Nano chelating agent capable of simulating tumor epithelial-mesenchymal transition process and inducing ferroptosis as well as preparation and application of nano chelating agent

Abstract

The invention belongs to the field of antitumor drugs and relates to a nano chelating agent capable of simulating a tumor epithelial-mesenchymal transition process and inducing ferroptosis as well as preparation and application of the nano chelating agent. The preparation method of the nano chelating agent is simple and low in cost, and specifically comprises the following steps: mixing and stirring MgSO4 and ethylenediamine tetraacetic acid disodium salt (EDTA) in a molar ratio of (0.5-2): 1, continuously stirring for 2-14 hours at the reaction temperature of 4 DEG C, and centrifuging to obtain the EDTA-Mg nano chelating agent. The EDTA-Mg nano chelating agent can destroy the connection between tumor cells, so that the adhesive force of the tumor cells is weakened, the cytoskeleton is remodeled, the polarity disappears, and the tumor cells are changed into a round cell form from an epithelial cell form, thereby realizing the simulation of the tumor epithelial-mesenchymal transition process. In addition, Fe < 2 + > of ferritin can be swept, and EDTA-Fe is formed. And EDTA-Fe reacts with rich H2O2 in the tumor to generate. OH with strong oxidizing property, so that mitochondria damage and lipid peroxidation are caused, and the tumor ferroptosis is induced.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and particularly relates to a preparation method and application of an ethylenediaminetetraacetic acid-Mg nano chelator that can simulate the tumor epithelial-mesenchymal transition process and induce ferroptosis. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Cancer metastasis is a dynamic multi-step process that involves complex pathways and dispersed sites. Currently, there is a lack of effective means in clinical practice to completely prevent tumor metastasis. Epithelial-mesenchymal transition (EMT) is a key stage of cancer metastasis. During this process, epithelial cells transform into mesenchymal cells, losing polarity and adhesiveness, and gaining enhanced migratory ability, which contributes to tumor metastasis and the development of drug resistance. Researchers are exploring methods to inhibit EMT to prevent tumor spread. However, the EMT process also makes tumor cells more vulnerable to external pressures such as immune recognition and blood shear stress, which provides opportunities for treatment. However, how to precisely control the timing of EMT occurrence in tumors is a huge challenge.

[0004] In addition, tumors in the EMT stage show higher sensitivity to ferroptosis. Ferroptosis is a form of cell death triggered by iron accumulation and lipid peroxidation. Cancer cells often exhibit an "iron addiction" metabolic characteristic. Compared with normal cells, cancer cells can enhance intracellular iron accumulation by altering iron metabolism to meet their growth needs. Ferritin, as the main iron storage protein in cells, is overexpressed in a variety of malignant tumors, including pancreatic cancer, breast cancer, hepatocellular carcinoma, etc. As the iron storage depot of cancer cells, ferritin is similar to an "explosive warehouse". Once detonated, the excessive accumulation of iron ions in tumors will lead to ferroptosis. Therefore, ferritin is a potential target for treating tumors. However, regulating the occurrence of EMT in tumors while releasing iron ions from ferritin to induce ferroptosis is a difficult and challenging obstacle.

[0005] Currently, there are studies on inducing ferroptosis and epithelial-mesenchymal transition (EMT) through gene regulation techniques, mainly including CRISPR-Cas9 technology, RNA interference technology, histone modification technology, transcription factor recruitment technology, etc. These gene regulation methods undoubtedly require professional synthesis equipment, technical talents, and have high technical route thresholds. Moreover, the biosafety of these gene regulation methods is currently unknown, and they may face problems such as low transformation efficiency and gene off-targeting. In addition, the popularization and implementation of these technologies face difficulties such as high professionalism, high costs, and a shortage of talents, and they cannot be widely used for the time being, being limited to the use in small-scale high-precision biotechnology enterprises and universities. Summary of the Invention

[0006] To solve the above problems, the present invention provides a preparation method and application of an ethylenediaminetetraacetic acid-magnesium (EDTA-Mg) nano-chelating agent that can simulate the process of tumor epithelial-mesenchymal transition and induce ferroptosis. The EDTA-Mg nano-chelating agent provided by the present invention precisely regulates the EMT process of tumors through metal chelation therapy, and at the same time "hijacks" iron ions in ferritin to induce ferroptosis. Specifically, the EDTA-Mg nano-chelating agent first chelates Ca 2+ in E-cadherin on the surface of tumor cells to form EDTA-Ca and simultaneously disrupt the connections between tumor cells. At this time, the adhesion of tumor cells weakens, the cytoskeleton is remodeled, the polarity disappears, and the tumor cells change from an epithelial cell morphology to a round cell morphology. At the same time, the expression of E-cadherin in tumor cells is down-regulated, while the expressions of vimentin and matrix metalloproteinase 2 (MMP-2) are up-regulated, and these indicators confirm that EMT has occurred in the tumors. Next, EDTA-Ca is internalized by tumor cells and enters the lysosome. In the lysosome, EDTA-Ca robs Fe 2+ from ferritin to form EDTA-Fe and release Ca 2+ , which causes the lysosome to rupture and release its contents. Finally, the released EDTA-Fe reacts with the abundant H2O2 in the tumor to generate strongly oxidizing ·OH, resulting in mitochondrial damage and lipid peroxidation (LPO), thereby inducing tumor ferroptosis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided a preparation method of a nano-chelating agent that can simulate the process of tumor epithelial-mesenchymal transition and induce ferroptosis, including:

[0009] Performing a coordination reaction on disodium ethylenediaminetetraacetate and a magnesium salt to obtain an EDTA-Mg nano-chelating agent.

[0010] In some embodiments, the molar ratio of the magnesium salt to disodium ethylenediaminetetraacetate is (0.5 - 2):1. Within this range, the molar ratio has little effect on the morphology of the EDTA-Mg nano chelator. The optimal molar ratio of divalent magnesium ions to disodium ethylenediaminetetraacetate is preferably 1:1.

[0011] The chelating agent (disodium ethylenediaminetetraacetate) used in the present invention has been widely used in medical devices and has high biosafety. Nano chelators prepared using its analogs ethylenediaminetetraacetic acid and dipotassium ethylenediaminetetraacetate (EDTA-2K) have the same metal chelating properties. However, since ethylenediaminetetraacetic acid is highly soluble in alkaline solutions and slightly soluble in water, it is less commonly used in the biological field, and its sodium or potassium salt forms are commonly used. Therefore, in some embodiments, ethylenediaminetetraacetic acid or dipotassium ethylenediaminetetraacetate (EDTA-2K) can be used to replace disodium ethylenediaminetetraacetate.

[0012] In some embodiments, the magnesium salt is selected from at least one of MgSO4 and MgCl2. Different magnesium salts also have relatively little effect on the morphology of the EDTA-Mg nano chelator. The preferred magnesium salt is MgSO4.

[0013] In some embodiments, the temperature of the coordination reaction is 4°C - 80°C, preferably 4°C, 25°C, and 80°C, and more preferably 4°C.

[0014] In some embodiments, the time of the coordination reaction is 2 - 14 h, preferably 6 h.

[0015] In some embodiments, the solvent for the coordination reaction is water.

[0016] In some embodiments, it further includes: after the coordination reaction is completed, solid-liquid separation and washing are carried out.

[0017] In some embodiments, centrifugal separation is used for solid-liquid separation. The rotation speed of centrifugal separation is 2000 - 8000 rpm, and the centrifugation time is 5 - 15 min; preferably, the rotation speed of centrifugation is 5000 rpm, and the centrifugation time is 10 min.

[0018] In some embodiments, after centrifugally separating the EDTA-Mg nano chelator, it further includes a step of washing it, and the number of washing times is 2 - 3 times.

[0019] In the second aspect of the present invention, there is provided a nano chelator prepared by the above method that can simulate the tumor epithelial-mesenchymal transition process and induce ferroptosis. The EDTA-Mg nano chelator has a crystalline structure, wherein the coordination ratio of Mg 2+ to EDTA is 1:1. In an ethanol solution, EDTA-Mg can react with Ca 2+Chelation forms EDTA-Ca while releasing Mg 2+ In addition, EDTA-Ca can chelate with Fe 2+ to form EDTA-Fe and release Ca 2+ And EDTA-Fe can react with H2O2 to generate ·OH to oxidize TMB.

[0020] In a third aspect of the present invention, there is provided the use of the above-mentioned nano chelating agent in simulating the process of tumor epithelial-mesenchymal transition and inducing ferroptosis.

[0021] In a fourth aspect of the present invention, there is provided the use of the above-mentioned nano chelating agent in the preparation of anti-cancer drugs.

[0022] The EDTA-Mg nano chelating agent chelates Ca on the surface of breast cancer cells (4T1) 2+ to form EDTA-Ca, promoting the dissociation of tumor cells. This process down-regulates the expression of E-cadherin and up-regulates the expression of Vimentin and MMP-2, which confirms the occurrence of EMT. In addition, once EDTA-Ca is internalized by tumor cells, it will separate Fe 2+ from ferritin to form EDTA-Fe, which induces LPO through the Fenton-like reaction to trigger ferroptosis. The EMT simulation amplifies the therapeutic effect of ferroptosis on tumor cells.

[0023] Advantages of the present invention

[0024] (1) Traditional chelation therapy consumes the overloaded metal elements in the host body by intravenous injection of chelating agents. Due to the lack of targeting, it is prone to systemic toxicity. The EDTA-Mg nano chelating agent provided by the present invention effectively solves the challenge of insufficient accumulation of chelating agents at the tumor site, and at the same time promotes the enrichment of local high-concentration iron ions in tumor cells.

[0025] (2) The EDTA-Mg nano chelating agent provided by the present invention has a milder chelating effect compared to the chelating agent EDTA, and can reduce the damage to normal cells.

[0026] (3) The EDTA-Mg nano chelating agent artificially simulates the EMT process of tumors through metal chelation therapy. By using the spatio-temporal sequence of the contact between the EDTA-Mg nano chelating agent and tumor cells, not only can the occurrence of tumor EMT be regulated, but also iron ions can be hijacked from ferritin to induce ferroptosis in tumor cells. This method has a synergistic therapeutic effect, achieving a 1+1>2 effect.

[0027] (4) The EDTA-Mg nano chelating agent provided by the present invention has better biocompatibility than Zn-penicillamine (Zn-PEN), and its damage to normal tissues is less than that of Zn-PEN. Although Zn-PEN releases Zn by chelating Cu 2+ to release Zn 2+ , but Zn 2+ is highly toxic and will also have certain side effects on normal cells. The EDTA-Mg nano chelating agent releases biotoxic Mg 2+ , which has no damage to normal cells. The EDTA-Mg nano chelating agent exerts its therapeutic effect by using the endogenous metal ions of tumor cells, without introducing metal ions with cytotoxicity. In addition, the biotoxic EDTA-Mg nano chelating agent can achieve the same therapeutic effect as Zn-PEN by cleverly using endogenous metal ions.

[0028] (5) The preparation method of the present invention is simple, the raw materials are cheap and easy to obtain, the product yield is large, the biological safety is high, the practicability is strong, and it is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention, and the exemplary embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0030] Figure 1 It is a transmission electron microscope (TEM) image of the EDTA-Mg nano chelating agent prepared in Example 1.

[0031] Figure 2 It is a particle size distribution diagram of the EDTA-Mg nano chelating agent described in Example 1.

[0032] Figure 3 It is an X-ray diffraction (XRD) characterization of the EDTA-Mg nano chelating agent described in Example 1.

[0033] Figure 4 It is a Fourier transform infrared spectroscopy (FT-IR) characterization of the EDTA-Mg nano chelating agent described in Example 1.

[0034] Figure 5 It is an X-ray photoelectron spectroscopy (XPS) characterization of the EDTA-Mg nano chelating agent described in Example 1.

[0035] Figure 6 It is a characterization of the EDTA-Mg nano chelating agent described in Example 1 chelating Ca 2+ and releasing Mg 2+ .

[0036] Figure 7Characterization of the EDTA-Ca chelate formed by chelating Ca with the EDTA-Mg nano chelating agent described in Example 1 2+ and Fe 2+ after chelation

[0037] Figure 8 Characterization of the oxidation of TMB by EDTA-Fe formed by chelating Fe with the EDTA-Mg nano chelating agent described in Example 1 in the presence of H2O2 2+ after chelation

[0038] Figure 9 Characterization of the release of Mg by chelating Ca in 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 2+ and releasing Mg 2+ after chelation

[0039] Figure 10 Characterization of the morphology of 4T1 cells after treatment with the EDTA-Mg nano chelating agent described in Example 1 for different times

[0040] Figure 11 Morphological changes caused by treating 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 and untreated materials

[0041] Figure 12 Detection of the expression of EMT-related proteins caused by treating 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 and untreated materials

[0042] Figure 13 Detection of the induction of intracellular reactive oxygen species (ROS) by treating 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 and untreated materials

[0043] Figure 14 Detection of the induction of intracellular LPO by treating 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 and untreated materials

[0044] Figure 15 Detection of the expression of ferroptosis-related proteins caused by treating 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 and untreated materials

[0045] Figure 16 Detection of the induction of apoptosis by treating 4T1 cells with the EDTA-Mg nano chelating agent described in Example 1 and untreated materials Detailed implementation mode

[0046] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0047] Glossary of terms:

[0048] In the present invention, the EDTA mentioned in the preparation, synthesis and experiments is in the form of disodium ethylenediaminetetraacetate.

[0049] The following will further elaborate on the present invention with specific embodiments. It should be noted that the specific embodiments are for the explanation rather than the limitation of the present invention.

[0050] Example 1

[0051] The preparation method of the EDTA-Mg nano chelating agent is as follows:

[0052] 1) Weigh 1 mmol of MgSO4 and EDTA-2Na respectively into a 50 mL high-speed centrifuge tube;

[0053] 2) Add 20 mL of deionized water and dissolve by ultrasonic treatment;

[0054] 3) Stir at 4 °C for 6 h, centrifuge at 5000 rpm for 10 min to obtain a white EDTA-Mg nano chelating agent. Then wash it 2 - 3 times with deionized water and centrifuge again to finally obtain the EDTA-Mg nano chelating agent.

[0055] The EDTA-Mg nano chelating agent prepared in Example 1 was characterized by transmission electron microscopy. As Figure 1 shown, it can be seen that the prepared EDTA-Mg nano chelating agent is a spherical nano material. Figure 2 The particle size distribution shows that the average particle size of the EDTA-Mg nano chelating agent is 36.1 ± 10.2 nm.

[0056] Example 2

[0057] XRD characterization of the EDTA-Mg nano chelating agent

[0058] 1) Dry the EDTA-Mg nano chelating agent prepared in Example 1 in a vacuum drying oven at 70 °C;

[0059] 2) Grind the solid EDTA-Mg nano chelating agent into powder using an agate mortar;

[0060] 3) Finally, prepare a sample for XRD testing.

[0061] As Figure 3 shown, the XRD pattern shows that compared with EDTA-2Na (PDF#29 - 1914), new diffraction peaks appear while the original diffraction peaks are retained for EDTA-Mg, confirming that Mg 2+ chelates with EDTA to form EDTA-Mg.

[0062] Example 3

[0063] FT-IR Characterization of EDTA-Mg Nano-Chelating Agent

[0064] 1) The pretreatment is the same as that in Example 2;

[0065] 2) Finally, prepare the sample for FT-IR testing.

[0066] As Figure 4 shown, the peaks between 2990 and 3060 cm -1 are the stretching vibrations of C-H, and the peaks between 1587 and 1652 cm -1 are the stretching vibrations of C=O. The peaks between 1365 and 1427 cm -1 may be caused by the stretching vibrations of carboxyl C-O. The peaks in the range of 1083 - 1205 cm -1 are the stretching vibrations of C-N.

[0067] Example 4

[0068] XPS Characterization of EDTA-Mg Nano-Chelating Agent

[0069] 1) The pretreatment is the same as that in Example 2;

[0070] 2) Finally, prepare the sample and test the elements of the EDTA-Mg nano-chelating agent by XPS.

[0071] As Figure 5 shown, the XPS spectrum confirms the presence of Mg element in EDTA-Mg.

[0072] Example 5

[0073] Characterization of Chelation of Ca 2+ Release of Mg 2+ by EDTA-Mg Nano-Chelating Agent

[0074] 1) Prepare an ethanol solution of 5 mM CaCl2;

[0075] 2) Add different concentrations of EDTA-Mg (0, 100, 200, 400 μg mL -1 ) to the ethanol solution containing Ca 2+ ;

[0076] 3) After mixing evenly, let it stand for 2 h;

[0077] 4) After centrifugation, use the Mg 2+ and Ca 2+ detection kits to detect the concentrations of Mg 2+ and Ca 2+ in the supernatant.

[0078] As shown Figure 6 in the figure, EDTA-Mg can effectively chelate Ca 2+ and release Mg 2+ . As its concentration increases, the content of Ca 2+ in the solution gradually decreases, and the concentration of Mg 2+ gradually increases.

[0079] Example 6

[0080] Characterization of the chelation of Fe by the EDTA-Ca nano chelating agent 2+

[0081] 1) Prepare an ethanol solution of 5 mM FeCl2;

[0082] 2) Add different concentrations of EDTA-Ca (0, 100, 200, 400 μg mL 2+ ) obtained by chelating Ca with EDTA-Mg -1 to the ethanol solution containing Fe 2+ ;

[0083] 3) After mixing evenly, let it stand for 2 h;

[0084] 4) After centrifugation, use an Fe 2+ detection kit to detect the concentration of Fe 2+ in the supernatant.

[0085] As shown Figure 7 in the figure, EDTA-Ca can chelate Fe 2+ , thereby releasing Ca 2+ .

[0086] Example 7

[0087] Characterization of the Fenton-like reaction of the EDTA-Fe nano chelating agent

[0088] 1) Disperse the EDTA-Fe nano chelating agent obtained by chelating Ca with EDTA-Mg 2+ in 18 mL of deionized water, divide it into 6 equal parts, and the solution in each tube is 3 mL;

[0089] 2) Then add 50 μL of 10% H2O2 solution and 80 μL of TMB solution to each tube respectively;

[0090] 3) React the above solutions for 0, 2, 5, 10, 20, 30 min respectively, and take the supernatant to detect the absorption of TMB by a UV-visible spectrophotometer.

[0091] As shown Figure 8 ​As shown, in the presence of H2O2, EDTA-Fe can oxidize colorless TMB to blue oxTMB. As time extends, the characteristic absorption peak of oxTMB at 650 nm gradually increases.

[0092] Example 8

[0093] Test of EDTA-Mg nano chelating agent chelating Ca 2+ and releasing Mg 2+ in 4T1 cells

[0094] 1) Seed 4T1 cells in a 6-well plate (10 5 cells / well) and culture them in an incubator at 37 °C and 5% CO2 for 24 h;

[0095] 2) Set up 2 groups of experiments, namely: (1) PBS; (2) EDTA-Mg (500 μg mL -1 );

[0096] 3) After different treatments for 12 h, collect the cell culture media of each group and use a Ca 2+ and Mg 2+ detection kit to detect the contents of Ca 2+ and Mg 2+ in the cell culture media of different treatments.

[0097] As Figure 9 shown, compared with PBS, after EDTA-Mg treatment, the Ca 2+ in the cell culture media decreases and the Mg 2+ content increases, indicating that EDTA-Mg can still effectively chelate Ca 2+ and release Mg 2+ in cells.

[0098] Example 9

[0099] Changes in the morphology of 4T1 cells after treatment with EDTA-Mg nano chelating agent

[0100] 1) Seed 4T1 cells in a 6-well plate (10 5 cells / well) and culture them in an incubator at 37 °C and 5% CO2 for 24 h;

[0101] 2) Treat with EDTA-Mg (500 μg mL -1 ) for 0, 2, 4, 8 h respectively;

[0102] 3) After different treatments, wash three times with PBS and then fix 4T1 cells with 4% paraformaldehyde.

[0103] 4) Use an inverted optical microscope to observe the morphology of 4T1 cells treated for different times.

[0104] As Figure 10 shown, after co - culturing 4T1 cells with EDTA - Mg, as the culture time extended, 4T1 cells showed the characteristics of EMT. Specifically, tumor cells were separated, cell polarity disappeared, and the cell morphology became round.

[0105] Example 10

[0106] Changes in the morphology of 4T1 cells after treatment with different materials

[0107] 1) Seed 4T1 cells in a 6 - well plate (10 5 cells / well) and culture them in an incubator at 37 °C and 5% CO2 for 24 h;

[0108] 2) Set up 4 groups of experiments, which are respectively: (1) PBS; (2) EDTA; (3) Mg 2+ ; (4) EDTA - Mg (500 μg mL -1 );

[0109] 3) After treating 4T1 cells for 8 h, wash them three times with PBS, and then fix 4T1 cells with 4% paraformaldehyde.

[0110] 4) Observe the morphology of 4T1 cells in different experimental groups using an inverted optical microscope.

[0111] As Figure 11 shown, the 4T1 cells in the control group and the Mg 2+ treatment group were still closely connected, and the cell morphology did not change. The cells in the EDTA and EDTA - Mg treatment groups were separated from each other, and the cell morphology changed. These results confirmed that EDTA - Mg disrupted the connections between cells by chelating Ca 2+ .

[0112] Example 11

[0113] Expression of EMT - related proteins in 4T1 cells after treatment with different materials

[0114] 1) Seed 4T1 cells in a 10 - cm culture dish (10 6 cells / dish) and culture them in an incubator at 37 °C and 5% CO2 for 24 h;

[0115] 2) Set up 6 groups of experiments, which are respectively: (1) PBS; (2) EDTA; (3) Mg 2+ ; (4) EDTA - Mg (500 μg mL -1 ); (5) EDTA - Mg + Fe 2+ ; (6) EDTA - Mg + Fe 2+ + DFO (deferoxamine);

[0116] 3) After treating 4T1 cells for 8 h, wash them three times with PBS;

[0117] 4) Extract the proteins of 4T1 cells in different treatment groups, and detect the expressions of E-cadherin, Vimentin and MMP-2 by Western blot assay.

[0118] As Figure 12 shown, the expression of E-cadherin decreased in tumor cells of the treatment groups containing EDTA and EDTA-Mg, while the expressions of Vimentin and MMP-2 increased, indicating that the EMT process occurred in tumor cells. These results suggest that EDTA-Mg can 2+ artificially mimic the EMT process of tumors.

[0119] Example 12

[0120] Changes in ROS in 4T1 cells after treatment with different materials

[0121] 1) Seed 4T1 cells in a 6-well plate (10 5 cells / well) and culture them in an incubator at 37 °C and 5% CO2 for 24 h;

[0122] 2) Set up 6 groups of experiments, namely: (1) PBS; (2) EDTA; (3) Mg 2+ ; (4) EDTA-Mg (500 μg mL -1 ); (5) EDTA-Mg + Fe 2+ ; (6) EDTA-Mg + Fe 2+ + DFO;

[0123] 3) After different treatments for 12 h, wash them three times with PBS;

[0124] 4) Incubate 4T1 cells in different groups with 2,7-dichlorofluorescein diacetate (DCFH-DA) probe for 30 min, and wash them three times with PBS;

[0125] 5) Detect the production of ROS in 4T1 cells of different experimental groups by flow cytometry.

[0126] As Figure 13 can be seen, tumor cells treated with EDTA and Mg 2+ alone did not produce ROS, which may be due to the fact that free EDTA cannot maintain a high concentration of Fe 2+ inside tumor cells. However, EDTA-Mg and EDTA-Mg + Fe 2+The tumor cells of the group showed obvious ROS production, which was due to the fact that EDTA-Mg nanoparticles could enrich Fe 2+ , thereby generating ·OH through a Fenton-like reaction. To determine whether the ROS production in tumor cells was due to the deprivation of endogenous iron by EDTA-Mg, the present invention used the iron chelator deferoxamine (DFO) to eliminate iron ions in tumor cells. After DFO treatment, the ROS produced by EDTA-Mg decreased significantly, confirming that EDTA-Mg induced oxidative stress in tumor cells by depriving endogenous iron and through a Fenton-like reaction.

[0127] Example 13

[0128] Detection of LPO in 4T1 cells after treatment with different materials

[0129] 1) 4T1 cells were seeded in 6-well plates (10 5 cells / well) and cultured in an incubator at 37°C and 5% CO2 for 24 h;

[0130] 2) Six groups of experiments were set up, namely: (1) PBS; (2) EDTA; (3) Mg 2+ ; (4) EDTA-Mg (500 μg mL -1 ); (5) EDTA-Mg + Fe 2+ ; (6) EDTA-Mg + Fe 2+ + DFO;

[0131] 3) After different treatments for 12 h, the cells were washed three times with PBS;

[0132] 4) The Liperfluo fluorescent probe was co-incubated with 4T1 cells in different groups for 30 min, and then the cells were washed three times with PBS;

[0133] 5) Flow cytometry was used to detect the LPO levels of 4T1 cells in different experimental groups.

[0134] As Figure 14 shown, consistent with the ROS results, the proportion of green fluorescence in tumor cells of the EDTA-Mg and EDTA-Mg + Fe 2+ groups increased, while the proportion of green fluorescence in tumor cells decreased after DFO treatment, indicating that EDTA-Mg induced LPO in tumor cells through endogenous iron deprivation and a Fenton-like reaction.

[0135] Example 14

[0136] Expression of ferroptosis-related proteins in 4T1 cells after treatment with different materials

[0137] 1) 4T1 cells were seeded in 10-cm culture dishes (10 6Cells / plate), and cultured in an incubator at 37°C and 5% CO2 for 24 h;

[0138] 2) Six groups of experiments were set up, namely: (1) PBS; (2) EDTA; (3) Mg 2+ ; (4) EDTA-Mg (500 μg / mL -1 ); (5) EDTA-Mg + Fe 2+ ; (6) EDTA-Mg + Fe 2+ + DFO;

[0139] 3) After treating 4T1 cells for 12 h, wash them three times with PBS;

[0140] 4) Extract the proteins of 4T1 cells in different treatment groups, and detect the expression of Ferritin, GPX4 and ACSL4 proteins by Western blot assay.

[0141] As Figure 15 shown, the expression level of Ferritin was higher in the tumor cells of the control group, while the expression levels of Ferritin in the tumor cells of the EDTA, EDTA-Mg and EDTA-Mg + Fe 2+ groups were significantly decreased. In addition, in the tumor cells treated with EDTA-Mg and EDTA-Mg + Fe 2+ , the expression of GPX4 was decreased, while the expression of ACSL4 was increased. After DFO treatment, the expression trends of the two proteins were reversed. These findings suggest that EDTA-Mg induces ferroptosis of tumor cells through endogenous iron deprivation and Fenton-like reaction.

[0142] Example 15

[0143] Detection of apoptosis of breast cancer cells (4T1) induced by EDTA-Mg nanochelator

[0144] 1) Seed 4T1 cells in a 6-well plate (10 5 cells / well), and culture them in an incubator at 37°C and 5% CO2 for 24 h;

[0145] 2) Six groups of experiments were set up, namely: (1) PBS; (2) EDTA; (3) Mg 2+ ; (4) EDTA-Mg (500 μg / mL -1 ); (5) EDTA-Mg + Fe 2+ ; (6) EDTA-Mg + Fe 2+ + DFO;

[0146] 3) After different treatments for 12 h, remove the old medium in the 6-well plate and wash it 3 times with PBS;

[0147] 4) Digest the cells in the 6-well plate with trypsin, centrifuge at 1000 rpm for 5 min, and collect the precipitate to obtain cells under different treatments;

[0148] 5) Add the prepared apoptosis detection kit to the cells obtained above, gently pipette to resuspend the cells, continue to incubate for 30 min, and detect the apoptosis effect of the cells by flow cytometry.

[0149] From Figure 16 It can be seen that the treatment with Mg alone 2+ has no obvious effect on the activity of tumor cells, while the treatment with a single EDTA has a certain degree of damage to tumor cells. The tumor cells in the EDTA-Mg+Fe 2+ group showed the most severe apoptosis, and the treatment with DFO reduced the toxicity of EDTA-Mg to tumor cells.

[0150] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis, characterized in that, Comprising: Performing a coordination reaction between disodium ethylenediaminetetraacetate and a magnesium salt to obtain an EDTA-Mg nano chelating agent.

2. The preparation method of the nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis according to claim 1, characterized in that, The molar ratio of the magnesium salt to disodium ethylenediaminetetraacetate is (0.5 - 2):1, or 1:

1.

3. The preparation method of the nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis according to claim 1, characterized in that, The magnesium salt is selected from at least one of MgSO4 and MgCl2; Or, using ethylenediaminetetraacetic acid or dipotassium ethylenediaminetetraacetate to replace disodium ethylenediaminetetraacetate.

4. The preparation method of the nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis as described in claim 1, characterized in that, The temperature of the coordination reaction is 4°C - 80°C, or 4°C.

5. The preparation method of the nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis according to claim 1, characterized in that The time of the coordination reaction is 2 - 14 h, or 6 h.

6. The preparation method of the nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis as described in claim 1, characterized in that, The solvent for the coordination reaction is water.

7. The preparation method of the nano chelator capable of simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis as described in claim 1, wherein, Also comprising: After the coordination reaction is completed, performing solid-liquid separation and washing.

8. A nano chelating agent prepared by the method according to any one of claims 1 - 7, which can simulate the tumor epithelial-mesenchymal transition process and induce ferroptosis.

9. Use of the nano chelating agent according to claim 8 in simulating the tumor epithelial-mesenchymal transition process and inducing ferroptosis.

10. Use of the nano chelating agent according to claim 9 in the preparation of an anticancer drug.

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