Preparation method and application of metal-organic nano complex for combined treatment of tumors

Abstract

The invention aims to provide a preparation method and application of a metal-organic nano complex for combined treatment of tumors, belongs to the technical field of antitumor drugs, and adopts a one-pot method to prepare copper-quercetin nanoparticles with uniform morphology. The obtained metal organic complex CQT nanoparticles can be used for X-ray induced sensitization radiotherapy / chemical power combined treatment to promote tumor cell apoptosis, meanwhile, due to introduction of copper ions, the steady state of the copper ions in tumor cells is broken, tumor cell copper death is induced, interaction is formed between copper death and the radiotherapy biological effect, and the tumor cell apoptosis is promoted. The synergistic tumor treatment effect is realized. The novel CQT nano complex for three-in-one treatment of sensitization radiotherapy / chemodynamic therapy / copper death has the function of obviously delaying the growth speed of tumors. The system can maintain good stability and biocompatibility, provides an innovative and efficient treatment potential scheme for intractable tumors clinically lacking specific targets, and improves the long-term prognosis of early metastatic tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and specifically relates to a preparation method and application of a metal-organic nano-complex for the combined treatment of tumors. Background Art

[0002] Worldwide, malignant tumors are the second leading cause of death, seriously threatening human life and health. Malignant tumors are characterized by rapid growth, invasion of surrounding tissues, distant metastasis, and a high recurrence rate. In most cases, relying solely on a single treatment method cannot completely eradicate malignant tumors. Currently, common combined treatments for clinical tumor treatment, such as surgery combined with radiotherapy, surgery combined with chemotherapy, neoadjuvant therapy, etc., still have many side effects. For example, radiotherapy can damage normal tissues in the irradiated area, and in severe cases, it can cause myelosuppression; systemic administration of chemotherapy drugs has poor targeting effects, may produce off-target effects, has the risk of developing drug resistance and resulting in poor treatment effects, and can suppress the body's immunity in severe cases.

[0003] In recent years, the application of nano-formulations in various tumors has been greatly developed. Relying on the enhanced permeability and retention (EPR) effect of nanomaterials, passive enrichment at the tumor site can be achieved, and tumor targeting can be comparably improved. Metal-organic complexes, as compounds formed by metal ions or atoms and organic ligands through coordination bonds, are simple to synthesize and can also be controlled at the nanoscale, and are often developed as new tumor treatment nano-platforms. Most metal-organic complexes not only have metal-related catalytic activities but also can maintain the chemical properties of organic complexes, and can achieve the combined treatment effect of tumors among different components within the same material.

[0004] Research shows that tumor radiotherapy can break the high-level redox homeostasis in tumor cells and the microenvironment, effectively reduce the level of reduced glutathione, and increase the hydrogen peroxide content at the tumor site, so as to provide more sufficient catalytic reaction substrates for metal-mediated chemodynamic therapy, and achieve more efficient tumor killing effects at the same radiation dose through radiotherapy combined with chemodynamic therapy. At the same time, the radiotherapy sensitization potential of quercetin, as a natural flavonoid compound, has received extensive attention, and the biological effects generated by tumor tissue radiotherapy are expanded through the inhibition of DNA damage repair and cell cycle arrest. In addition, the latest research shows that cuproptosis can overcome radiotherapy resistance and enhance radiotherapy sensitivity, improving the treatment effect of local tumors, providing a solid foundation for radiotherapy combined with cuproptosis.

[0005] CN116570760A discloses a multifunctional sustained-release dressing for promoting chronic wound healing, its method and application. The active ingredient in the sustained-release dressing is copper metal-organic framework material nanoparticles TAX@HKUST-1 with dihydroquercetin incorporated into the lumen, which has anti-inflammatory and antibacterial effects.

[0006] CN116806923A discloses a preparation method of a quercetin-Cu(II) complex feed additive. The complex can introduce copper ions into feed at a safe dose to promote animal growth and provide technical support for the replacement of antibiotics by flavonoids in aquaculture.

[0007] It can be seen that in recent years, reports on copper-quercetin metal-organic complexes have not involved the fields of tumor treatment and combined treatment. Similarly, there are few reports on an integrated diagnosis and treatment platform based on the combination of tumor radiotherapy, chemodynamic therapy, and cuproptosis.

[0008] Therefore, a tumor "triple-in-one" integrated treatment nanoplatform based on copper-quercetin metal-organic complexes can effectively solve the existing problems in tumor treatment and existing combined treatments. Summary of the Invention

[0009] Aiming at the above problems, the purpose of the present invention is to provide a preparation method and application of a metal-organic nanocomplex for the combined treatment of tumors. A copper-quercetin nanoparticle (Cu-Qu-TPGS, CQT) with uniform morphology is prepared by a one-pot method.

[0010] The present invention adopts the following technical solutions: A preparation method of a metal-organic nanocomplex for the combined treatment of tumors, comprising the following steps: The first step, preparation of copper-quercetin (Cu-Qu) nanoparticles: Weigh quercetin and copper chloride respectively, dissolve them in absolute ethanol respectively, drop the ethanol solution of copper chloride into the ethanol solution of quercetin, stir at 600 - 800 rpm for 5 min to obtain a mixed solution, drop the mixed solution into deionized water, stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, the obtained solution is washed with deionized water, centrifuged at 12000 rpm for 15 min, the precipitate is collected, and the operation is repeated three times. Finally, it is redispersed in deionized water to obtain a copper-quercetin (Cu-Qu) nanoparticle solution, which is stored at room temperature for standby; The second step, preparation of copper-quercetin-vitamin E polyethylene glycol succinate (Cu-Qu-TPGS) nanoparticles: Dissolve vitamin E polyethylene glycol succinate (TPGS) in absolute ethanol, drop it into the above copper-quercetin (Cu-Qu) nanoparticle solution, stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, the obtained solution is washed with deionized water, centrifuged at 12000 rpm for 15 min, the precipitate is collected, and the operation is repeated three times. Finally, it is redispersed in deionized water and stored at room temperature for standby.

[0011] Further, in the first step, the dosage of quercetin is 10 - 20 mg, the dosage of copper chloride is 10 - 60 mg, the dosage of absolute ethanol is 2 ml, and the dosage of deionized water is 25 ml.

[0012] Furthermore, the dosage of vitamin E polyethylene glycol succinate in the second step is 10 - 20 mg, and the dosage of absolute ethanol is 1 ml.

[0013] Application of a metal-organic nanocomplex for combined tumor treatment in the manufacture of and / or as an anti-tumor drug.

[0014] The beneficial effects of the present invention are as follows: The preparation method of the present invention is simple, and the synthesis conditions are green and pollution-free. The obtained metal-organic complex CQT nanoparticles can be used for X-ray-induced radiosensitization radiotherapy / chemodynamic combined therapy to promote tumor cell apoptosis. At the same time, the introduction of copper ions disrupts the copper ion homeostasis in tumor cells, inducing cuproptosis in tumor cells. An interaction is formed between cuproptosis and the biological effect of radiotherapy, achieving a synergistic effect in treating tumors.

[0015] The novel CQT nanocomplex with the "triple combination" of radiosensitization radiotherapy / chemodynamic therapy / cuproptosis of the present invention has the function of significantly delaying the tumor growth rate. This system can maintain good stability and biocompatibility, providing an innovative and efficient potential treatment plan for clinically refractory tumors lacking specific targets and improving the long-term prognosis of early metastatic tumors. Description of the Drawings

[0016] Figure 1 Transmission electron microscope image of the Cu-Qu-TPGS nanoparticles prepared in Example 1 of the present invention.

[0017] Figure 2 Ultraviolet-visible absorption spectrum of the Cu-Qu-TPGS nanoparticles prepared in Example 1 of the present invention.

[0018] Figure 3 XRD pattern of the Cu-Qu-TPGS nanoparticles prepared in Example 1 of the present invention.

[0019] Figure 4 Particle size distribution diagram of the aqueous solution system of the Cu-Qu-TPGS nanoparticles prepared in Example 1 of the present invention.

[0020] Figure 5 ESR spectrum for detecting hydroxyl radicals of the Cu-Qu-TPGS nanoparticles prepared in Example 1 of the present invention.

[0021] Figure 6 Effect of the Cu-Qu-TPGS nanoparticles prepared in Example 1 of the present invention on the viability of 4T1 cells.

[0022] Figure 7 Survival ratio of 4T1 cells in different experimental groups after receiving different doses of radiotherapy.

[0023] Figure 8 Apoptosis change diagram of 4T1 cells in different experimental groups.

[0024] Figure 9 Content change of copper ions in mitochondria after radiotherapy at different doses.

[0025] Figure 10 Expression of cuproptosis-related proteins in different experimental groups.

[0026] Figure 11 Enrichment of materials at the tumor site after intravenous injection of Cu-Qu-TPGS nanoparticles into the tail vein of tumor-bearing mice.

[0027] Figure 12 Tumor volume comparison after intravenous injection of Cu-Qu-TPGS nanoparticles combined with radiotherapy into the tail vein of tumor-bearing mice.

[0028] Figure 13 Tumor section staining in different experimental groups. Specific implementation mode

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1 Preparation of copper - quercetin - vitamin E polyethylene glycol succinate (Cu - Qu - TPGS) nanoparticles (1) Preparation of copper - quercetin (Cu - Qu) nanoparticles: Weigh 20 mg of quercetin and 20 mg of copper chloride respectively, dissolve them in 2 ml of absolute ethanol respectively, drop the ethanol solution of copper chloride into the ethanol solution of quercetin, and stir at 600 - 800 rpm for five minutes. Drop the above mixed solution into 25 ml of deionized water, and stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, the obtained solution is washed with deionized water, centrifuged at 12000 rpm for 15 min, the precipitate is collected, and the operation is repeated three times. Finally, it is redispersed in deionized water and stored at room temperature for standby.

[0031] (2)Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles: Dissolve 20 mg of Vitamin E Polyethylene Glycol Succinate (TPGS) in 1 ml of absolute ethanol, and drop it into the above - mentioned copper - quercetin (Cu - Qu) nanoparticles solution. Stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water for storage at room temperature for standby.

[0032] Example 2 Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles (1)Preparation of Copper - Quercetin (Cu - Qu) Nanoparticles: Weigh 20 mg of quercetin and 10 mg of copper chloride respectively, dissolve them in 2 ml of absolute ethanol respectively, drop the ethanol solution of copper chloride into the ethanol solution of quercetin, and stir at 600 - 800 rpm for five minutes. Drop the above - mentioned mixed solution into 25 ml of deionized water, and stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water for storage at room temperature for standby.

[0033] (2)Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles: Dissolve 20 mg of Vitamin E Polyethylene Glycol Succinate (TPGS) in 1 ml of absolute ethanol, and drop it into the above - mentioned copper - quercetin (Cu - Qu) nanoparticles solution. Stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water for storage at room temperature for standby.

[0034] Example 3 Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles (1)Preparation of Copper - Quercetin (Cu - Qu) Nanoparticles: Weigh 20 mg of quercetin and 60 mg of copper chloride respectively, dissolve them in 2 ml of absolute ethanol respectively, drop the ethanol solution of copper chloride into the ethanol solution of quercetin, and stir at 600 - 800 rpm for five minutes. Drop the above - mentioned mixed solution into 25 ml of deionized water, and stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water for storage at room temperature for standby.

[0035] (2) Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles: Dissolve 20 mg of Vitamin E Polyethylene Glycol Succinate (TPGS) in 1 ml of absolute ethanol, and drop it into the above - mentioned copper - quercetin (Cu - Qu) nanoparticle solution. Stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water and store it at room temperature for later use.

[0036] Example 4 Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles (1) Preparation of Copper - Quercetin (Cu - Qu) Nanoparticles: Weigh 20 mg of quercetin and 30 mg of copper chloride respectively, dissolve them in 2 ml of absolute ethanol respectively, drop the ethanol solution of copper chloride into the ethanol solution of quercetin, and stir at 600 - 800 rpm for five minutes. Drop the above - mentioned mixed solution into 25 ml of deionized water, and stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water and store it at room temperature for later use.

[0037] (2) Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles: Dissolve 20 mg of Vitamin E Polyethylene Glycol Succinate (TPGS) in 1 ml of absolute ethanol, and drop it into the above - mentioned copper - quercetin (Cu - Qu) nanoparticle solution. Stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water and store it at room temperature for later use.

[0038] Example 5 Preparation of Copper - Quercetin - Vitamin E Polyethylene Glycol Succinate (Cu - Qu - TPGS) Nanoparticles (1) Preparation of Copper - Quercetin (Cu - Qu) Nanoparticles: Weigh 10 mg of quercetin and 20 mg of copper chloride respectively, dissolve them in 2 ml of absolute ethanol respectively, drop the ethanol solution of copper chloride into the ethanol solution of quercetin, and stir at 600 - 800 rpm for five minutes. Drop the above - mentioned mixed solution into 25 ml of deionized water, and stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water and store it at room temperature for later use.

[0039] (2) Preparation of copper - quercetin - vitamin E polyethylene glycol succinate (Cu - Qu - TPGS) nanoparticles: Dissolve 10 mg of vitamin E polyethylene glycol succinate (TPGS) in 1 ml of absolute ethanol, and drop it into the above - mentioned copper - quercetin (Cu - Qu) nanoparticle solution. Stir at 600 - 800 rpm at room temperature for 24 h. After 24 h, wash the obtained solution with deionized water, centrifuge at 12000 rpm for 15 min, collect the precipitate, repeat three times, and finally redisperse it in deionized water and store it at room temperature for standby.

[0040] Example 6 Figure 1 It is the transmission electron microscope (TEM) image of the Cu - Qu - TPGS nanoparticles of the sample in Example 1. As shown in the image, the particle size of the material is between 30 - 40 nm.

[0041] Figure 2 It is the ultraviolet - visible absorption image of the Cu - Qu - TPGS nanoparticles of the sample in Example 1. As shown in the image, a new absorption peak appears at 420 nm for the material, indicating the successful synthesis of Cu - Qu - TPGS nanoparticles.

[0042] Figure 3 It is the XRD pattern of the Cu - Qu - TPGS nanoparticles of the sample in Example 1. As shown in the image, the peaks at 6.101, 10.548, 12.354, and 27.194 correspond to the crystal planes of quercetin respectively, indicating that the synthesized Cu - Qu - TPGS nanoparticles retain the crystal form of quercetin.

[0043] Figure 4 It is the particle size distribution diagram of the Cu - Qu - TPGS nanoparticles of the sample in Example 1. As shown in the image, the hydrated particle size of Cu - Qu - TPGS nanoparticles is 156.19 ± 0.2 nm, the PDI is 0.194 ± 0.005, and the potential of the aqueous solution system is measured. The result shows that its potential is - 26.32 ± 0.63 mV.

[0044] Figure 5 It is the ESR image of the Cu - Qu - TPGS nanoparticles of the sample in Example 1 using DMPO as a scavenger to detect hydroxyl radicals. As shown, Cu - Qu - TPGS nanoparticles have good ability to induce the generation of hydroxyl radicals.

[0045] Example 7 In this example, the effect of the Cu - Qu - TPGS nanoparticles of the sample in Example 1 on cell viability was measured as follows: (1)Cell culture Culture 4T1 cells in DMEM or 1640 medium containing 10% fetal bovine serum, and place them at 37℃, 5% CO2 in an incubator.

[0046] (2)Cell viability assay Seed 4T1 cells at a density of 3000 cells / well evenly into a 96-well plate. After adhering to the wall in the incubator for 24 h, add DMEM or 1640 medium (10% fetal bovine serum) containing Cu-Qu-TPGS nanoparticles at concentrations of 0, 1.95, 3.9, 7.8, 15.625, 31.25, 62.5, 125, 250, 500 μg / ml, and after treatment for 24 h, discard the supernatant medium, wash once with PBS, add 100 μl of basal medium containing 10% CCK-8 to each well, after incubation in the incubator for 1 h, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Subtract the absorbance of the blank PBS solution from the absorbance of each group, and divide the absorbance of each well by the absorbance of the control group as the cell viability. Set 6 parallel wells for each group.

[0047] Figure 6 To investigate the effect of the Cu-Qu-TPGS nanoparticles of Example 1 on the viability of 4T1 cells, as shown in the figure, at a concentration of 62.5 μg / ml, the cell viability was already below 50%, indicating that the Cu-Qu-TPGS nanoparticles have an obvious killing effect on 4T1 tumor cells, providing a basis for subsequent tumor treatment.

[0048] Example 8 In this example, we evaluated the effect of the radiotherapy (RT) alone group and the Cu-Qu-TPGS nanoparticles combined with radiotherapy (Cu-Qu-TPGS+RT) group prepared in Example 1 on the proliferation of 4T1 cells after radiotherapy at different doses through a colony formation assay in vitro. The method is as follows: Digest the 4T1 tumor cells in the logarithmic growth phase with trypsin, count them, and add them to a 6-well plate. The cell seeding density is 200 cells / well (0 Gy), 200 cells / well (2 Gy), 400 cells / well (4 Gy), 800 cells / well (6 Gy), 1600 cells / well (8 Gy). Shake the 6-well plate until the cells are fully dispersed, and then place the 6-well plate back into the incubator (37 °C, 5% CO 2 ) and culture overnight. Observe the cell adhesion situation with an inverted microscope the next day. After normal adhesion, add the nanomaterial to the Cu-Qu-TPGS+RT group to a final concentration of 200 μg / mL. After incubating the material for 4 h, irradiate the cells in each 6-well plate with different doses of X-rays from 2 to 8 Gy. After irradiation, continue to culture the 6-well plate in the cell incubator overnight. The next day, remove the old medium, wash once with sterile PBS stored at room temperature, and add fresh medium. Observe the cell culture status every 2 days. Change the culture medium every 4 days, continue culturing for 10 - 14 days. Wait until cell clusters visible to the naked eye grow, then stop culturing. Remove the old culture medium, wash the cells in the 6-well plate 3 times with sterile PBS placed at room temperature, fix the cells in the 6-well plate with 4% paraformaldehyde for 30 min, carefully remove the 4% paraformaldehyde, then wash 3 times with sterile PBS placed at room temperature. Then add 0.5 mL of 1% crystal violet staining solution to each well, after staining for 10 min, carefully remove the staining solution, wash the cells with pure water 2 - 4 times, air-dry the 6-well plate and then perform cell counting.

[0049] Figure 7 The survival ratios of 4T1 cells in different experimental groups after receiving different doses of radiotherapy. As shown in the image, the results indicate that the combination of Cu-Qu-TPGS nanoparticles and radiotherapy can reduce the mean lethal dose of 4T1 cells, significantly improve the sensitivity of 4T1 cells to radiotherapy. The radiotherapy sensitization ratio (SER10) of the combination of Cu-Qu-TPGS nanoparticles and radiotherapy is 1.234, showing a good radiotherapy sensitization effect. The above research shows that the Cu-Qu-TPGS nanoparticles prepared in Example 1 can significantly improve the sensitivity of 4T1 cells to X-rays.

[0050] Example 9 To further investigate whether the radiosensitization effect of Cu-Qu-TPGS nanoparticles on 4T1 cells is related to apoptosis, we detected the apoptosis changes in each group.

[0051] (1) Take 4T1 cells in the logarithmic growth phase cultured in a cell incubator (37 °C, 5% CO 2 )), after digesting into single cells, seed them in a 6-well plate at an appropriate density and continue culturing in the incubator.

[0052] (2) The next day, when the cells grow to an appropriate density, remove the old culture medium, add the nanomaterials diluted with DMEM or 1640 culture medium, adjust its final concentration to 200 μg / ml. Divide them into 4 groups, namely PBS group, X-ray group, Cu-Qu-TPGS (prepared in Example 1) group, and Cu-Qu-TPGS + X-ray group. After uptake for 4 h, wash 2 times with cold PBS, then add fresh DMEM or 1640 culture medium. The corresponding groups in the 6-well plate are irradiated with 6 Gy of X-rays and then placed back in the cell incubator to continue culturing for 24 h.

[0053] (3) Collect the old culture medium of the cells in each treatment group in the 6-well plate, wash 2 times with cold PBS, collect the PBS washing solution, digest the cells with trypsin for 3 - 4 min, terminate the digestion with the old culture medium and resuspend the cells, add the collected PBS washing solution, and centrifuge at 1000 rpm for 5 min using a 4 °C centrifuge.

[0054] (4) Collect the cell pellet, gently resuspend the cell pellet with 100 μL of Binding Buffer on ice, add 5 μL of Annexin V FITC and 10 μL of PI staining solution, and stain for 30 min in the dark.

[0055] (5) After staining, transfer the cells of each group to a flow tube and immediately perform flow cytometry analysis of cell apoptosis changes.

[0056] Figure 8 It is the apoptosis change diagram of 4T1 cells in different experimental groups. As shown in the image, when the cells were not treated with anything, the apoptosis ratio was only 4.36%. After the X-ray group treated the cells for 24 h, the apoptosis ratio was 11.69%, which was 2.68 times that of the PBS group. The apoptosis rate of the Cu-Qu-TPGS group was 40.5%. When the cells were incubated with the Cu-Qu-TPGS group first and then irradiated with 6 Gy of X-rays, the apoptosis ratio was as high as 60%, which was 5.13 times that of the X-ray group. Therefore, single X-ray treatment can promote cell apoptosis. Treating cells with Cu-Qu-TPGS alone will further induce obvious cell apoptosis. However, the combination of Cu-Qu-TPGS and X-ray treatment can greatly and significantly promote the apoptosis of tumor cells, which indicates that Cu-Qu-TPGS can enhance the sensitivity of 4T1 cells to radiation and can be further prepared into an adjuvant drug or reagent for RT treatment.

[0057] Example 10 In this example, we detected the change of copper ion content in mitochondria by radiotherapy with different doses. The method is as follows: (1) Take 4T1 cells in the logarithmic growth phase cultured in a cell incubator (37 °C, 5% CO 2 ) and digest them into single cells, then seed them in a confocal dish at an appropriate density and continue to culture in the incubator.

[0058] (2) The next day, when the cells grow to an appropriate density, remove the old culture medium, perform radiotherapy with different doses (0, 4, 6, 8 Gy), and then continue to culture in the incubator for 24 hours.

[0059] (3) After 24 hours, discard the supernatant culture medium, wash three times with cold PBS, dilute the copper ion probe (CS-1) to an appropriate concentration with basal medium, add it to the confocal dish, incubate at 37 °C for 30 min, and wash three times with cold PBS.

[0060] (4) Next, dilute the mitochondrial probe (Mito-Tracker) to an appropriate concentration with basal medium, add it to the confocal dish, incubate at 37 °C for 30 min, and wash three times with cold PBS.

[0061] (5) Finally, dilute Hoechst33342 to an appropriate concentration with the basal medium, add it to the confocal dish, incubate at 37 °C for 30 min, and wash three times with cold PBS.

[0062] (6) Add 1 ml of PBS to the confocal dish and immediately observe under confocal microscopy.

[0063] Figure 9 This shows the changes in the content of copper ions in mitochondria after radiotherapy at different doses. As shown in the images, the results indicate that the higher the radiation dose, the more copper ions are present in the mitochondria. Moreover, the more copper ions there are in the cells, the more prone they are to copper-induced cell death, laying the foundation for the subsequent induction of copper death by Cu-Qu-TPGS nanoparticles.

[0064] Example 11 In view of the above findings, we next investigated the effect of Cu-Qu-TPGS nanoparticles on inducing copper death, and the method is as follows: (1) Take 4T1 cells in the logarithmic growth phase cultured in a cell incubator (37 °C, 5% CO 2 )), digest them into single cells, and seed them in a 6-well plate at an appropriate density and continue culturing in the incubator.

[0065] (2) The next day, when the cells reach an appropriate density, remove the old medium, add the nanomaterials diluted with DMEM or 1640 medium, and adjust the final concentration to 200 μg / ml. Divide them into 4 groups, namely the PBS group, the X-ray group, the Cu-Qu-TPGS (prepared in Example 1) group, and the Cu-Qu-TPGS + X-ray group. After 4 h of uptake, wash twice with cold PBS, then add fresh DMEM or 1640 medium. The corresponding groups in the 6-well plate are irradiated with 6 Gy of X-rays and then returned to the cell incubator for continued culture for 24 h.

[0066] (3) Collect the old cell culture medium from each treatment group in the 6-well plate, wash twice with cold PBS, collect the PBS washings, digest the cells with trypsin for 3 - 4 min, terminate the digestion with the old culture medium and resuspend the cells, add the collected PBS washings, and centrifuge at 1000 rpm for 5 min using a 4 °C centrifuge.

[0067] (4) Collect the cell pellet, wash once with cold PBS, and centrifuge at 1000 rpm for 5 min using a 4 °C centrifuge.

[0068] (5) Gently resuspend the cell pellet with 100 μL of RIPA on ice and lyse on ice for 30 min.

[0069] (6) Centrifuge at 14000 rpm for 10 min using a 4 °C centrifuge. Collect the supernatant for BCA quantification.

[0070] (7)The expression of cuproptosis-related proteins was detected by Western Blot.

[0071] Figure 10 It was the expression of cuproptosis-related proteins in different experimental groups. As shown in the images, the results showed that the expressions of FDX1 and LIAS proteins decreased to varying degrees after treatment in different experimental groups, among which the Cu-Qu-TPGS + X-ray group had the most significant decrease in expression, and the oligomerization of DLAT protein further indicated the occurrence of cuproptosis.

[0072] Example 12 To observe the therapeutic effect of Cu-Qu-TPGS nanoparticles on 4T1 breast cancer, we first observed the enrichment of Cu-Qu-TPGS nanoparticles in tumors, and the method was as follows: (1)Cy5.5 was added dropwise to the aqueous solution of Cu-Qu-TPGS nanoparticles in proportion, stirred in the dark for 24 hours, washed once with deionized water after 24 hours, and finally the Cu-Qu-TPGS-Cy5.5 nanoparticles were redispersed in PBS.

[0073] (2)4T1 cells were inoculated into the right back of Balb / c mice at a density of 1×106 cells / mouse. After successful modeling, the concentration of Cu-Qu-TPGS-Cy5.5 nanoparticles was adjusted to 10 mg / ml with PBS, and 100 μl was injected into each mouse. Then, small animal in vivo imaging was performed at 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours to observe the enrichment of the nanomaterials in tumors.

[0074] (3)After 48 hours, the mice were euthanized, and the organs and tumors were taken for small animal in vivo imaging.

[0075] Figure 11 Regarding the enrichment of the materials at the tumor site after tail vein injection of Cu-Qu-TPGS nanoparticles into tumor-bearing mice, as shown in the images, the results showed that there was good enrichment in tumors within 48 hours after tail vein injection of Cu-Qu-TPGS nanoparticles, reaching the peak at 2 hours, and little enrichment of Cu-Qu-TPGS nanoparticles in organs other than the liver, indicating good biosafety of Cu-Qu-TPGS nanoparticles.

[0076] Example 13 Next, the therapeutic effect of Cu-Qu-TPGS nanoparticles on 4T1 cell-bearing mice was studied, and the method was as follows: (1)4T1 cells were at a density of 1×10 6Cells / mouse were inoculated on the right back of Balb / c mice. After successful modeling, the mice were randomly divided into 4 groups of 5 mice each, namely the PBS group, the X-ray group, the Cu-Qu-TPGS group, and the Cu-Qu-TPGS + X-ray group. The length (L) and width (W) of the tumor were measured with vernier calipers, and the tumor volume was estimated according to the formula V = 0.5 × L × W 2 When the subcutaneous tumors of Balb / c mice in each group grew to a volume of about 60 - 100 mm 3 treatment experiments were started. The concentration of Cu-Qu-TPGS was adjusted to 10 mg / mL with PBS, and 100 μL was injected into the tail vein of each mouse. The control group was injected with an equal volume of PBS. After 8 h, radiotherapy of 2 Gy was given. The drug was administered again every 3 days after the first treatment, and radiotherapy was given after 8 h, for a total of 4 treatments.

[0077] The growth of tumors in Balb / c mice was observed every other day after the start of treatment, the length and width of the tumors were measured, and the body weights of the mice were weighed. To ensure animal welfare, the experiment was terminated before the tumor volume exceeded 2000 mm 3 The tumor-bearing Balb / c mice were euthanized, and the organs and tumors of Balb / c mice in each treatment group were collected. Figure 12 The tumor growth curves of each experimental group during the treatment process are shown in the figure. As shown in the figure, Figure 12 the curves from top to bottom are the PBS group, the Cu-Qu-TPGS group, the X-ray group, and the Cu-Qu-TPGS + X-ray group. It can be seen from the figure that compared with the untreated control group, each treatment group had a therapeutic effect on the growth of tumors in 4T1 cell-bearing Balb / c mice. The inhibition rates of the tumor volume treated with Cu-Qu-TPGS alone were 13.79%, and the inhibition rates of the tumor volume treated with X-ray alone were 35.27%. The therapeutic effect of radiotherapy alone was more obvious than that of the Cu-Qu-TPGS group. The inhibition rates of the tumor volume treated with Cu-Qu-TPGS + X-ray were 66.5%. The tumor inhibition effect of the combined treatment was significantly stronger than that of radiotherapy alone. The above results indicate that Cu-Qu-TPGS nanoparticles can significantly enhance the inhibitory effect of radiotherapy on tumor growth.

[0078] Example 14 1. HE staining was performed as follows: On the 14th day after treatment, the mice were euthanized, and the tumors were removed. After fixation in 4% paraformaldehyde for 24 hours, they were dehydrated, embedded in paraffin, and subsequent paraffin sections were prepared. The paraffin sections were placed in xylene I for 10 minutes, xylene II for 10 minutes, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes to dewax to water. Then, they were washed with water for 1 minute, stained with hematoxylin for 2 minutes, washed with water for 1 minute, differentiated with differentiating solution for 7 seconds, washed with water for 1 minute, blued with bluing solution for 15 seconds, washed with water for 1 minute, placed in 95% ethanol for 20 seconds, stained with eosin for 12 seconds, placed in 95% ethanol for 1 minute, 100% ethanol for 1 minute, 100% ethanol for 1 minute, xylene for 1 minute, and xylene for 1 minute. The sections were taken out of the xylene, air-dried, mounted with neutral balsam, incubated in an oven at 37°C overnight, taken out and placed in a cool place to air-dry, and scanned with a high-throughput slide scanner.

[0079] 2. Tunel staining was performed as follows: On the 14th day after treatment, the mice were euthanized, and the tumors were removed. After fixation in 4% paraformaldehyde for 24 hours, they were dehydrated, embedded in paraffin, and subsequent paraffin sections were prepared. The paraffin sections were placed in xylene I for 10 minutes, xylene II for 10 minutes, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes to dewax to water. Subsequently, they were washed 3 times with PBS, stained using a Tunel cell apoptosis detection kit. After staining was completed, they were counterstained with hematoxylin for 15 seconds, washed with water for 1 minute, differentiated with differentiating solution for 7 seconds, washed with water for 1 minute, blued with bluing solution for 15 seconds, washed with water for 1 minute, placed in 95% ethanol for 20 seconds, stained with eosin for 12 seconds, placed in 95% ethanol for 1 minute, 100% ethanol for 1 minute, 100% ethanol for 1 minute, xylene for 1 minute, and xylene for 1 minute. The sections were taken out of the xylene, air-dried, mounted with neutral balsam, incubated in an oven at 37°C overnight, taken out and placed in a cool place to air-dry, and scanned with a high-throughput slide scanner.

[0080] 3. Ki67 staining was performed as follows: On the 14th day after treatment, the mice were euthanized, and the tumors were removed. After being fixed in 4% paraformaldehyde for 24 hours, they were dehydrated, paraffin-embedded, and subsequent paraffin sections were made. The paraffin sections were placed in xylene I for 10 minutes, xylene II for 10 minutes, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, and 85% ethanol for 5 minutes for dewaxing to water. Subsequently, they were washed 3 times with PBS, antigen retrieval was performed using a citrate retrieval solution. After the retrieval was completed, they were placed in a cool place to cool naturally to room temperature. Then, endogenous peroxidase was blocked with a 3% hydrogen peroxide methanol solution for 25 minutes, washed 3 times with PBS, blocked with normal goat serum for 30 minutes, the primary antibody diluted to an appropriate concentration was added dropwise, and incubated overnight at 4°C. The next day, the sections were taken out and rewarmed for 30 minutes, the corresponding rabbit / mouse secondary antibody was added, and incubated at 37°C for 60 minutes. Then, DAB color development was performed. After the staining was completed, counterstained with hematoxylin for 15 seconds, washed with water for 1 minute, differentiated with differentiation solution for 7 seconds, washed with water for 1 minute, blued with bluing solution for 15 seconds, washed with water for 1 minute, 95% ethanol for 20 seconds, eosin for 12 seconds, 95% ethanol for 1 minute, 100% ethanol for 1 minute, 100% ethanol for 1 minute, xylene for 1 minute, and xylene for 1 minute. The sections were taken out of the xylene, air-dried, sealed with neutral balsam, incubated overnight in an oven at 37°C, taken out and placed in a cool place to air-dry, and scanned with a high-throughput slide scanner.

[0081] 4. DLAT tissue immunofluorescence was performed as follows: On the 14th day after treatment, the mice were euthanized, and the tumors were removed. After being fixed in 4% paraformaldehyde for 24 hours, they were dehydrated, paraffin-embedded, and subsequent paraffin sections were made. The paraffin sections were placed in xylene I for 10 minutes, xylene II for 10 minutes, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, and 85% ethanol for 5 minutes for dewaxing to water. Subsequently, they were washed 3 times with PBS, antigen retrieval was performed using a citrate retrieval solution. After the retrieval was completed, they were placed in a cool place to cool naturally to room temperature. Then, endogenous peroxidase was blocked with a 3% hydrogen peroxide methanol solution for 25 minutes, washed 3 times with PBS, blocked with normal goat serum for 30 minutes, the primary antibody diluted to an appropriate concentration was added dropwise, and incubated overnight at 4°C. The next day, the sections were taken out and rewarmed for 30 minutes, the corresponding rabbit / mouse fluorescent secondary antibody was added, and incubated at 37°C for 60 minutes. Then, DAPI diluted to an appropriate concentration was added dropwise, incubated at room temperature for 10 minutes, sealed with an anti-fluorescence quenching mounting medium, and photographed using a confocal microscope.

[0082] Figure 13 Staining for tumor sections, as shown in the image, Figure 13From top to bottom are HE staining, Tunel staining, Ki67 staining and DLAT staining. The results show that the Cu-Qu-TPGS+X-ray group has the most significant cell damage as seen from HE staining. Tunel staining indicates that apoptosis occurred to varying degrees in each experimental group, and the Cu-Qu-TPGS+X-ray group had the highest apoptosis ratio. Ki67 staining also shows that the Cu-Qu-TPGS+X-ray group has the highest degree of inhibiting cell proliferation. Finally, DLAT tissue immunofluorescence indicates that the Cu-Qu-TPGS nanoparticles did induce the occurrence of cuproptosis.

[0083] The above only elaborates on the preferred embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention, and all such changes should be included within the protection scope of the present invention.

Claims

1. A method for preparing a metal-organic nanocomplex for combined tumor treatment, characterized in that: The steps include: The first step is to prepare copper-quercetin nanoparticles: quercetin and copper chloride are weighed separately, and they are dissolved in anhydrous ethanol respectively, and the ethanol solution of copper chloride is added dropwise to the ethanol solution of quercetin, and stirred at 600-800rpm for 5min to obtain a mixed solution, and the mixed solution is added dropwise to deionized water, and stirred at 600-800rpm for 24h at room temperature. After 24h, the obtained solution is washed with deionized water, centrifuged at 12000rpm for 15min, and the precipitate is collected, and the process is repeated three times, and finally, it is redispersed in deionized water to obtain a copper-quercetin nanoparticle solution, which is stored at room temperature for later use; The second step is the preparation of copper-quercetin-vitamin E polyethylene glycol succinate nanoparticles: vitamin E polyethylene glycol succinate is dissolved in anhydrous ethanol, added dropwise to the above copper-quercetin nanoparticle solution, stirred at 600-800 rpm for 24 hours at room temperature, washed with deionized water after 24 hours, centrifuged at 12000 rpm for 15 minutes, collected the precipitate, repeated three times, and finally redispersed in deionized water and stored at room temperature for later use.

2. The method for preparing a metal-organic nanocomplex for combined tumor treatment according to claim 1, characterized in that: In the first step, the amount of quercetin is 10-20 mg, the amount of copper chloride is 10-60 mg, the amount of anhydrous ethanol is 2 ml, and the amount of deionized water is 25 ml.

3. The method for preparing a metal-organic nanocomplex for combined tumor treatment according to claim 1, characterized in that: In the second step, the dosage of the vitamin E polyethylene glycol succinate is 10-20 mg, and the dosage of the anhydrous ethanol is 1 ml.

4. Use of the metal-organic nanocomplex for combined tumor treatment prepared by the preparation method as claimed in claim 1 as and / or in the preparation of anti-tumor drugs.

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