A Ru(II) complex with anti-tumor immune activity and a preparation method and application thereof

Abstract

This invention belongs to the field of antitumor immunotherapy, specifically relating to a Ru(II) complex with antitumor immunomodulatory activity, its preparation method, and its application. The chemical formula of the complex in this invention is [Ru(C-N)(N-N)L](PF6), where N-N = 2,2-bipyridine (bpy), C-N = 2-(p-tolyl)pyridine, and the ligand L = 2-(6-quinoxalinyl)-1H-imidazo[4,5-f][1,10]o-phenanthroline. Experiments have confirmed that this complex is effective against IC50 in MDA-MB-231 and 4T1 tumor cells. 50 The values ​​were 2.34±0.02 μM and 4.25±0.01 μM, respectively, with a selectivity coefficient of 7.19. It can kill tumor cells through ferroptosis, induce immunogenic cell death, promote dendritic cell maturation, and downregulate T cells. reg Cell count, activation of the body's anti-tumor immunity, suitable for the preparation of anti-tumor immunotherapies.

Description

Technical Field

[0001] This invention belongs to the field of antitumor immunotherapy technology, specifically relating to a Ru(II) complex with antitumor immunotherapy activity, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human life and health. Their incidence and mortality rates are rising year by year, placing a heavy burden on global healthcare systems and patients' families. Currently, traditional clinical treatments for malignant tumors mainly include surgical resection, chemotherapy, and radiotherapy. While these treatments can inhibit tumor progression to some extent, they generally have significant drawbacks: surgical treatment is difficult to completely eliminate micrometastases, easily leading to tumor recurrence; chemotherapy drugs lack the ability to specifically recognize tumor cells, severely damaging normal tissue cells while killing tumor cells, causing a series of toxic side effects such as bone marrow suppression and gastrointestinal reactions; radiotherapy suffers from radiation damage and limited local tolerance. Furthermore, none of these treatments can effectively activate the body's own immune system, failing to form long-term anti-tumor immune memory and fundamentally solve the problems of tumor metastasis and recurrence.

[0003] In recent years, the discovery of immunogenic cell death (ICD) has provided a novel approach to cancer treatment. Unlike traditional passive cell death, ICD is a special regulatory cell death mode. Its core mechanism lies in the active release of a series of damage-associated molecular patterns (DAMPs) during tumor cell death. These mainly include calreticulin (CRT) exposed on the cell surface, high-mobility group box 1 (HMGB1) released extracellularly, and adenosine triphosphate (ATP). These signaling molecules can effectively recruit dendritic cells (DCs) to the tumor site and promote DC maturation. Mature DCs can then present processed tumor antigen peptides to naïve CD8 cells via major histocompatibility complex (MHC) class I molecules. + T cells can be activated into cytotoxic T lymphocytes (CTLs) with cytotoxic functions. Activated CTLs can specifically recognize and kill residual tumor cells in the body. Simultaneously, some activated T cells differentiate into memory T cells, which can survive long-term in the body and play a role in immune surveillance, thereby effectively preventing tumor recurrence and metastasis. Therefore, drugs that can induce ICD in tumor cells hold promise as a new generation of anti-tumor drugs with both direct tumor cell killing and anti-tumor immune activation functions, and are currently a research hotspot in the field of cancer treatment.

[0004] In the development of antitumor drugs, metal complexes have attracted much attention due to their unique structure and physicochemical properties. Among them, Ru(II) complexes exhibit more significant advantages compared to traditional platinum-based antitumor drugs: Ruthenium has multiple stable redox states, enabling it to participate in intracellular redox reactions and regulate tumor cell signaling pathways; Ru(II) complexes have flexible and diverse coordination configurations, allowing for the modification of ligand structures to regulate their targeting and biological activity; simultaneously, Ru(II) complexes exhibit good selectivity for tumor cells, reducing toxic side effects on normal cells. Based on these advantages, Ru(II) complexes are considered to be highly promising candidates for antitumor drugs.

[0005] However, currently reported Ru(II) complexes still have many shortcomings. Most Ru(II) complexes can only kill tumor cells individually and cannot effectively induce ICD in tumor cells, making it difficult to activate the body's systemic anti-tumor immune response. The few Ru(II) complexes that can induce ICD have low tumor cell killing efficiency and insufficient selectivity for normal cells, limiting their clinical application. Therefore, developing a Ru(II) complex that possesses both high tumor cell killing activity and high selectivity, and can kill tumor cells through ferroptosis and effectively induce immunogenic cell death, thereby reshaping the tumor immune microenvironment and activating the body's anti-tumor immune memory, has significant scientific and clinical application value. Summary of the Invention

[0006] The purpose of this invention is to provide a Ru(II) complex with anti-tumor immune activity, its preparation method and application. This complex can help the body form long-term anti-tumor immune memory, which is expected to reduce the risk of tumor recurrence and metastasis, and provide a novel drug candidate with both direct killing and immune activation functions for clinical tumor treatment.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a Ru(II) complex with antitumor immunomodulatory activity, the chemical formula of which is [Ru(CN)(NN)L](PF6), wherein NN = 2,2-bipyridine (bpy), CN = 2-(p-tolyl)pyridine, and the ligand L = 2-(6-quinoxalinyl)-1 H -imidazo[4,5-f][1,10]o-diazanthroline, the structural formula of the complex is shown in formula (I): Formula (I).

[0008] The present invention also provides a method for preparing the Ru(II) complex, comprising the following steps: taking [(η 6The Ru(II) complex was refluxed with 2-(p-tolyl)pyridine at 140 °C for 18 h, and then the ligand 2-(quinoxalo-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline was added and reacted for 1 h. Finally, an excess of saturated NH4PF6 solution was added, the mixture was filtered, and the precipitated solid was collected to obtain the Ru(II) complex.

[0009] Furthermore, the solvent used in the preparation process is an N,N-dimethylformamide solution.

[0010] The present invention also provides the application of the Ru(II) complex in the preparation of antitumor immunotherapeutic drugs.

[0011] Furthermore, the drug targets human triple-negative breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 cells.

[0012] Furthermore, the Ru(II) complex exerts its antitumor effect by inducing ferroptosis in tumor cells.

[0013] Furthermore, the Ru(II) complex can induce immunogenic death in human triple-negative breast cancer MDA-MB-231 cells.

[0014] Furthermore, the drug enables female BALB / c mice inoculated with 4T1 cells treated with Ru(II) complex to acquire immunity against 4T1 tumors.

[0015] Furthermore, the method of acquiring immunity is to administer three doses of a 4T1 cell vaccine treated with Ru(II) complex to female BALB / c mice.

[0016] The beneficial effects of this invention are as follows: The Ru(II) complex with antitumor immunomodulatory activity provided by this invention is a metal complex with phenanthroline derivatives as ligands, exhibiting significant structural advantages compared to traditional small-molecule organic antitumor drugs. This complex carries an internal charge, enhancing its penetration and retention effects in vivo and increasing drug accumulation at tumor sites. Simultaneously, ruthenium possesses flexible coordination mechanisms and abundant redox states, allowing for further optimization of its biological activity through ligand structure modification. Experimental data confirm that this complex demonstrates excellent killing effects against human triple-negative breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 cells, with an IC50 value of [missing information]. 50 The values ​​were as low as 2.34±0.02 μM and 4.25±0.01 μM, respectively, while the IC50 for MCF-10A in normal human mammary epithelial cells was... 50With a concentration as high as 16.83±0.02 μM and a selectivity coefficient of 7.19, this complex can selectively kill tumor cells and significantly reduce toxic side effects on normal cells. It specifically targets the endoplasmic reticulum of tumor cells, triggering cell death through ferroptosis. Its mechanism of action is well-defined and unlikely to induce drug resistance, addressing the shortcomings of traditional chemotherapy drugs, such as poor selectivity and strong side effects.

[0017] The Ru(II) complex of this invention not only possesses the ability to directly kill tumor cells, but also exhibits outstanding anti-tumor immunomodulatory activity, effectively compensating for the shortcomings of traditional tumor treatments. This complex can induce immunogenic cell death in tumor cells, promote the exposure of calreticulin on the cell surface, and simultaneously promote the extracellular release of high-mobility group box 1 (HMP-B1) and adenosine triphosphate (ATP). These signaling molecules can recruit and promote the maturation of dendritic cells, thereby activating naïve CD8+. + T cells form cytotoxic T lymphocytes, enabling precise elimination of residual tumor cells. Animal experiments show that this complex can downregulate regulatory T cells in tumor tissue. reg The increased cell number effectively reshapes the tumor immune microenvironment and activates the body's systemic anti-tumor immune response. Female BALB / c mice vaccinated with tumor cell vaccines treated with this complex acquired immunity against 4T1 tumors, with the three-dose vaccine group showing particularly significant tumor volume inhibition, and no significant organic damage to major organs was observed. This complex can help the body form long-term anti-tumor immune memory, potentially reducing the risk of tumor recurrence and metastasis, and providing a novel drug candidate with both direct killing and immune activation functions for clinical cancer treatment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a diagram showing the effect of the complex Ru1 on the ferroptosis-associated protein GPX4 in this invention. Figure 2 The diagram shows the interaction between the complex Ru1 and MDA-MB-231 cells for 24 hours, where (A) cell surface exposure of calreticulin (CRT); (B) extracellular release of high-mobility group box 1 (HMGB1); and (C) extracellular release of adenosine triphosphate (ATP). Figure 3The image shows the effect of female BALB / c mice on tumor immunity after being inoculated with Ru1-treated 4T1 cells in this invention. In the image, A is the curve of mouse body weight change; B is the curve of mouse tumor volume change; C is the solid tumor image of each group 30 days after tumor inoculation; and D is the HE staining result image of each experimental group. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention provides a Ru(II) complex [Ru(CN)(NN)L](PF6) that can specifically target the endoplasmic reticulum and induce immunogenic death in MDA-MB-231 cells, wherein NN = 2,2-bipyridine (bpy), CN = 2-(p-tolyl)pyridine, and ligand L = 2-(6-quinoxalinyl)-1 H-Imidazolo[4,5-f][1,10]o-diazanthroline, complex Ru1, has the structural formula shown in formula (I):

[0026] Formula (I).

[0027] The preparation method of the Ru(II) complex in this invention includes the following steps: Preparation of complexes: [(η 6 [C6H6]Ru(bpy)Cl]Cl was refluxed with 2-(p-tolyl)pyridine at 140 °C for 18 h, then the ligand 2-(quinoxalo-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline was added and reacted for 1 h. Finally, excess saturated NH4PF6 solution was added, and the mixture was filtered. The precipitated solid was the ruthenium complex. The preparation method of the above ruthenium complex can be expressed by the following reaction formula:

[0028] Preferably, the reaction process for preparing complex Ru1 is carried out by reflux in N,N-dimethylformamide solution. After the reaction is completed, an excess of saturated NH4PF6 solution is added, and the precipitated solid is the ruthenium(II) complex.

[0029] The ruthenium complexes of the present invention can be used to prepare drugs with antitumor immunomodulatory activity.

[0030] The antitumor activity of the ruthenium complex of this invention was detected mainly through the following steps: After cells proliferated to the logarithmic growth phase, they were digested into a single-cell suspension using 0.25% trypsin, and viable cell counting was performed. Cells were seeded at a density of 8000 cells (100 μL volume) per well into a 96-well plate and incubated in a CO2 incubator for 24 h. Subsequently, 100 μL of different concentrations of the drug diluted with culture medium was added to each well, and incubation continued for 44 h. Afterward, 10 μL of the drug diluted with culture medium was added to each well. MTT solution dissolved in L PBS was reacted for 4 h, and then the culture medium was discarded. 100 μL of the solution was added to each well. L DMSO was incubated in the dark and shaken on a shaker for approximately 10 minutes. The absorbance at 490 nm was then measured using a microplate reader. Cell viability was calculated using a formula, and the half-maximal inhibitory concentration (IC50) was determined by fitting a curve. 50 (Value). This method is based on the classic MTT assay, has good reliability, and can be directly used to evaluate whether a compound has antitumor activity.

[0031] Survival rate % = (Average OD value of dosing wells / Average OD value of control wells) × 100% In vivo experimental procedures for inducing ICD in tumor cells using the complex: 4T1 cells were treated with Ru1 at a concentration of 8 μM for 24 h. The cell pellet was collected, washed three times with PBS, and resuspended to obtain the vaccine. The vaccine was administered to female BALB / c mice, followed by inoculation with 4T1 cells one week later. The mice's condition was observed, and changes in body weight and tumor volume were monitored. T cells in the tumor and spleen tissues were analyzed.

[0032] Therefore, the Ru(II) complex of the present invention can be used to prepare antitumor immunotherapies. Preferably, the tumor is a solid tumor, and more preferably, the tumor is 4T1 tumor cells. More preferably, the mice used in the experiment are female BALB / c mice.

[0033] The present invention will be further illustrated below through examples.

[0034] Example 1: Preparation of Ru(II) Complex 0.17 g (0.4 mmol) [(η 6 [C6H6)Ru(bpy)Cl]Cl and 68.4 μL (0.4 mmol) of 2-(p-tolyl)pyridine were added to 16 mL of N,N-dimethylformamide solution, refluxed at 140 °C for 18 h, and then 0.14 g (0.4 mmol) of ligand 2-(quinoxaloline-6-yl)-1- H -Imidazolo[4,5-f][1,10]phenanthroline continued to react for 1 h. After the reaction was completed, it was cooled to room temperature, and excess NH4PF6 solution was added. The mixture was stirred at room temperature until a precipitate formed. The precipitate was filtered, washed several times with pure water and anhydrous ethanol, and dried to obtain complex Ru1 0.26 g, with a yield of 70.3%.

[0035] Elemental analysis C 43 H 30 F6N9PRu (molecular weight 919.13), theoretical values: C, 56.21%; H, 3.29%; N, 13.72%; experimental values: C, 55.98%; H, 3.25%; N, 13.85%. ESI-MS: [(M-PF6)] + Theoretical value: m / z = 774.17, experimental value: m / z = 774.10.

[0036] Example 2: In vitro antitumor activity assay of Ru(II) complex. Antitumor activity assay (MTT method): The antitumor activity of ruthenium complexes was mainly determined by the MTT method: 8000 cells per well (100 μL) were seeded into 96-well plates and incubated in a CO2 incubator for 24 h. Then, 100 μL of different concentrations of the drug diluted with culture medium were added, and the plates were incubated for another 44 h. Finally, 10 μL of the drug diluted with culture medium was added to each well. Add L of MTT (dissolved in PBS), remove the culture medium after 4 hours, and add 100 μL of MTT solution to each well. In a dark place, mix L of DMSO with a shaker for about 10 minutes, and immediately measure the OD value at 490 nm using an ELISA reader.

[0037] The test results are shown in Table 1 below: Table 1 Test Results

[0038] a IC 50 This represents the concentration of the complex that inhibits cell growth by 50%. The experimental data are the average values ​​obtained from three parallel experiments.

[0039] The experimental results show that the complex Ru1 not only has good antitumor activity but also exhibits a certain degree of selectivity, particularly against the IC50 of MDA-MB-231 cells. 50 =2.34±0.02, IC50 for MCF-10A in normal human mammary epithelial cells 50 =16.83±0.02.

[0040] Western Blot Analysis: MDA-MB-231 cells were cultured in 100 mm culture dishes. When the cell density reached 70%, a specified concentration of the complex Ru1 was added. After incubation for 24 h, protein samples were collected. The prepared protein samples were denatured at 95°C, followed by SDS-PAGE gel electrophoresis to separate the desired proteins. The samples were then transferred to a membrane, blocked, and incubated with primary antibody GPX4 and secondary antibody for protein detection. GPX4 protein is one of the main indicators for detecting ferroptosis in cells. Figure 1 As shown, compared with the control group, Ru1 can reduce GPX4 protein expression, indicating that Ru1 can induce ferroptosis in MDA-MB-231 cells.

[0041] Detection of extracellular release of calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP) from cell surface exposure: (1) Detection of calreticulin (CRT): Cells were seeded at a density of 25,000 in 15 mm confocal dishes. After the cells adhered, different concentrations of drugs diluted with culture medium were added for 24 h. The cells were then washed twice with PBS solution, fixed with 4% paraformaldehyde solution for 30 minutes, washed three times with PBS solution, blocked with immunostaining blocking solution for 30 minutes, washed three times with PBS solution, incubated with primary antibody CRT, washed three times with immunostaining detergent, incubated with secondary antibody, washed three times with immunostaining detergent, stained with DAPI for 15 minutes, washed three times with immunostaining detergent, and then observed on the instrument.

[0042] (2) Detection of high-mobility group box 1 (HMGB1): Cells were seeded at a density of 8000 per well in a 96-well plate. After the cell density reached 70%, different concentrations of drug diluted with culture medium were added for 24 h. Cell supernatant was then collected. Different concentrations of standard were prepared according to the instructions of the HMGB1 ELISA kit. Then, 100 μL of sample and standard were added to each well and incubated at room temperature for 2 h. After washing the plate 5 times, 100 μL of horseradish peroxidase-conjugated HMGB1 antibody was added to each well and incubated at room temperature for 1 h. After washing the plate 5 times, 100 μL of chromogenic reagent TMB solution was added to each well and incubated at room temperature in the dark for 20 minutes. Finally, 50 μL of stop solution was added to each well and mixed. The value at A450 was detected using an ELISA reader.

[0043] (3) Detection of extracellular release of adenosine triphosphate (ATP): Cells were seeded at a density of 8000 cells in 96-well plates and cultured in a CO2 incubator for 24 h. Then, different concentrations of drugs diluted with culture medium were added for 24 h. Cells were collected and centrifuged at 1000 r / min for 5 min to obtain supernatant. ATP standard with concentration gradient was prepared using an ATP detection kit. Finally, the fluorescence of the samples was detected using a multifunctional microplate reader.

[0044] The final result is as follows Figure 2 As shown, after 24 h of treatment with different concentrations of Ru1, MDA-MB-231 cells showed increased CRT exposure on the cell surface and increased extracellular release of HMGB1 and ATP. This indicates that Ru1 can induce immunogenic cell death in MDA-MB-231 cells.

[0045] Example 3: In vivo antitumor immunomodulatory activity assay of Ru(II) (1) Tumor cell vaccine preparation: Tumor cells in the logarithmic growth phase were washed with PBS and then digested with trypsin to collect the cell suspension. The suspension was then diluted with 1×10⁻⁶ PBS. 6Cells were seeded at a density of [insert density here] in cell culture dishes and incubated overnight. Different drugs were administered to different groups: CDDP (cisplatin) group: cells were incubated with CDDP solution for 24 hours; drug group: cells were incubated with drug solution for 24 hours. After drug treatment, cells were collected, centrifuged, and the supernatant was discarded. Cells were washed with PBS and resuspended in PBS in each tube, thus obtaining the tumor cell vaccine. Among them, the Ru1 single-dose vaccine group (Ru1 1 [insert specific vaccine name here])... st One dose of vaccine (vaccine), Ru1 three-dose vaccine group (Ru1 3) rd (vacc) requires three doses of the vaccine.

[0046] (2) Mouse tumor vaccine injection: Thirty-six healthy 4-week-old female BALB / c mice were selected and divided into four groups (control group, cisplatin group, single-dose vaccine group, and triple-dose vaccine group), and were housed at a constant temperature of (22 ± 1) ℃ with sufficient water and food. The area from the left groin to the midpoint of the dorsal thigh was disinfected, and the mice were injected subcutaneously in this area using a syringe: Control group: 100 μL PBS was injected only; CDDP group: cell suspension treated with CDDP was injected; Vaccine group: cell suspension treated with the drug was injected.

[0047] (3) Mouse tumorigenesis experiment: Thirty-six healthy 4-week-old BALB / c mice were selected. One week after vaccination, the mice were disinfected from the midpoint between the left groin and the back of the thigh. 1×10⁶ mouse tumor cells in the logarithmic growth phase were injected into the mice using a syringe. 7 The mice in the four groups were then observed, and their body weight and tumor volume were monitored every two days. T cells in the tumor and spleen tissues were also analyzed.

[0048] The final result is as follows Figure 3 As shown, the body weight of mice in all groups showed a slow upward trend. The tumor volume in the Ru1 three-dose vaccine group was significantly inhibited, followed by the Ru1 one-dose vaccine group, while the tumor volume in the Control group and CDDP group grew rapidly. HE staining results revealed no obvious organic damage to the major organs in each group. This indicates that the Ru1 three-dose vaccine group has excellent anti-tumor immune activity.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A Ru(II) complex with antitumor immunomodulatory activity, characterized in that, The chemical formula of the complex is [Ru(CN)(NN)L](PF6), where NN = 2,2-bipyridine (bpy), CN = 2-(p-tolyl)pyridine, and the ligand L = 2-(6-quinoxalinyl)-1 H -imidazo[4,5-f][1,10]o-diazanthroline, the structural formula of the complex is shown in formula (I): Equation (I).

2. A method for preparing the Ru(II) complex as described in claim 1, characterized in that, Includes the following steps: [(η 6 The Ru(II) complex was refluxed with 2-(p-tolyl)pyridine at 140 °C for 18 h, and then the ligand 2-(quinoxalo-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline was added and reacted for 1 h. Finally, an excess of saturated NH4PF6 solution was added, the mixture was filtered, and the precipitated solid was collected to obtain the Ru(II) complex.

3. The preparation method according to claim 2, characterized in that, The solvent used in the preparation process is an N,N-dimethylformamide solution.

4. The use of the Ru(II) complex as described in claim 1 in the preparation of antitumor immunotherapeutic drugs.

5. The application according to claim 4, characterized in that, The drug targets human triple-negative breast cancer MDA-MB-231 cells and murine breast cancer 4T1 cells.

6. The application according to claim 4, characterized in that, The Ru(II) complex exerts its antitumor effect by inducing ferroptosis in tumor cells.

7. The application according to claim 4, characterized in that, The Ru(II) complex can induce immunogenic death in human triple-negative breast cancer MDA-MB-231 cells.

8. The application according to claim 4, characterized in that, The drug enables female BALB / c mice inoculated with 4T1 cells treated with Ru(II) complex to acquire immunity against 4T1 tumors.

9. The application according to claim 8, characterized in that, The method for acquiring immunity is to administer three doses of a 4T1 cell vaccine treated with Ru(II) complex to female BALB / c mice.

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