminate the culture. The cells were incubated at 37℃ in the dark for 10 min, and the absorbance was measured at 490 nm. The figures show that NCM460 cells had a high survival rate, while 4T1 cells had a lower survival rate. The survival rate of 4T1 cells with added H2O2 was lower than both of the above. Furthermore, the higher the concentration of He@CeBTB, the more cells died. These phenomena indicate that the material has good biocompatibility. The low survival rate of tumor cells suggests that the material has a good anti-tumor effect on tumor cells. By comparing the cytotoxicity of NCM460 cells and tumor cells, the good biocompatibility and bioefficacy of the two-dimensional metal-organic nanolayer multi-enzyme mimicking nanozyme were confirmed.

[0057] The above description is merely an exemplary embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis, characterized in that, Includes the following steps: Step 1: Dissolve cerium ammonium nitrate in pure water and disperse it evenly by ultrasonication to obtain cerium ammonium nitrate solution; dissolve H3BTB in N,N-dimethylformamide and disperse it evenly by ultrasonication to obtain H3BTB solution; Step 2: Mix the cerium ammonium nitrate solution from Step 1 with the H3BTB solution and react them. After centrifugation and washing, collect the precipitate to obtain two-dimensional metal-organic nanosheets, denoted as Ce-BTB. The mass ratio of cerium ammonium nitrate to H3BTB is 5.85~58.5 mg: 7.5~75 mg. Step 3: Disperse the two-dimensional metal-organic nanosheets obtained in step 2 in ethanol, add heme chloride to react, centrifuge and wash the resulting reaction solution, and collect the precipitate to obtain the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme, denoted as He@Ce-BTB; wherein, the mass ratio of two-dimensional metal-organic nanosheets to heme chloride is 50~500 mg: 32~320 mg.

2. The method for preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis according to claim 1, characterized in that: In step 2, the H3BTB solution is added dropwise to the cerium ammonium nitrate solution under ultrasonic conditions.

3. The method for preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis according to claim 1, characterized in that, The reaction conditions in step 2 are as follows: first, ultrasonically heat at 70~90℃ for 10~20 min, then bathe in a water bath at room temperature for 3~5 min, and finally continue ultrasonically heating at 70~90℃ for 10~20 min.

4. The method for preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis according to claim 1, characterized in that, The reaction conditions in step 3 are: first, magnetic stirring in an oil bath at 50~70℃ for 10-14 h, and then sonication at 50~70℃ for 2~4 h.

5. The method for preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis according to claim 1, characterized in that: In both steps 2 and 3, the centrifugation speed is 9000~9500 rpm.

6. The method for preparing two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozymes for inducing ferroptosis according to claim 1, characterized in that: In steps 2 and 3, the organic solvents used for washing are N,N-dimethylformamide and dimethyl sulfoxide.

7. A two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the two-dimensional metal-organic nanolayer multi-enzyme mimicking active nanozyme according to claim 7, characterized in that: Used to prepare antitumor nanozymes that induce ferroptosis.

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