A nanomaterial for inducing ferroptosis of tumor cells and a preparation method and application thereof

Abstract

The application belongs to the technical field of nanomedicine, and particularly relates to a kind of nanometer material for inducing tumor cell ferroptosis and its preparation method and application.The nanometer material is GZHMU-1 and GZHMU-1@Mo, contains mixed valence copper ions, contains monovalent copper and divalent copper, has a similar Fenton effect, is more sensitive to GSH, the effect is more obvious under the condition of containing hydrogen peroxide, and the latter also contains guest molecule MoO4 2‑ , increases the effect of generating hydroxyl radicals, and if hydrogen peroxide exists, the effect of generating ROS is more obvious, and triaminoguanidine hydrochloride is used as a frame connector, which has good biological safety;The nanometer material of the application can be oxidized and reduced in response to high concentration of GSH and hydrogen peroxide in tumor microenvironment to induce tumor cell ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicines, and particularly relates to a nanomaterial for inducing ferroptosis of tumor cells, a preparation method thereof, and an application thereof. Background Art

[0002] Ferroptosis, a non-apoptotic cell death method that is highly dependent on ferroptosis, was proposed by Dixon et al. in 2012. This cell death method plays a key role in cancer treatment. Studies have shown that it can inhibit tumor cell growth and induce tumor cell death in numerous cancers. Furthermore, lipid peroxidation is a key indicator of ferroptosis, a process that occurs largely due to the depletion of the reducing substance GSH within tumor cells. Therefore, disrupting the redox balance within tumors and inducing ferroptosis is a promising approach for cancer treatment.

[0003] Ferroptosis can be induced through two pathways: exogenous and endogenous. Many researchers have studied nanomedicines that induce ferroptosis primarily through the following mechanisms: First, inhibition of the transport function of xCT (SLC7A11) impairs GSH synthesis and induces ferroptosis in tumor cells by increasing the production of ROS. Second, targeting intracellular iron ions, the concentration of intracellular iron ions is affected by transferrin, inducing ferroptosis from an exogenous pathway. Third, the inhibitor RSL3 is introduced against the downstream substance GPX4 of GSH, and iron ions that produce the Fenton effect are introduced simultaneously to promote ferroptosis in tumor cells. Much research is focused on how to reduce GSH in tumor cells, thereby affecting the activity of glutathione peroxidase 4 in ferroptosis, leading to a weakening of the antioxidant capacity of tumor cells. Simply introducing ions that produce the Fenton effect can cause an increase in ROS, thereby causing lipid peroxidation in tumor cells and triggering ferroptosis.

[0004] At present, the above-mentioned treatment methods are relatively simple, and the development of a new model of nanomedicine that induces ferroptosis is more important for the treatment of tumors. Summary of the Invention

[0005] In view of the above problems, the present invention aims to provide a nanomaterial that induces ferroptosis of tumor cells, and a preparation method and application thereof.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides a nanomaterial for inducing ferroptosis of tumor cells. The nanomaterial is named GZHMU-1 and GZHMU-1@Mo. The nanomaterial contains copper ions in mixed valence states.

[0008] The structure of the nanomaterial is to use bio-friendly guanidine as a linker and Cu3L three-cluster copper as a framework node;

[0009] The chemical structure of GZHMU-1 is as follows:

[0010]

[0011] Furthermore, the guest molecule MoO4 2- , whose chemical structure is shown below:

[0012]

[0013] The GZHMU-1@Mo can induce ferroptosis by consuming GSH, catalyzing the production of NADH and ROS, and simultaneously causing redox responses to the three active substances, thereby avoiding the action mode of single induction of ferroptosis leading to poor therapeutic effect. Among them, the consumption mode of GSH is mainly: the divalent copper in the mixed valence state in the nanomaterial GZHMU-1@Mo can undergo redox reaction with the reducing substance GSH, oxidizing GSH to GSSG, and the content of GSH decreases, thereby leading to GPX4 protein expression. Secondly, NADH, another bioactive substance in tumor cells, is also an antioxidant substance. GZHMU-1@Mo can catalyze NADH to NAD+, thereby affecting the content of the antioxidant substance NADH; in addition, hydrogen peroxide in tumor cells participates in the occurrence of the above-mentioned total process, and the monovalent copper in the mixed valence state can produce hydroxyl radicals by Fenton effect with hydrogen peroxide, while the guest molecule MoO4 2 The introduction of hydroxyl radicals promotes the production of hydroxyl radicals during this process. The simultaneous consumption of the three bioactive substances inhibits GPX4 expression, reduces the antioxidant capacity of cells, and accumulates ROS, causing widespread lipid peroxidation and triggering ferroptosis in tumor cells.

[0014] The present invention also provides a method for preparing a nanomaterial that induces ferroptosis of tumor cells, comprising the following steps:

[0015] Using guanidine and copper tricrystal as raw materials, under the action of benzenesulfonate ion compound and PEG material, the nanomaterial GZHMU-1 is generated through mixed reaction;

[0016] The benzenesulfonate ion compound includes one or more of p-toluenesulfonic acid, benzenesulfonic acid, p-hydroxybenzenesulfonic acid, and nitrobenzenesulfonic acid;

[0017] The guanidine includes one or more of triaminoguanidine, metformin and aminoguanidine;

[0018] The PEG material includes one or more of PEG (200), PEG (400), PEG (800), PEG (1000) and PEG (2000);

[0019] The molar ratio of the guanidine and the copper tricluster is (1-2): (1-2);

[0020] The amount of the benzenesulfonate ion compound used is 8 to 12 equivalents of guanidine;

[0021] The amount of the PEG material used is 10 to 30 times the sum of the mass of guanidine and the copper tricluster;

[0022] The temperature of the mixing reaction is 160-180°C.

[0023] The present invention also provides a method for preparing a nanomaterial that induces ferroptosis of tumor cells, comprising the following steps:

[0024] Guanidine, molybdate ion compound sodium molybdate dihydrate and copper tricrystal are used as raw materials. Under the action of benzenesulfonate ion compound and PEG material, the mixture undergoes Schiff reaction between aldehyde and amine to produce nanomaterial GZHMU-1@Mo.

[0025] The guanidine includes one or more of triaminoguanidine, metformin and aminoguanidine;

[0026] The molybdate ion compound includes one or more of sodium molybdate dihydrate and ammonium molybdate;

[0027] The benzenesulfonate ion compound includes one or more of p-toluenesulfonic acid, benzenesulfonic acid, p-hydroxybenzenesulfonic acid, and nitrobenzenesulfonic acid;

[0028] The PEG material includes one or more of PEG (200), PEG (400), PEG (800), PEG (1000) and PEG (2000);

[0029] The molar ratio of the molybdate ion compound to guanidine and copper tricluster is (1-2):1:1, preferably 1.5:1:1;

[0030] The amount of p-toluenesulfonic acid used is 8 to 12 equivalents of guanidine;

[0031] The amount of the PEG material is 10 to 30 times the sum of the mass of guanidine and the copper tricluster;

[0032] The temperature of the mixed reaction is 160-180°C;

[0033] Furthermore, in order to obtain higher active sites for GZHMU-1@Mo, exchange was performed in an organic solvent after the synthesis (24 h or more);

[0034] The present invention also provides an application of a nanomaterial that induces ferroptosis in tumor cells, wherein the nanomaterial is used to prepare anti-tumor nanomedicines;

[0035] The tumor includes lung cancer, breast cancer, pancreatic cancer or prostate cancer;

[0036] The lung cancer includes targeting A549 cells;

[0037] The application of the nanomaterial is that the nanomaterial can be used in anti-tumor drugs to induce ferroptosis by catalyzing three bioactive substances in tumors, thereby improving the therapeutic effect on tumors.

[0038] The beneficial effects of the present invention are as follows:

[0039] The nanomaterials of the present invention are GZHMU-1 and GZHMU-1@Mo, which contain mixed-valence copper ions, including monovalent copper and divalent copper, and have a similar Fenton effect. They are relatively sensitive to GSH and the effect is more obvious under conditions containing hydrogen peroxide. The latter also contains the guest molecule MoO4 2- , which increases the effect of generating hydroxyl radicals. If hydrogen peroxide is present, the effect of generating ROS is more obvious. Using triaminoguanidine hydrochloride as a framework connector has good biosafety. The nanomaterial of the present invention can induce ferroptosis of tumor cells by redox response to high concentrations of GSH and hydrogen peroxide in the tumor microenvironment, and this process can continuously induce the mutual conversion of copper mainly through mixed valence states.

[0040] The preparation method of the nanomaterial of the present invention uses guanidine as a linker, which has good biosafety and is easily metabolized by the body and excreted from the body in the form of urine. In addition, the unique amino group in its structure can react with the aldehyde group in the copper tricrystal to form a Schiff base reaction. The reaction type is relatively simple, so that the prepared nanomaterial has good biocompatibility, dispersibility, and redox responsiveness. The preparation method is simple, low-cost, and bio-environmentally friendly, making it suitable for large-scale promotion.

[0041] The nanomaterial of the present invention is used in the preparation of anti-tumor nanomedicines. The GZHMU-1@Mo of the present invention can utilize its own redox properties to catalyze the entry of biologically active substances into tumor cells, thereby causing lysosomal dysfunction, thereby causing "lysosomal escape". It also produces a redox response to the high concentrations of glutathione, hydrogen peroxide, and the reducing substance NADH in tumor cells, inducing ferroptosis. The simultaneous oxidation and reduction further enhances the therapeutic efficacy of the nanomedicine, providing a new option for nanomedicines that induce ferroptosis in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 GZHMU-1 and GZHMU-1@Mo were characterized by XRD and SEM;

[0043] Figure 2 EDS-mapping of GZHMU-1@Mo;

[0044] Figure 3 GZHMU-1@Mo Fenton effect evaluation results chart;

[0045] Figure 4 GZHMU-1@Mo GSH consumption and NADH catalysis evaluation results chart;

[0046] Figure 5 GZHMU-1 and GZHMU-1@Mo in vitro anti-tumor activity evaluation results chart;

[0047] Figure 6 GZHMU-1 and GZHMU-1@Mo anti-tumor mechanism research results chart;

[0048] Figure 7 GZHMU-1 and GZHMU-1@Mo in vivo anti-tumor activity evaluation results chart;

[0049] Figure 8 GZHMU-1 and GZHMU-1@Mo in vivo anti-tumor safety evaluation results chart. DETAILED DESCRIPTION

[0050] The application will be described in further detail below with specific examples and accompanying drawings. It should be understood that these examples are only used to illustrate the application and are not intended to limit the scope of protection of the application. After reading the application, those skilled in the art can make various equivalent modifications to the application, which fall within the scope of the appended claims.

[0051] Unless otherwise specified, all raw materials and reagents of the application are conventional market raw materials and reagents.

[0052] Example 1

[0053] Preparation of a nanomaterial GZHMU-1

[0054] 1) Cu3L synthesis: weigh copper nitrate (1 mmoL, 0.185 g), 1H-pyrazole-4-carboxaldehyde (1 mmoL, 0.096 g) into a 5 mL pressure tube, add 6.7 mL of DMF and anhydrous ethanol, and 5 mL of deionized water, mix uniformly, then heat at 100°C for 12 h, after the reaction is completed, filter, get light yellow crystals, then exchange with deionized water for 3 days, finally, wash the crystals with acetone three times, vacuum dry, and grind for use;

[0055] 2) Synthesis of GZHMU-1: Triaminoguanidine hydrochloride (0.16 mmol, 0.023 g), Cu3L (0.16 mmol, 0.076 g), and p-toluenesulfonic acid (1.6 mmol, 0.275 g) were weighed separately and 10 mL of a 1:1 volume ratio of PEG-400 and PEG-1000 was added. The mixture was then blended in a crusher until complete, and air bubbles were removed by ultrasonication. The mixture was then placed in an oven and heated at 170°C for 30 minutes. After the reaction, the mixture was washed twice with THF, water, DMF, and ethanol, respectively, and dried to obtain GZHMU-1. The reaction process is shown below:

[0056]

[0057] Example 2

[0058] Preparation of a Nanomaterial GZHMU-1@Mo

[0059] Weigh triaminoguanidine hydrochloride (0.16 mmol, 0.0226 g), sodium molybdate dihydrate (0.24 mmol, 0.058 g), Cu3L (0.16 mmol, 0.0763 g), and p-toluenesulfonic acid (1.6 mmol, 0.275 g). Add 10 mL of a 1:1 volume ratio of PEG-400 and PEG-1000 to the mixture. Mix the mixture in a crusher until uniform. Remove bubbles by ultrasonication and heat in an oven at 170°C for 30 minutes. Wash the mixture twice with THF, water, DMF, and ethanol, respectively, and dry to obtain GZHMU-1@Mo. The reaction process is as follows:

[0060]

[0061] The nanomaterials obtained in Example 1 and Example 2 were scanned by electron microscope. Figure 1 It can be seen that these are the SEM and XRD patterns of the two nanomaterials. From the SEM patterns, it can be seen that both (a) and (b) represent GZHMU-1 and GZHMU-1@Mo. The nanomaterials have good dispersibility, uniform morphology, and a particle size of about 200 nm. The reason why the particle size of the GZHMU-1@Mo nanomaterial has not increased may be because the molybdate is obtained by in situ synthesis. In addition, the molybdate may exist in the pores of the entire material; Figure (c) also further shows that the GZHMU-1@Mo nanomaterial synthesized in situ still maintains the GZHMU-1 framework structure.

[0062] Figure 2The EDS-mapping diagram of the nanomaterial GZHMU-1@Mo shows the presence of Mo elements, indicating that the nanomaterial is successfully synthesized, and the in-situ synthesis method of the nanomaterial is feasible.

[0063] Example 3

[0064] Preparation of a nanomaterial GZHMU-1

[0065] 1) Cu3L synthesis: weigh copper nitrate (1.2 mmoL, 0.222 g), 1H-pyrazole-4-carboxaldehyde (1 mmoL, 0.096 g) into a 5 mL pressure tube, add 7.2 mL of DMF and anhydrous ethanol, and 5 mL of deionized water, mix uniformly, then heat at 100°C for 12 h, after the reaction is completed, filter, get light yellow crystals, then exchange with deionized water for 3 days, finally, wash the crystals with acetone three times, and dry in vacuum, and grind for use;

[0066] 2) GZHMU-1 synthesis: weigh metformin hydrochloride (0.2 mmol, 0.029 g), Cu3L (0.15 mmol, 0.0713 g) and nitrobenzenesulfonic acid (2 mmol, 0.344 g) respectively, and add a mixed reaction solvent of PEG-800 and PEG-2000 15 mL, the volume ratio is 1:1, then uniformly crush the above-mentioned by a crusher, remove the bubbles by ultrasonic, and place in an oven for heating, react at 160°C for 40 minutes. After the reaction is completed, wash with THF, water, DMF and ethanol in turn, and dry to obtain GZHMU-1.

[0067] Example 4

[0068] Preparation of a nanomaterial GZHMU-1@Mo

[0069] Weigh metformin hydrochloride (0.2 mmol, 0.0283 g), sodium molybdate dihydrate (0.4 mmol, 0.097 g), Cu3L (0.2 mmol, 0.0954 g) and nitrobenzenesulfonic acid (2 mmol, 0.344 g), and add a mixed reaction solvent of PEG-800 and PEG-2000 16 mL, the volume ratio is 1:1, then uniformly crush the above-mentioned by a crusher, remove the bubbles by ultrasonic, and place in an oven for heating, react at 160°C for 40 minutes. Wash with THF, water, DMF and ethanol in turn, and dry to obtain GZHMU-1@Mo.

[0070] Example 5

[0071] Preparation of a nanomaterial GZHMU-1

[0072] 1) Synthesis of Cu3L: Copper nitrate (1.5 mmol / L, 0.278 g) and 1H-pyrazole-4-carboxaldehyde (1.3 mmol / L, 0.125 g) were weighed into a 5 mL pressure tube. 8.0 mL of DMF and anhydrous ethanol were added, along with 5 mL of deionized water. The mixture was mixed thoroughly, and the mixture was heated at 100°C for 12 h. After the reaction, the mixture was filtered to obtain pale yellow crystals. The crystals were then exchanged with deionized water for 3 days. Finally, the crystals were washed three times with acetone, dried under vacuum, and ground for later use.

[0073] 2) Synthesis of GZHMU-1: Aminoguanidine hydrochloride (0.18 mmol, 0.026 g), Cu3L (0.18 mmol, 0.086 g), and p-hydroxybenzenesulfonic acid (2.16 mmol, 0.371 g) were weighed separately and 15 mL of a 1:1 volume ratio of PEG-200 and PEG-400 was added. The mixture was then blended using a crusher. After completion, air bubbles were removed by ultrasonication and the mixture was placed in an oven at 180°C for 30 minutes. After completion of the reaction, the mixture was washed twice with THF, water, DMF, and ethanol, respectively, and dried to obtain GZHMU-1.

[0074] Example 6

[0075] Preparation of a Nanomaterial GZHMU-1@Mo

[0076] Aminoguanidine hydrochloride (0.18 mmol, 0.025 g), ammonium molybdate (0.3 mmol, 0.073 g), Cu3L (0.18 mmol, 0.0858 g), and p-hydroxybenzenesulfonic acid (2.2 mmol, 0.378 g) were weighed and 17 mL of a 1:1 volume ratio of PEG-200 and PEG-400 was added. The mixture was then blended in a crusher. After completion, air bubbles were removed by ultrasonication and the mixture was heated in an oven at 180°C for 30 minutes. The mixture was then washed twice with THF, water, DMF, and ethanol, respectively, and dried to obtain GZHMU-1@Mo.

[0077] The nanomaterials GZHMU-1 (Example 1) and GZHMU-1@Mo (Example 2) synthesized above were evaluated as follows:

[0078] Test Example 1

[0079] Evaluation of Chemical Properties of GZHMU-1 and GZHMU-1@Mo Nanomaterials

[0080] 1. Verification of the Fenton effect

[0081] Weigh 3,3',5,5'-tetramethylbenzidine (1 mmol, 0.0024 g) and dissolve it in 5 mL of N,N-dimethylformamide to prepare a 2 mM TMB solution. Then, prepare two portions of GZHMU-1 and GZHMU-1@Mo simultaneously using PBS (pH = 5) at concentrations of 0, 12.5, 25, 50, 100, and 200 μg mL, respectively. -1 Solution. One group consisted of TMB + material, and the other group consisted of TMB + H₂O₂ + material. The TMB concentration was 200 μM, and the H₂O₂ concentration was 100 μM. After a 10-minute reaction, the maximum absorbance at 652 nm was measured using a UV-visible spectrophotometer.

[0082] The results are as follows Figure 3 As shown, Figure 3 (a) is the evaluation of the introduction of heteropoly acid MoO4 2- The ability to produce ·OH was demonstrated by ultraviolet absorption spectroscopy, where the characteristic absorption peak of the GZHMU-1@Mo nanomaterial at 650nm was detected. ·OH was used to oxidize the colorless TMB to produce the blue substance OX-TMB. As shown in the figure, with the increase of material concentration (from bottom to top), GZHMU-1@Mo exhibited a good ability to oxidize TMB. Figure 3 (b) Introduced heteropoly acid MoO4 2- The ability of GZHMU-1@Mo nanomaterials to oxidize TMB was enhanced, and the generation of ·OH was confirmed by EPR.

[0083] 2. GSH Consumption Experiment Verification

[0084] Reduced glutathione (1.0 mmol, 0.0031 g) was weighed and added to DI water to prepare a 1 mM GSH solution. Phosphate buffer solution was prepared using 1.425 g of disodium hydrogen phosphate, 0.0037 g of EDTA, and 100 mL of DI water. This solution was used to prepare a DTNB working solution with a concentration of 2 mM. Two portions of GZHMU-1 and GZHMU-1@Mo were prepared simultaneously with DI water, with concentrations of 0, 12.5, 25, 50, 100, and 200 μg mL, respectively. -1 One group consisted of GSH + material, and the other group consisted of GSH + H2O2 + material. The hydrogen peroxide concentration was 0.1 mM, and the volume of GSH solution added to the reaction solution was 0.1 mM. The order of adding reagents and materials was irrelevant. After 30 minutes of reaction with GSH, 0.1 mL of DTNB colorimetric solution was added for color development, followed by detection using a UV spectrophotometer.

[0085] The results are as follows Figure 4As shown in (a), after GZHMU-1@Mo nanomaterials reacted with GSH (1 mM) for 30 minutes, the DTNB probe was used to detect the consumption of GSH by GZHMU-1@Mo. When the concentration of H2O2 introduced was 100 μM, the lowest concentration of GZHMU-1@Mo nanomaterials at 12.5 μg / mL could deplete glutathione at this concentration by 74.5%.

[0086] 3. Catalyze NADH

[0087] Prepare a buffer solution containing 90% PBS and 10% DMSO. Use this solution to prepare a 2 mM NADH solution. Then use this buffer solution to prepare 1 mg / mL nanomaterial GZHMU-1 and GZHMU-1@Mo solutions. The total reaction system solution is 5 mL, and the nanomaterial concentration is 10 μg mL -1 A blank group without nanomaterials, a GZHMU-1 group, and a GZHMU-1@Mo group were set up. The reaction was stirred at 37°C for 5-7 hours. 1 mL of the solution was sampled at 1.5, 3, and 6 hours for detection using a UV spectrophotometer. To investigate whether hydrogen peroxide promotes the reaction between GZHMU-1@Mo and NADH, a NADH+H2O2 group was designed. The experimental design and grouping were as above, with a hydrogen peroxide concentration of 0.1 mM and a reaction time of 6 hours. 1 mL of the solution was sampled at 1.5, 3, and 6 hours for detection using a UV spectrophotometer.

[0088] The results are as follows Figure 4 As shown in (b), GZHMU-1@Mo catalyzes the bioactive substance NADH. When the H2O2 concentration in tumor cells is about 100 μM, the GZHMU-1@Mo nanomaterial significantly decreases at 339 nm after 6 hours, and the generated NAD+ significantly increases at 260 nm, indicating that the GZHMU-1@Mo nanomaterial can catalyze NADH.

[0089] Test Example 2

[0090] Evaluation of the antitumor activity of GZHMU-1 and GZHMU-1@Mo in vitro

[0091] The cytotoxicity of nanomaterials was analyzed by MTT assay: cells were seeded in 96-well plates (5×10 3The cells were cultured overnight in 4% CO2 (0.5 μg / mL) of 1% DMSO (150 μL / well). The next day, the cells were treated with varying concentrations of the nanomaterial (200 μL, 0-200 μg / mL) and incubated at 37°C in a 5% CO2 incubator for 48 hours. The cells were washed with PBS, and 200 μL of fresh serum-free medium containing MTT (0.5 mg / mL) was added and incubated at 37°C for 4 hours. The supernatant was discarded, and the formazan precipitate was dissolved in DMSO (150 μL / well). The cells were shaken in the dark for 10 minutes, and the absorbance was measured at 492 nm using a microplate reader to calculate the relative cell viability.

[0092] MTT colorimetric method was used to evaluate the cytotoxicity and biocompatibility of nanomaterials GZHMU-1 and GZHMU-1@Mo. Figure 5 As shown, different concentrations of nanomaterials GZHMU-1 and GZHMU-1@Mo are Figure 5 (a) A549 cell line and Figure 5 (b) After incubation with normal lung epithelial cell line BEAS-2B for 48h, the introduced heteropoly acid MoO4 2- IC of GZHMU-1@Mo nanomaterials on A549 cell lines 50 The inhibitory activity concentration was about 50μg / mL, and the survival rate of both on the normal cell line BEAS-2B was as high as more than 80%. The above results showed that the introduced heteropoly acid MoO4 2- The GZHMU-1@Mo nanomaterial as a drug has good anti-proliferative activity against the A549 tumor cell line. Secondly, the results from the normal cell line BEAS-2B showed that GZHMU-1 and GZHMU-1@Mo have no obvious toxic side effects on normal cells.

[0093] Test Example 3

[0094] Study on the anti-tumor mechanism of GZHMU-1 and GZHMU-1@Mo

[0095] 1. Lysosomal membrane permeability assay

[0096] Acridine orange (AO) is a lysosomal metachromatic fluorescent dye used to detect lysosomal membrane permeability in living cells. A549 cells were seeded in six-well plates (2.5×10 4 / well) and cultured overnight. On the second day, different nanomaterials were added for treatment. After treatment, the cells were stained with AO (20μM), incubated at 37°C for 10 minutes, and washed three times with PBS. Finally, images were collected using a laser confocal microscope. When the lysosomal membrane is intact, protonated oligomeric AO exhibits red fluorescence, using an excitation wavelength of 561nm and an emission wavelength range of 610±20nm. When the lysosomal membrane permeability increases and the lysosomal contents are released into the cytoplasm, deprotonated monomeric AO exhibits green fluorescence, using an excitation wavelength of 488nm and an emission wavelength range of 510±20nm.

[0097] The results are as follows Figure 6 As shown in (a), AO is known to penetrate the cytoplasm and nucleus, staining DNA or RNA (green fluorescence). Compared with the control cells, the average red fluorescence of cells treated with the nanomaterials GZHMU-1 and GZHMU-1@Mo decreased, with GZHMU-1@Mo showing the most significant effect. This suggests that the nanomaterials GZHMU-1 and GZHMU-1@Mo alter the membrane permeability of A549 cells. The results of the extracellular oxidative microbial growth factor (TMB) assay indicate that the nanomaterial GZHMU-1@Mo can generate ROS in a slightly acidic tumor microenvironment.

[0098] 2. Detection of Intracellular Reactive Oxygen Species

[0099] DCFH-DA was used to detect the intracellular ROS level. A549 cells were seeded in six-well plates (2.5×10 4 Cells were cultured overnight (100 μM per well) and treated the next day. After drug treatment, cells were stained with DCFH-DA (10 μM), incubated at 37°C for 20 minutes, and washed three times with PBS. Finally, images were acquired using a laser confocal microscope. An excitation wavelength of 488 nm was used, and the emission wavelength range was 510 ± 20 nm.

[0100] 3. Determination of Intracellular Lipid Peroxides

[0101] C11-BODIPY 581 / 591 A549 cells were seeded in confocal microplates (2.5×10 4 After drug treatment, cells were cultured with C11BODIPY 581 / 591 (2μM) staining, incubated at 37℃ for 30min. After washing 3 times with PBS, counterstained with Hoechst33342 for 10min, washed again with PBS, and images were collected using a laser confocal microscope. Oxidized C11 BODIPY 581 / 591 Green, excited by 488nm laser, emission wavelength range is 510±20nm; unoxidized C11-BODIPY 581 / 591It is red, excited by 561nm laser, and has an emission wavelength range of 591±20nm.

[0102] The results are as follows Figure 6 As shown in (b), ROS fluorescence probe (DCFH-DA) was used to evaluate the generation of ROS in cells. After 15 hours of treatment of A549 cells with nanomaterials GZHMU-1 and nanomaterials GZHMU-1@Mo, the green fluorescence of both was increased compared with the untreated control cells (black). 2- The green fluorescence of the nanomaterial GZHMU-1@Mo group was stronger. The results showed that the nanomaterials GZHMU-1 and GZHMU-1@Mo induced excessive production of ROS in cells. Due to the large surge in ROS and the consumption of GSH, the key indicator of ferroptosis, LPO, was further evaluated. As shown in the figure, C11-BODIPY 581 / 591 The probe detects LPO, where unoxidized lipids are red. After 15 hours of action of nanomaterials GZHMU-1 and nanomaterials GZHMU-1@Mo on A549 cells, both turn into green fluorescence, while the control group shows red fluorescence. The introduced guest molecule MoO4 2- The green fluorescence of the nanomaterial GZHMU-1@Mo group was stronger. The results showed that the nanomaterials GZHMU-1 and GZHMU-1@Mo caused the production of LPO in cells.

[0103] Test Example 4

[0104] In vivo antitumor activity / safety evaluation of GZHMU-1 and GZHMU-1@Mo In vivo antitumor activity / safety evaluation

[0105] Take A549 cells in the logarithmic growth phase and adjust the cell suspension concentration to 5×10 7 Each nude mouse was inoculated subcutaneously with 0.2 mL of the suspension in the axilla of the forelimb to establish A549 subcutaneous tumor-bearing mice. After the nude mice were tumor-bearing, the length (L) and width (W) of the tumor were measured every two days using a digital vernier caliper. The formula V (mm3) = π / 6 × L × W was used. 2 Calculate tumor volume. When the tumor volume reached approximately 100 mm3, mice were randomly divided into 4 groups (n=5) and injected intravenously with PBS, COF-1 (30 mg / kg), GZHMU-12 (30 mg / kg), or GZHMU-12@Mo (30 mg / kg). The drugs were injected every two days for a total of 7 times. Tumor size and mouse body weight were monitored simultaneously, and the relative tumor volume (V) was calculated as V d= / V0, where V0 is the tumor volume measured on the first day. After treatment, all mice were euthanized, and tumor tissues were collected, weighed, and photographed. Major organs, including the heart, liver, spleen, lungs, and kidneys, were also collected. Furthermore, tissues were fixed, sectioned, and analyzed by H&E staining.

[0106] like Figure 7 As shown in (a), based on tumor tissue photos, the tumors in the PBS and COF-1 groups were significantly larger than those in the GZHMU-1 and GZHMU-1@Mo groups, with the GZHMU-1@Mo group having the smallest volume.

[0107] In addition, the relative tumor volume Figure 7 (b) The results also showed that compared with the other groups, the tumor growth in the GZHMU-1@Mo group was significantly inhibited and significantly lower than that in the other groups, indicating that GZHMU-1@Mo has the best in vivo anti-tumor effect. After treatment, tumor tissues were obtained for HE staining and GPX4 immunohistochemistry analysis.

[0108] like Figure 7 (c) Results showed that the tumor tissue in the treatment group showed loose gaps and a decrease in tumor cells. The GZHMU-1@Mo group showed nuclear condensation and plasma membrane rupture, and GPX4 expression was significantly reduced. Combined with in vitro experimental results, this suggests that GZHMU-1@Mo may exhibit good anti-tumor efficacy by inducing and amplifying oxidative stress, downregulating GPX4 expression, and leading to ferroptosis.

[0109] For biosafety evaluation, Figure 8 As shown, compared with the PBS group, no obvious inflammation, lesions or organ damage were detected in the groups treated with nanomaterials, indicating that the prepared nanomaterials have good biocompatibility and no obvious toxic side effects on major organs.

Claims

1. A nanomaterial that induces ferroptosis in tumor cells, characterized in that: The nanomaterial is GZHMU-1 or GZHMU-1@Mo; The preparation method of GZHMU-1 comprises the following steps: Using triaminoguanidine and copper tricrystal as raw materials, GZHMU-1 is generated through mixed reaction under the action of p-toluenesulfonic acid and PEG materials; The temperature of the mixed reaction is 160-180°C; The preparation method of GZHMU-1@Mo comprises the following steps: Using triaminoguanidine, molybdate ion compounds and copper tricluster as raw materials, under the action of p-toluenesulfonic acid and PEG materials, the mixture undergoes Schiff reaction between aldehyde and amine to produce GZHMU-1@Mo; The molybdate ion compound includes one or more of sodium molybdate dihydrate and ammonium molybdate; The mixed reaction temperature is 160~180℃; The structural formula of the copper tricluster is .

2. The nanomaterial for inducing tumor cell ferroptosis according to claim 1, characterized in that: The PEG material includes one or more of PEG200, PEG400, PEG800, PEG1000 and PEG2000.

3. The nanomaterial for inducing ferroptosis of tumor cells according to claim 1, characterized in that: The molar ratio of the molybdate ion compound to guanidine and copper tricluster is (1-2):1:

1.

4. A method for preparing the nanomaterial for inducing ferroptosis of tumor cells according to any one of claims 1 to 3.

5. Use of the nanomaterial for inducing ferroptosis of tumor cells according to any one of claims 1 to 3, characterized in that: The nanomaterial is used to prepare anti-tumor nanomedicine; the tumor includes lung cancer, breast cancer, pancreatic cancer or prostate cancer.

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