A kind of nanoparticle for radiotherapy-copper death combined treatment breast cancer and its preparation method and application

Abstract

The application provides a kind of nanoparticle for radiotherapy-copper death combined treatment breast cancer and its preparation method and application, it is suitable for the preparation field of nanoparticle, wherein the nanoparticle includes the following raw materials: 10mg precipitate A, 3~8mg CuCl2, 10mg DA.HCl, 2~6mg BSO, 100~250ul Tris-HCl buffer solution and 30~40mg BSA;The precipitate A includes the following raw materials: the mass ratio of hafnium tetrachloride and polyvinylpyrrolidone is 8:5;With can be through exogenous delivery copper ion, make intracellular copper ion accumulation, induce copper death effect, combined radiotherapy and BSO, produce strong copper death, enhance the beneficial effect of tumor cell radiation absorption and antitumor immune effect, can effectively reverse radioresistance and tumor immunosuppression microenvironment.

Description

Technical Field

[0001] This application relates to the field of nanoparticle preparation, specifically to a nanoparticle for radiotherapy-copper death combined therapy for breast cancer, its preparation method, and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women. Statistics show that in 2020, a total of 2.26 million new cases of breast cancer were reported globally, and 685,000 women died from it. In my country, the incidence of new breast cancer cases is increasing significantly; in 2020 alone, 82,100 women died from breast cancer, and this number continues to rise. Globally, the incidence rate of breast cancer in women was 11.6% in 2022, and the mortality rate was 6.9%. This indicates that breast cancer has seriously threatened women's health, causing significant harm to their mental health, families, and society, and impacting patients' normal work and lives. Currently, breast cancer treatment methods mainly include surgical treatment, radiotherapy, chemotherapy, hormone therapy, immunotherapy, targeted therapy, and palliative care.

[0003] Radiotherapy (RT) is a form of treatment that exposes tumor cells to ionizing radiation sources such as X-rays, electrons, or protons. It is one of the most powerful treatment methods for breast cancer. The principle behind RT is to directly damage the DNA of tumor cells (DNA is the main target determining radiobiological effects) using high-energy ionizing radiation, or indirectly react with water molecules to generate reactive oxygen species (ROS), such as superoxide anion radicals (O2). - Hydrogen radicals (H), hydroxyl radicals (OH), H₂O₂, and singlet oxygen 1 Radiation irradiation (RT) uses oxygen (O2, etc.) to damage DNA or other cellular components, thereby inducing tumor cell apoptosis and necrosis. RT increases ROS levels in the endoplasmic reticulum, leading to the exposure of calreticulin (CRT), which in turn causes immunogenic cell death (ICD), releasing specific antigens. Antigen-presenting cells (dendritic cells, DCs) stimulate the recruitment and differentiation of T lymphocytes (CTLs), promoting T cell recognition and killing of tumor cells. However, high doses of RT can damage normal tissues, while lower doses are less effective at inhibiting tumor growth. Furthermore, RT can induce radioresistance in tumor cells, resulting in poor radiotherapy efficacy and limiting its use.

[0004] In recent years, the application of nanoparticles in various tumors has seen significant development. Therefore, it is possible to develop "green," multifunctional nanoparticles that can be combined with radiotherapy (RT) to improve therapeutic efficacy. Summary of the Invention

[0005] To address one of the aforementioned technical deficiencies, this application provides nanoparticles for radiotherapy-copper death combined therapy of breast cancer, their preparation method, and their applications.

[0006] According to the first aspect of this application, nanoparticles for radiotherapy-copper death combined therapy for breast cancer are provided, comprising the following raw materials: 10 mg precipitate A, 3-8 mg CuCl2, 10 mg DA.HCl, 2-6 mg BSO, 100-250 μl Tris-HCl buffer, and 30-40 mg BSA; wherein precipitate A comprises the following raw materials: hafnium tetrachloride and polyvinylpyrrolidone in a mass ratio of 8:5.

[0007] Preferably, the concentration of the Tris-HCl buffer is 1M and the pH value of the Tris-HCl buffer is 8.8.

[0008] Preferably, the molecular weight of the polyvinylpyrrolidone is 10000 kDa.

[0009] According to a second aspect of this application, a method for preparing nanoparticles for radiotherapy-copper death combined therapy of breast cancer is provided, comprising:

[0010] Wash precipitate A three times with deionized water, and dissolve 10 mg of precipitate A in 1-10 ml of deionized water to obtain solution B;

[0011] Dissolve 3-8 mg CuCl2, 10 mg DA.HCl and 2-6 mg BSO in 0.1-1 ml of deionized water to prepare CuCl2 solution, DA.HCl solution and BSO solution respectively;

[0012] Solution B was sonicated, and then CuCl2 solution, DA.HCl solution and BSO solution were added to solution B. After stirring evenly, 100~250μl of Tris-HCl buffer was added to obtain a mixed solution.

[0013] The mixed solution was ultrasonically treated for 15-40 min, then stirred for the first time, and the precipitate C was collected after the second centrifugation. After washing the precipitate C, HfO2-Cu-BSO-PDA was obtained.

[0014] Dissolve 30-40 mg of BSA in 1 ml of deionized water to prepare a BSA solution.

[0015] HfO2-Cu-BSO-PDA was dissolved in 1~10 ml of deionized water, then BSA solution was added, and the mixture was stirred a second time. After a third centrifugation, the precipitate was collected to obtain HfO2-Cu-BSO-PDA-BSA nanoparticles.

[0016] HfO2-Cu-BSO-PDA-BSA nanoparticles were dissolved in 5 ml of physiological saline for preservation at a temperature of 4 °C.

[0017] More preferably, in the HfO2-Cu-BSO-PDA-BSA nanoparticles, the valence states of Hf are +2 and +3, the valence state of copper is +2, the valence state of sulfur is +4, the valence state of nitrogen is +1 and -3, and the valence state of phosphorus is +5.

[0018] Preferably, the HfO2-Cu-BSO-PDA-BSA nanoparticles have a particle size of 300~340nm, and the hydrated particle size of the HfO2-Cu-BSO-PDA-BSA nanoparticles after being dissolved in 5ml of physiological saline is 100~350nm.

[0019] Preferably, the method for preparing precipitate A includes: dissolving 160 mg hafnium tetrachloride and 100 mg polyvinylpyrrolidone in 10 ml of deionized water, then placing them in a hydrothermal reactor for hydrothermal treatment, and collecting precipitate A after a first centrifugation treatment.

[0020] More preferably, the centrifugation speed of the first centrifugation treatment, the second centrifugation treatment, and the third centrifugation treatment are all 9000~12000 rpm, the time is all 15~30 min, and the centrifugation temperature is all 25℃.

[0021] Preferably, the stirring speed of the first stirring and the second stirring are both 700~1000 rpm, the stirring time is 24h, and the stirring temperature is 4~25℃.

[0022] According to a third aspect of this application, the application of nanoparticles prepared according to the preparation method of nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to any one of the above claims in a drug for treating breast cancer is provided.

[0023] The beneficial effects of this application are as follows:

[0024] The nanoparticles provided in this application are HfO2-Cu-BSO-PDA-BSA nanoparticles, in which the radiosensitizer hafnium oxide is used as a carrier to load copper ions and BSO. The surface is modified by polydopamine (PDA) polymerization coating, and further modified with BSA to improve the dispersibility of the nanoparticles. A Tris-HCl buffer solution is used to maintain pH balance by binding Tris and HCl to form a buffer system. In the treatment of breast cancer, the HfO2-Cu-BSO-PDA-BSA nanoparticles can exogenously deliver copper ions, causing intracellular copper ion accumulation and inducing cerebral apoptosis. Combined with radiotherapy and BSO, this produces a powerful cerebral apoptosis effect, enhances radiation absorption by tumor cells, and exerts anti-tumor immune effects, effectively reversing radioresistance and the tumor immunosuppressive microenvironment.

[0025] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of what is pointed out in the written description and the accompanying drawings. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0027] Figure 1 An elemental scan of nanoparticles for radiotherapy-copper death combined therapy for breast cancer, provided in Embodiment 1 of this application;

[0028] Figure 2 XRD patterns of nanoparticles provided in Embodiment 1 and Comparative Example 1 of this application;

[0029] Figure 3 TEM image of nanoparticles for radiotherapy-copper death combined therapy for breast cancer provided in Embodiment 1 of this application;

[0030] Figure 4 A particle size diagram of nanoparticles for radiotherapy-copper death combined therapy for breast cancer provided in Embodiment 1 of this application;

[0031] Figure 5 Potential diagram of nanoparticles for radiotherapy-copper death combined therapy for breast cancer provided in Embodiment 1 of this application;

[0032] Figure 6 The image shows the effect of nanoparticles used in radiotherapy-copper death combined therapy for breast cancer on the viability of 4T1 tumor cells, as provided in Embodiment 1 of this application.

[0033] Figure 7 The figure shows the effect of nanoparticles provided in Example 1 and Comparative Example 1 of this application on the expression of copper death-related proteins in 4T1 tumor cells.

[0034] Figure 8 The survival rate of 4T1 tumor cells after receiving different doses of radiotherapy using the nanoparticles provided in Example 1 of this application;

[0035] Figure 9 Figures showing changes in apoptosis in 4T1 tumor cells after treatment in each group provided in this application;

[0036] Figure 10 The following are images of the hemolysis results after treatment provided in this application;

[0037] Figure 11A comparison of tumor volumes in tumor-bearing mice after intratumoral administration of nanoparticles combined with radiotherapy.

[0038] Figure 12 CD8 in mouse spleen + Statistical chart of T lymphocyte infiltration;

[0039] Figure 13 CD8 in mouse tumors + Statistical chart of T lymphocyte infiltration results. Detailed Implementation

[0040] To make the technical solutions and advantages of the embodiments of this application clearer, the exemplary embodiments of this application will be described in further detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not an exhaustive list of all embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0041] Unless otherwise specified, all reagents used in this application are commercially available. Mouse breast cancer cells (4T1 tumor cells, Wuhan Sewell Life Science & Technology Co., Ltd.); albino laboratory mice (Balb / c mice, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0042] To address the aforementioned issues, this application provides nanoparticles for combined radiotherapy-copper death therapy of breast cancer, comprising the following raw materials: 10 mg precipitate A, 3-8 mg copper chloride (CuCl2), 10 mg dopamine hydrochloride (DA.HCl), 2-6 mg sulfoxide butyrate (BSO), 100-250 μl tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl buffer), and 30-40 mg bovine serum albumin (BSA).

[0043] The concentration of the Tris-HCl buffer is 1M, and the pH value of the Tris-HCl buffer is 8.8;

[0044] The precipitate A comprises the following raw materials: hafnium tetrachloride and polyvinylpyrrolidone (PVP) in a mass ratio of 8:5, wherein the molecular weight of the polyvinylpyrrolidone is 10000kD.

[0045] The nanoparticles provided in this application are HfO2-Cu-BSO-PDA-BSA nanoparticles, in which the radiosensitizer hafnium oxide is used as a carrier to load copper ions and BSO. The surface is modified by polydopamine (PDA) polymerization coating, and further modified with BSA to improve the dispersibility of the nanoparticles. A Tris-HCl buffer solution is used to maintain pH balance by binding Tris and HCl to form a buffer system. In the treatment of breast cancer, the HfO2-Cu-BSO-PDA-BSA nanoparticles can exogenously deliver copper ions, causing intracellular copper ion accumulation and inducing cerebral apoptosis. Combined with radiotherapy and BSO, this produces a powerful cerebral apoptosis effect, enhances radiation absorption by tumor cells, and exerts anti-tumor immune effects, effectively reversing radioresistance and the tumor immunosuppressive microenvironment.

[0046] Hafnium oxide, as a radiosensitizer, combined with radioactive irradiation (RT), can improve the killing efficiency of ionizing radiation. Through sustained or even controlled release, hafnium oxide can improve blood circulation time and increase accumulation at the tumor site. This increases the sensitivity of tumor cells to radiation-induced killing while reducing damage to the structure and function of surrounding healthy tissue cells. Radiosensitizers can also act as inhibitors of intracellular thiol groups, cytotoxic substances formed by radiolysis, inhibitors of biomolecular repair, thymine analogs that can bind to DNA, and electrophilic oxygen mimics. Metal-based nanoparticles, due to their excellent photoelectric and Compton effects, can effectively absorb energy from X-rays or gamma rays, directly transferring it to tumor cells or enhancing the radioactive decomposition of water to generate reactive oxygen species (ROS).

[0047] Copper death is a copper-dependent immunogenic cell death (ICD) that involves reversing the immunosuppressive tumor microenvironment and enhancing anti-tumor immunity through the release of damage-associated molecular patterns (DAMPs) and tumor-associated antigens. Mechanistically, firstly, ferroreduction protein 1 (FDX1) and lipoic acid synthase (LIAS), as key regulators of protein lipoylation, participate in the regulation of protein lipoylation, including dihydrolipoamide S-acetyltransferase (DLAT). Lipoylated proteins directly bind to copper ions to form oligomers. Secondly, FDX1... 2+ Reduced to the more cytotoxic Cu + Excessive copper leads to instability in Fe-S cluster proteins, ultimately resulting in increased protein toxicity stress and copper death. Furthermore, excess copper causes oxidative damage through free radical-mediated pathways. Copper dysregulation affects the antioxidant defense system, leading to increased ROS levels, depletion of deoxyribonucleotides, inability to synthesize DNA, and cell cycle arrest, ultimately inducing cell death. In tumor cells, Cu... + Subject to glutathione (GSH, a natural copper chelator), Cu 2+Restrictions on transport and metabolic proteins are major obstacles to inducing effective copper death. Therefore, further enhancing intracellular copper uptake in tumor cells via carriers is crucial. 2+ The accumulation of copper-dependent glutathione in the tumor microenvironment, while simultaneously reducing glutathione levels, is essential for enhancing copper-based antitumor therapy. Furthermore, part of the effectiveness of radiotherapy is due to the induction of copper death in cells, and radiotherapy-resistant cells exhibit low copper death induction, indicating that copper death can overcome radioresistance and enhance radiosensitivity, improving the therapeutic effect on local tumors and providing a solid foundation for combining radiotherapy with copper death.

[0048] BSO is a cell-permeable, potent, and irreversible G-glutamate-cysteine ​​synthase inhibitor that can reduce intracellular glutathione levels, inhibit intracellular GSH function, and promote the accumulation of copper ions in cells. Therefore, BSO can be used to reduce GSH in tumor cells and enhance copper death.

[0049] In summary, this application achieves a highly efficient combined therapy of copper death and radiotherapy by scientifically designing metal nanoparticles based on copper death. This approach can address issues such as tumor radioresistance, low efficacy of single RT therapy, and the characteristics of "cold" breast cancer tumors, providing a new combined therapy strategy for breast cancer treatment. It has scientific value and social significance.

[0050] Example 1

[0051] This application also provides a method for preparing nanoparticles for radiotherapy-copper death combined therapy for breast cancer, comprising:

[0052] 160 mg hafnium tetrachloride and 100 mg polyvinylpyrrolidone were dissolved in 10 ml of deionized water and then placed in a hydrothermal reactor for hydrothermal treatment. After a first centrifugation treatment, precipitate A (HfO2-PVP) was collected. The hydrothermal treatment temperature was 160 °C and the time was 10 h.

[0053] The precipitate A was washed three times with deionized water. 10 mg of precipitate A was dissolved in 9.4 ml of deionized water to obtain solution B. Solution B was stored at 4°C for later use.

[0054] 6 mg CuCl2, 10 mg DA.HCl and 3 mg BSO were dissolved in 200 μl of deionized water to prepare CuCl2 solution, DA.HCl solution and BSO solution respectively;

[0055] Solution B was sonicated, and then CuCl2 solution, DA.HCl solution and BSO solution were added to solution B. After stirring evenly, 200 μl of Tris-HCl buffer was added to obtain a mixed solution.

[0056] The mixed solution was sonicated for 30 min, then stirred and centrifuged for the first time to collect precipitate C. After washing precipitate C, HfO2-Cu-BSO-PDA was obtained.

[0057] A BSA solution was prepared by dissolving 40 mg of BSA in 1 ml of deionized water.

[0058] HfO2-Cu-BSO-PDA was dissolved in 9 ml of deionized water, then BSA solution was added. After a second stirring and a third centrifugation, the precipitate was collected to obtain HfO2-Cu-BSO-PDA-BSA nanoparticles. In the HfO2-Cu-BSO-PDA-BSA nanoparticles, hafnium has a valence state of +2 and +3, copper has a valence state of +2, sulfur has a valence state of +4, nitrogen has a valence state of +1 and -3, and phosphorus has a valence state of +5.

[0059] HfO2-Cu-BSO-PDA-BSA nanoparticles were dissolved in 5 ml of physiological saline for preservation at a temperature of 4 °C.

[0060] The preparation method provided in this application is simple and easy to operate. The prepared nanoparticles can be directly stored and used. The reaction conditions are mild, easy to implement, and exhibit significant anti-tumor effects. The prepared HfO2-Cu-BSO-PDA-BSA nanoparticles possess good stability, dispersibility, and biocompatibility, and show significant killing power against tumor cells, promoting tumor cell apoptosis. Using the radiosensitizer hafnium oxide as a carrier, copper ions and BSO are loaded, and the surface is modified by polydopamine (PDA) polymerization coating, followed by further modification with BSA to improve the dispersibility of the nanoparticles. The Tris-HCl buffer solution maintains pH balance by forming a buffer system through the binding of Tris and HCl.

[0061] Furthermore, the centrifugation speed for the first, second, and third centrifugation processes was 9500 rpm, the time was 20 min, and the centrifugation temperature was 25℃.

[0062] Furthermore, the first and second stirring speeds were both 1000 rpm, the stirring time was 24 hours, and the stirring temperature was 25℃.

[0063] In this application, "rpm" means "revolutions per minute".

[0064] This application also provides the application of nanoparticles prepared according to the above-described method for preparing nanoparticles for radiotherapy-copper death combined therapy of breast cancer in the treatment of breast cancer.

[0065] Comparative Example 1

[0066] Comparative Example 1 of this application provides an HfO2-Cu-PDA-BSA nanomaterial, which differs from Example 1 in that: no butyrosine sulfoxide solution was prepared or added, while the remaining steps are the same as in Example 1.

[0067] To further demonstrate the beneficial effects of the HfO2-Cu-BSO-PDA-BSA nanoparticles prepared in Example 1 of this application, the nanoparticles prepared in Example 1 and Comparative Example 1 were measured, as shown below:

[0068] 1. Performance determination of HfO2-Cu-BSO-PDA-BSA nanoparticles:

[0069] 1) Elemental scanning of HfO2-Cu-BSO-PDA-BSA nanoparticles, such as... Figure 1 As shown, the elemental scan results of HfO2-Cu-BSO-PDA-BSA nanoparticles include copper, hafnium, sulfur, nitrogen, and phosphorus, indicating that HfO2-Cu-BSO-PDA-BSA nanoparticles were successfully prepared.

[0070] 2) Draw the XRD patterns of HfO2-PVP, HfO2-Cu-PDA-BSA, and HfO2-Cu-BSO-PDA-BSA nanoparticles respectively, as follows: Figure 2 As shown, the peaks of these three nanoparticles correspond to the standard card of hafnium oxide lattice, therefore the hafnium oxide crystal form is retained in the HfO2-PVP, HfO2-Cu-PDA-BSA, and HfO2-Cu-BSO-PDA-BSA nanoparticles.

[0071] 3) The morphology and particle size of HfO2-Cu-BSO-PDA-BSA nanoparticles were observed using transmission electron microscopy, and a particle size diagram was plotted, such as... Figure 3 and Figure 4 As shown, the nanomaterial consists of spindle-shaped hafnium oxide and PDA covering the surface of hafnium oxide. After dissolving in physiological saline, the hydrated particle size is 269.24±0.2nm.

[0072] 4) Analyze the potential of the aqueous solution system of HfO2-Cu-BSO-PDA-BSA nanoparticles dissolved in physiological saline, such as... Figure 5 As shown, the potential is -7.64 ± 0.6 mV. This indicates that the HfO2-Cu-BSO-PDA-BSA nanoparticles possess good stability and dispersibility.

[0073] In this application, 4T1 tumor cells (mouse breast cancer cells) were cultured in DMEM or 1640 medium containing 10% fetal bovine serum and various amino acids and glucose, and placed in a cell culture incubator at 37°C and 5% CO2. This was to further determine the effect of the HfO2-Cu-BSO-PDA-BSA nanoparticles prepared in Example 1 on breast cancer cells.

[0074] 2. The effect of HfO2-Cu-BSO-PDA-BSA nanoparticles on the cell viability of 4T1 tumor cells was determined using the following methods:

[0075] 4T1 tumor cells were seeded uniformly at a density of 5000 cells / well in 96-well plates and divided into 10 groups. After 12 h of cell culture, DMEM or 1640 medium (10% fetal bovine serum) containing HfO2-Cu-BSO-PDA-BSA nanoparticles was added to each of the 10 groups. The concentration of HfO2-Cu-BSO-PDA-BSA nanoparticles was quantified according to the hafnium oxide content, with concentrations of 0, 3.9, 7.8, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg / ml, respectively. After 24 h of treatment, the supernatant medium was discarded, and the cells were washed once with phosphate-buffered saline (PBS) solution. Then, 100 μl of basal medium containing 10% cell counting reagent (CCK-8) was added to each well.

[0076] In this study, well plates with a concentration of 0 HfO2-Cu-BSO-PDA-BSA nanoparticles were used as the control group, while well plates with other concentrations of HfO2-Cu-BSO-PDA-BSA nanoparticles were used as the experimental group, with 6 parallel wells in each group.

[0077] After 1 hour of incubation, the absorbance values ​​of the control group and the experimental group at 450 nm were measured using an ELISA reader. The actual absorbance value of each group was obtained by subtracting the absorbance value of the blank PBS solution from the absorbance value of each group. The cell viability of the corresponding experimental group was obtained by dividing the actual absorbance value of the experimental group by the actual absorbance value of the control group.

[0078] like Figure 6 As shown, when the concentration of HfO2-Cu-BSO-PDA-BSA nanoparticles was 250 μg / ml, the viability of 4T1 tumor cells was already below 50%, indicating that HfO2-Cu-BSO-PDA-BSA nanoparticles have a significant killing effect on 4T1 tumor cells, providing a foundation for subsequent tumor treatment.

[0079] 3. The effect of HfO2-Cu-BSO-PDA-BSA nanoparticles on inducing copper death was investigated:

[0080] 1) Logarithmic growth phase 4T1 tumor cells cultured in a cell culture incubator (37℃, 5% CO2) were digested into single cells and divided into 4 groups: Control group, HfO2-PVP group, HfO2-Cu-PDA-BSA group and HfO2-Cu-BSO-PDA-BSA group. The cells were then seeded into 6-well plates at a density of 200,000 cells / well and cultured in an incubator.

[0081] 2) Remove the old culture medium after 12 hours;

[0082] HfO2-PVP, HfO2-Cu-PDA-BSA, and HfO2-Cu-BSO-PDA-BSA nanoparticles diluted with DMEM or 1640 medium were added to the HfO2-PVP group, HfO2-Cu-PDA-BSA group, and HfO2-Cu-BSO-PDA-BSA group, respectively. The concentration of nanoparticles in each group was quantified according to the hafnium oxide content, and the concentration was 200 μg / ml. The control group was not treated with any medium.

[0083] Continue culturing for 24 hours.

[0084] 3) After 24 hours, collect the culture medium from each group, wash three times with PBS solution, collect the PBS wash solution, digest the cells with trypsin for 3-4 minutes, stop the digestion with complete culture medium and resuspend the cells, add the collected PBS wash solution, and centrifuge at 4°C and 1000 rpm for 5 minutes.

[0085] 4) Collect the cell pellet, wash once with cold PBS, and centrifuge at 4°C and 1000 rpm for 5 min.

[0086] 5) Collect the cell pellet and gently resuspend it on ice with 100 μl of RIPA lysis buffer. Lyse on ice for 30 min. The RIPA lysis buffer is a radioimmunoprecipitation lysis buffer.

[0087] 6) Centrifuge at 4°C and 14,000 rpm for 10 min, collect the supernatant and quantify the water-soluble complex (BCA) for protein quantification.

[0088] 7) The expression of copper death-related proteins was detected by Western blotting.

[0089] The effects of copper death-related protein expression on the results are as follows: Figure 7As shown, both the HfO2-Cu-PDA-BSA and HfO2-Cu-BSO-PDA-BSA groups showed varying degrees of decreased expression of FDX1 and LIAS proteins after treatment. The HfO2-Cu-BSO-PDA-BSA group showed the most significant decrease in expression, and the HfO2-Cu-BSO-PDA-BSA group also showed the highest oligomerization of DLAT protein and the greatest decrease in GPX4, further indicating that BSO can enhance copper death.

[0090] 4. The effects of radiotherapy alone (RT) and combined radiotherapy with HfO2-Cu-BSO-PDA-BSA nanoparticles (HfO2-Cu-BSO-PDA-BSA+RT) on the proliferation of 4T1 tumor cells after different doses of radiotherapy were evaluated using a plate clone assay. The methods are as follows:

[0091] 4T1 tumor cells in logarithmic growth phase were digested with trypsin, counted, and then added to two 6-well plates. The cell seeding density in each 6-well plate was 400 cells / well, 400 cells / well, 800 cells / well, 1600 cells / well, and 2000 cells / well. The 6-well plates were shaken until the cells were fully dispersed, and then placed back into an incubator (37°C, 5% CO2) for overnight incubation. The two 6-well plates corresponded to the RT group and the HfO2-Cu-BSO-PDA-BSA+RT group, respectively.

[0092] The following day, the cell adhesion was observed using an inverted microscope. After normal adhesion, HfO2-Cu-BSO-PDA-BSA nanoparticles diluted with DMEM or 1640 medium were added to the HfO2-Cu-BSO-PDA-BSA+RT group. The concentration of the added nanoparticles was quantified according to the hafnium oxide content, and the final concentration was 200 μg / ml.

[0093] After 6 hours of incubation, cells in the RT group and the HfO2-Cu-BSO-PDA-BSA+RT group were irradiated with different doses of X-rays: 400 cells / well (0 Gy), 400 cells / well (2 Gy), 800 cells / well (4 Gy), 1600 cells / well (6 Gy), and 2000 cells / well (8 Gy). After irradiation, the 6-well plates were returned to the cell culture incubator and cultured overnight. The next day, the old culture medium was removed, and the cells were washed once with sterile PBS solution stored at room temperature, and fresh culture medium was added. The cell culture status was observed every 2 days, and culture was continued for 7 days until visible cell clusters were formed, at which point culture was stopped. Remove the old culture medium, wash three times with sterile PBS solution at room temperature, fix the cells in the 6-well plate with 4% paraformaldehyde for 30 min, carefully remove the 4% paraformaldehyde, wash three more times with sterile PBS solution at room temperature, then add 0.5 ml of 1% crystal violet staining solution to each well, stain for 10 min, carefully remove the staining solution, wash the cells 2-4 times with pure water, air dry the 6-well plate, count the cells and take pictures for recording.

[0094] like Figure 8 As shown, the results indicate that combined radiotherapy with HfO2-Cu-BSO-PDA-BSA nanoparticles can reduce the mean lethal dose of 4T1 tumor cells and significantly improve the sensitivity of 4T1 tumor cells to radiotherapy. The radiosensitization ratio (SER10) of combined radiotherapy with HfO2-Cu-BSO-PDA-BSA nanoparticles is 1.625753676, demonstrating a good radiosensitization effect. These studies indicate that the HfO2-Cu-BSO-PDA-BSA nanoparticles prepared in Example 1 can significantly improve the sensitivity of 4T1 tumor cells to X-rays. This solves the problem in existing technologies where high RT doses can damage normal tissues, while low doses are ineffective in inhibiting tumor growth. Furthermore, RT can induce radioresistance in tumor cells, leading to poor radiotherapy efficacy and limiting the use of radiotherapy.

[0095] 5. The radiosensitizing effect of HfO2-Cu-BSO-PDA-BSA nanoparticles on 4T1 tumor cells is related to apoptosis, and changes in apoptosis were detected.

[0096] 1) Take 4T1 tumor cells in the logarithmic growth phase cultured in a cell culture incubator (37℃, 5% CO2), digest them into single cells, and divide them into 4 groups: Control group, X-ray group, HfO2-Cu-BSO-PDA-BSA group and HfO2-Cu-BSO-PDA-BSA+X-ray group. Each group is cultured at 200,000 cells / well in a 6-well plate in an incubator.

[0097] 2) After 12 hours, remove the old culture medium from the HfO2-Cu-BSO-PDA-BSA group and the HfO2-Cu-BSO-PDA-BSA+X-ray group, and add HfO2-Cu-BSO-PDA-BSA nanoparticles diluted with DMEM or 1640 medium. The concentration of the added nanoparticles was quantified according to the hafnium oxide content, and the final concentration was 200 μg / ml for all groups. The culture medium for the Control group and X-ray group was not treated. After 6 hours, they were washed twice with cold PBS and then fresh DMEM or 1640 medium was added.

[0098] Then, the X-ray group and the HfO2-Cu-BSO-PDA-BSA+X-ray group were irradiated with 6 Gy of X-rays and then returned to the cell culture incubator for 24 h of further culture.

[0099] 3) Collect the old cell culture medium from each group in the 6-well plate, wash twice with cold PBS, collect the PBS wash buffer, digest the cells with trypsin for 3-4 min, stop the digestion with the old culture medium and resuspend the cells, add the collected PBS wash buffer, and centrifuge at 4℃ and 1000 rpm for 5 min.

[0100] 4) Collect the cell pellet, gently resuspend the cell pellet on ice with 100 μl of binding buffer, add 5 μl of fluorescent labeling reagent (Annexin V FITC) and 10 μl of nuclear fluorescent dye (PI staining solution), and stain in the dark for 30 min.

[0101] 5) After staining, transfer each group of cells to flow cytometry tubes and immediately perform flow cytometry analysis on changes in cell apoptosis.

[0102] like Figure 9 As shown, the apoptosis rate of cells in the Control group without any treatment was only 5.36%. After 24 hours of X-ray treatment, the apoptosis rate in the X-ray group was 5.26%, and in the HfO2-Cu-BSO-PDA-BSA group, it was 19.23%. When cells were first incubated with HfO2-Cu-BSO-PDA-BSA before irradiation with 6 Gy of X-rays, the apoptosis rate reached 35.8%, which was 6.8 times that of the X-ray group. Therefore, HfO2-Cu-BSO-PDA-BSA treatment alone can promote cell apoptosis, but the combination of HfO2-Cu-BSO-PDA-BSA and X-ray treatment can significantly promote tumor cell apoptosis. This indicates that HfO2-Cu-BSO-PDA-BSA can enhance the sensitivity of 4T1 tumor cells to radiation and could be further developed into an adjuvant drug or reagent for RT therapy.

[0103] 6. Biosafety:

[0104] 1) Take mouse red blood cells and wash them with PBS solution until the supernatant is no longer red. Then, use physiological saline to prepare a red blood cell solution with a concentration of 4%. Set up a blank group, a positive control group, a negative control group and 6 experimental groups. Take 500 μl of red blood cell solution into each of the positive control group, the negative control group and the 6 experimental groups and put it into a 1.5ml EP tube. Take 1ml of pure water into a 1.5ml EP tube for the blank group.

[0105] 2) Six experimental groups: The concentration of hafnium oxide in HfO2-Cu-BSO-PDA-BSA was prepared with physiological saline to be 15.625, 31.25, 62.5, 125, 250, and 500 μg / ml, respectively, and 500 μl of each was added to the corresponding EP tube;

[0106] Positive control group: Add 500 μl of pure water to the corresponding EP tube;

[0107] Negative control group: Add 500 μl of physiological saline to the corresponding EP tube;

[0108] After being left at room temperature for 3 hours, the sample was centrifuged at 3000 rpm for 5 minutes and photographed.

[0109] 3) Take 100 μl of the supernatant from each group after centrifugation and place it in a 96-well plate. Measure the absorbance (OD value) at 545 nm using a microplate reader. Calculate the hemolysis rate using the following formula:

[0110] ;

[0111] from Figure 10 The results show that the hemolysis rate of all experimental groups was less than 5%, proving that HfO2-Cu-BSO-PDA-BSA nanoparticles have good biocompatibility.

[0112] 7. Therapeutic effect on 4T1 tumor-bearing mice:

[0113] 4T1 tumor cells were injected at a rate of 1×10 6 One cell per mouse was in situ injected into the right mammary gland of a leukovar laboratory mouse (Balb / c mouse). After successful modeling, the mice were randomly divided into 5 groups of 5 mice each: Control group, X-ray group, HfO2-Cu-BSO-PDA-BSA group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group.

[0114] The length (L) and width (W) of the tumor are measured using vernier calipers, and the result is calculated using the formula V = 0.5 × L × W. 2The tumor volume was estimated when the subcutaneous tumors in each group of Balb / c mice grew to a volume of 50-100 mm. 3 The treatment experiment will begin at that time. The specific details of the treatment experiment are as follows:

[0115] Control group: 50 μl of normal saline was injected into the tumor on days 0, 5, and 10;

[0116] X-ray group: 50 μl of normal saline was injected into the tumor on days 0, 5, and 10;

[0117] HfO2-PVP+X-ray group: 50 μl of treatment solution was added on days 0, 5, and 10. The treatment solution was HfO2-PVP nanoparticles diluted with physiological saline, in which the concentration of hafnium oxide was 2 mg / mL.

[0118] HfO2-Cu-BSO-PDA-BSA group and HfO2-Cu-BSO-PDA-BSA+X-ray group: 50 μl of treatment solution was added on days 0, 5 and 10. The treatment solution was HfO2-Cu-BSO-PDA-BSA nanoparticles diluted with physiological saline, in which the concentration of hafnium oxide was 2 mg / mL.

[0119] The X-ray group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group received 2 Gy of radiotherapy 24 hours after each dose. The HfO2-Cu-BSO-PDA-BSA group and the Control group did not receive radiotherapy.

[0120] After treatment began, Balb / c mice were observed for tumor growth every other day, with tumor length and width measured and mouse weight assessed. To ensure animal welfare, tumor volume exceeding 2000 mm² was considered acceptable. 3 In the pre-termination experiment, tumor-bearing Balb / c mice were euthanized, and organs and tumors of Balb / c mice in each treatment group were collected for analysis and detection.

[0121] like Figure 11 As shown, Figure 11 The curves in the image, from top to bottom, represent the Control group, HfO2-Cu-BSO-PDA-BSA group, X-ray group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group, respectively. Figure 11It was found that, compared to the Control group, the other groups showed therapeutic effects on the growth of 4T1 tumor cells in Balb / c mice. The HfO2-PVP+X-ray group had a better therapeutic effect than the X-ray group, demonstrating the radiosensitizing effect of hafnium oxide. The HfO2-Cu-BSO-PDA-BSA+X-ray group showed a significantly stronger tumor-suppressing effect than the other groups, and it could significantly inhibit tumor growth. These results indicate that HfO2-Cu-BSO-PDA-BSA nanoparticles can significantly enhance the inhibitory effect of radiotherapy on tumor growth.

[0122] After treatment, the spleens of mice in the Control group, HfO2-Cu-BSO-PDA-BSA group, X-ray group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group were collected and prepared into single-cell suspensions. Then, flow cytometry was used to detect the infiltration of immune cells in the tumor to evaluate the effect of nanoparticles combined with radiotherapy on anti-tumor immunity. Figure 12 CD8+ levels in the spleen of mice in the Control group, HfO2-Cu-BSO-PDA-BSA group, X-ray group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group were measured. + A statistical chart of T lymphocyte infiltration, from... Figure 12 As can be seen, the HfO2-Cu-BSO-PDA-BSA+X-ray group had the highest immune cell infiltration, proving that the combination of HfO2-Cu-BSO-PDA-BSA and radiotherapy can significantly enhance anti-tumor immunity in mice and reverse the immunosuppressive microenvironment.

[0123] After treatment, tumor cells from mice in the Control group, HfO2-Cu-BSO-PDA-BSA group, X-ray group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group were collected and prepared into single-cell suspensions. Flow cytometry was then used to detect the infiltration of immune cells in the spleen and tumors to evaluate the effect of combining nanoparticles and radiotherapy on anti-tumor immunity. Figure 13 The control group, HfO2-Cu-BSO-PDA-BSA group, X-ray group, HfO2-PVP+X-ray group, and HfO2-Cu-BSO-PDA-BSA+X-ray group showed CD8+ in tumors of mice. + Statistical graph of T lymphocyte infiltration results, from Figure 13It can be seen that the HfO2-Cu-BSO-PDA-BSA+X-ray group had the most lymphocyte infiltration in the tumor, which was significantly higher than other groups. This indicates that HfO2-Cu-BSO-PDA-BSA+X-ray can significantly reverse the tumor immunosuppressive microenvironment, enhance anti-tumor immunity, and effectively achieve anti-tumor effects through immunity.

[0124] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature.

[0125] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0126] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A nanoparticle for radiotherapy-copper death combined therapy of breast cancer, characterized in that, The following raw materials are included: 10 mg precipitate A, 3-8 mg CuCl2, 10 mg DA.HCl, 2-6 mg BSO, 100-250 μl Tris-HCl buffer, and 30-40 mg BSA; precipitate A is HfO2-PVP; Methods for preparing nanoparticles include: After washing 10 mg of precipitate A three times with deionized water, dissolve it in 1-10 ml of deionized water to obtain solution B; Dissolve 3-8 mg CuCl2, 10 mg DA.HCl and 2-6 mg BSO in 0.1-1 ml of deionized water to prepare CuCl2 solution, DA.HCl solution and BSO solution respectively; Solution B was sonicated, and then CuCl2 solution, DA.HCl solution and BSO solution were added to solution B. After stirring evenly, 100~250μl of Tris-HCl buffer was added to obtain a mixed solution. The mixed solution was ultrasonically treated for 15-40 min, then stirred for the first time, and the precipitate C was collected after the second centrifugation. After washing the precipitate C, HfO2-Cu-BSO-PDA was obtained. Dissolve 30-40 mg of BSA in 1 ml of deionized water to prepare a BSA solution. HfO2-Cu-BSO-PDA was dissolved in 1~10 ml of deionized water, then BSA solution was added, and the mixture was stirred a second time. After a third centrifugation, the precipitate was collected to obtain HfO2-Cu-BSO-PDA-BSA nanoparticles. HfO2-Cu-BSO-PDA-BSA nanoparticles were dissolved in 5 ml of physiological saline for storage at a temperature of 4°C. The preparation method of precipitate A includes: dissolving 160 mg hafnium tetrachloride and 100 mg polyvinylpyrrolidone in 10 ml of deionized water, then placing them in a hydrothermal reactor for hydrothermal treatment, and collecting precipitate A after a first centrifugation treatment.

2. The nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to claim 1, characterized in that, The concentration of the Tris-HCl buffer is 1M, and the pH value of the Tris-HCl buffer is 8.

8.

3. The nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to claim 1, characterized in that, The molecular weight of the polyvinylpyrrolidone is 10000 kDa.

4. The nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to claim 1, characterized in that, In the HfO2-Cu-BSO-PDA-BSA nanoparticles, Hf has a valence state of +2 and +3, copper has a valence state of +2, sulfur has a valence state of +4, nitrogen has a valence state of +1 and -3, and phosphorus has a valence state of +5.

5. The nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to claim 1, characterized in that, The particle size of the HfO2-Cu-BSO-PDA-BSA nanoparticles is 300~340nm; When HfO2-Cu-BSO-PDA-BSA nanoparticles are dissolved in 5 ml of physiological saline, their hydrated particle size is 100~350 nm.

6. The nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to claim 1, characterized in that, The centrifugation speeds for the first, second, and third centrifugation processes were all 9000~12000 rpm, the time was 15~30 min, and the centrifugation temperature was 25℃.

7. The nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to claim 1, characterized in that, The stirring speed for both the first and second stirring operations was 700~1000 rpm, the stirring time was 24 hours, and the stirring temperature was 25℃.

8. The use of the nanoparticles for radiotherapy-copper death combined therapy for breast cancer according to any one of claims 1 to 7 in the preparation of a medicament for treating breast cancer.

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