BiMn / BSA nano-sensitizer, its preparation and application in preparing radiotherapy sensitizing drugs for triple-negative breast cancer

Abstract

The present invention belongs to the field of medical biomaterials, and specifically relates to a preparation method of a BiMn / BSA nano-sensitizer. The Bi<supgt;3+< / supgt; source, Mn<supgt;2+< / supgt> source and BSA are subjected to mineralization treatment to obtain the BiMn / BSA nano-sensitizer. The present invention also relates to the application of the BiMn / BSA nano-sensitizer prepared by the preparation method in the preparation of radiosensitization for triple-negative breast cancer radiotherapy. The material of the present invention can adapt to the treatment characteristics of triple-negative breast cancer, and excellent radiotherapy preparation effects can be obtained at a lower radiotherapy dose.

Description

Technical Field

[0001] The present invention belongs to the field of medical biomaterials, and particularly relates to the field of anti-tumor technology. Background Art

[0002] There are indeed differences in sensitivity to radiotherapy for different types of cancers. Some tumors show high sensitivity to radiotherapy. For example, nasopharyngeal carcinoma, small cell lung cancer, etc. are usually relatively sensitive to radiotherapy. At the same time, there are also some tumors that are less sensitive to radiotherapy, such as liposarcoma and triple-negative breast cancer.

[0003] Triple-negative breast cancer is a subtype of breast cancer with strong invasiveness, high recurrence risk and difficult to cure. The current main treatment methods are surgical treatment and chemotherapy. Radiotherapy is mainly used to assist in improving the overall efficacy and is generally not used alone for radiotherapy. The main reasons are as follows: one is that triple-negative breast cancer has low sensitivity to radiotherapy and requires a large dose of irradiation to effectively inhibit the proliferation of triple-negative breast cancer cells, but the side effects of a large dose of radiation on normal tissues limit the application of radiotherapy. The other is the hypoxia resistance of tumors, which enables the damaged tumor cell DNA to be repaired, resulting in recurrence and metastasis.

[0004] Triple-negative breast cancer (TNBC) is insensitive to traditional hormonal therapy and targeted therapy due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). In terms of radiotherapy sensitization regimens, the treatment strategy for TNBC is to use chemotherapy drugs as radiosensitizers for radiotherapy. Commonly used chemotherapy drugs include cisplatin and paclitaxel. These drugs can increase the sensitivity of tumor cells to radiotherapy, but due to the lack of targeting, they often bring relatively large side effects to the body. With the progress of nanotechnology, many high-Z element nanoparticles, such as heavy metal nanoparticles of gold, silver or platinum, can enhance the effect of radiation. They increase the radiation dose of tumor cells by improving the absorption of radiation and generating secondary radiation (such as the photoelectric effect and Compton scattering). Due to their very small size, these nanoparticles can more easily cross the blood vessel wall and accumulate in tumor tissues. Some nanoparticles can be designed to have multiple functions, such as simultaneous drug delivery, imaging and treatment sensitization, but it is still difficult to be applied clinically. Insufficient biocompatibility and biodegradability are the biggest obstacles to their clinical application. Summary of the Invention

[0005] The first object of the present invention is to provide a preparation method of a BiMn / BSA nano-sensitizer, aiming to prepare a sensitizer suitable for radiotherapy sensitization of triple-negative breast cancer.

[0006] The second object of the present invention is to provide the BiMn / BSA nano-sensitizer prepared by the preparation method and its application in the preparation of radiosensitizing drugs for triple-negative breast cancer radiotherapy.

[0007] The third object of the present invention is to provide a radiosensitizing drug for triple-negative breast cancer radiotherapy containing the BiMn / BSA nano-sensitizer.

[0008] A preparation method of a BiMn / BSA nano-sensitizer, which mineralizes a Bi 3+ source, an Mn 2+ source and BSA (bovine serum albumin) to obtain the BiMn / BSA nano-sensitizer.

[0009] The research of the present invention shows that the BiMn / BSA nano-sensitizer obtained by mineralizing the Bi 3+ source, the Mn 2+ source and BSA can unexpectedly adapt to the characteristics of triple-negative breast cancer cells, and can obtain excellent selective killing effects on triple-negative breast cancer cells and normal cells at a lower radiotherapy dose. Moreover, it can effectively weaken the toxic side effects caused by the radiotherapy process.

[0010] In the present invention, the Bi 3+ source is a water-soluble salt that can ionize Bi 3+ , preferably bismuth nitrate;

[0011] Preferably, the Mn 2+ source is a water-soluble salt that can ionize Mn 2+ , preferably at least one of manganese nitrate and manganese chloride;

[0012] Preferably, the molar ratio of the Bi 3+ source to the Mn 2+ source is 1:3 to 5.

[0013] In the present invention, there is no special requirement for the dosage ratio of the Bi 3+ source to BSA, and it can be controlled according to needs. For example, the molar amount of the Bi 3+ source and the weight ratio of BSA are 0.1 to 1 mmol / g, and further can be 0.1 to 0.3 mmol / g.

[0014] In the present invention, using the Bi 3+ source, the Mn 2+ source in combination and BSA for mineralization treatment is the key to synergistically improving its adaptability to triple-negative breast cancer radiotherapy and improving its low-dose radiotherapy effect and selectivity.

[0015] In the present invention, BSA is pre-dissolved in water, and then the Bi 3+ source and the Mn 2+The source is mixed evenly, the pH of the system is adjusted to 10-13, and mineralization treatment is carried out. Subsequently, dialysis and freeze-drying are performed to obtain the BiMn / BSA nano-sensitizer described above.

[0016] In the present invention, the mineralization treatment time is above 20 h, and more preferably 20-30 h.

[0017] The present invention also provides a BiMn / BSA nano-sensitizer prepared by the above preparation method.

[0018] In the present invention, there is also provided an application of the BiMn / BSA nano-sensitizer prepared by the above preparation method in the preparation of a radiotherapy sensitizing drug for treating triple-negative breast cancer.

[0019] For conventional radiotherapy of triple-negative breast cancer, the dose mostly needs to be above 5 Gy and has a large dose dependence. However, the research of the present invention shows that the BiMn / BSA nano-sensitizer prepared by the above preparation method can unexpectedly adapt to the pathogenesis of triple-negative breast cancer and can unexpectedly obtain excellent killing effect and selectivity for triple-negative breast cancer at a low radiotherapy dose (such as below 5 Gy).

[0020] In the present invention, based on conventional ideas, the BiMn / BSA nano-sensitizer described in the present invention can be prepared into any preparation that meets the requirements of conventional radiotherapy sensitization.

[0021] For example, in the present invention, it is combined with pharmaceutically acceptable excipients to prepare the above-mentioned radiotherapy sensitizing drug for treating triple-negative breast cancer;

[0022] Preferably, the radiotherapy sensitizing drug for treating triple-negative breast cancer has a pharmaceutically acceptable dosage form, and further can be an injection.

[0023] Preferably, it is prepared into a radiotherapy sensitizing drug for treating triple-negative breast cancer with a radiotherapy dose below 5 Gy, more preferably 2-4.5 Gy.

[0024] The present invention also provides a radiotherapy sensitizing drug for treating triple-negative breast cancer, which contains a pharmaceutically effective amount of the BiMn / BSA nano-sensitizer prepared by the above preparation method.

[0025] The radiotherapy sensitizing drug for treating triple-negative breast cancer described in the present invention further includes pharmaceutically acceptable excipients;

[0026] The radiotherapy sensitizing drug for treating triple-negative breast cancer described in the present invention is a pharmaceutically acceptable injection preparation, more preferably an intravenous injection; even more preferably a powder for injection or an injection solution.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The BiMn / BSA prepared by the preparation method described in the present invention can unexpectedly adapt to the disease type of triple-negative breast cancer, can unexpectedly exhibit excellent adaptation synergy, can exhibit excellent selective radiotherapy poisoning effect on triple-negative breast cancer at a lower radiotherapy dose, and can significantly reduce other radiotherapy toxicities on the premise of obtaining excellent anti-cancer effects; BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a synthesis diagram of BiMn / BSA and a combined radiotherapy mode diagram;

[0030] Figure 2 It is the characterization of BiMn / BSA: (A) Transmission electron microscope (TEM) image of BiMn / BSA, (B) Hydrodynamic size of BiMn / BSA, (C) Element mapping of BiMn / BSA. (D) X-ray photoelectron spectroscopy (XPS) full spectrum of BiMn / BSA, (E) XPS spectrum of Bi 4f orbit, (F) XPS spectrum of Mn 2p orbit. (G) Element content of BiMn / BSA measured by inductively coupled plasma - optical emission spectrometry (ICP-OES). (H) Dynamically monitor the particle size of BiMn / BSA in PBS and DMEM containing FBS. (I) Oxygen generation after adding different formulations in H2O2 solution, (J) Oxygen generation of different concentrations of BiMn / BSA in H2O2 solution.

[0031] Figure 3 It is the killing test of BiMn / BSA on breast cancer and its in vitro radiotherapy sensitization ability. Effects of different treatment methods on the survival rates of (A) 4T1 cells and (B) MDA-MB-231 cells. Survival rates of (C) 4T1 cells and (D) MDA-MB-231 cells under different doses of X-ray radiation and different concentrations of BiMn / BSA treatment. Detection of reactive oxygen species (ROS) by (E) confocal fluorescence imaging and (F) flow cytometry analysis of the ROS level in 4T1 cells after different treatments. Confocal fluorescence imaging of γ-H2AX after different treatments. Evaluation of the (H) expression levels of STING and p-STING proteins and (I) quantitative analysis after different treatments by Western blot. Flow cytometry analysis of the proportions of (J) 4T1 cell death and (K) survival, early apoptosis, late apoptosis and necrosis cells after different treatment methods.

[0032] Figure 4Biological distribution and MRI sensitization ability of BiMn / BSA. Among them, (A) Dynamic biodistribution map shows the distribution in vivo after injection of BiMn / Cy 5.5-BSA nanoparticles, and (B) Ex vivo fluorescence imaging of tumors and major organs 24 hours after drug administration. (C) Bismuth (Bi) and (D) Manganese (Mn) contents in major organs and tumors 24 hours after drug administration measured by ICP-MS. (E) Penetration and distribution of BiMn / BSA in tumors 24 hours after drug administration. Scale bar = 1 mm. (F) T1-weighted contrast-enhanced MRI images of BiMn / BSA at different concentrations in PCR tubes. (G) MRI T1 images of tumors in mice after different treatments;

[0033] Figure 5 Therapeutic effect in vivo of the combination of BiMn / BSA and radiotherapy; among them, (A) Overall treatment plan for in vivo anti-tumor experiments (n = 7), (B) Changes in tumor volume during different treatments, (C) Photos of tumors harvested after different treatments, (D) Evaluation of tumor necrosis, apoptosis, hypoxia, and nuclear damage after different treatments by staining with H&E, TUNEL, caspase 3, HIF-1α, and γ-H2AX, (E) Levels of STING and p-STING proteins after treatment, and (F) Quantitative analysis;

[0034] In each panel, G1 represents PBS; G2 represents radiotherapy (RT); G3 represents Bi / BSA; G4 represents RT + Bi / BSA; G5 represents BiMn / BSA; G6 represents RT + BiMn / BSA.

[0035] Figure 6 Serum biochemical parameters and blood cell levels in mice after treatment;

[0036] Figure 7 H&E staining of important organs after different treatments; Detailed implementation manners

[0037] The present invention will be further described below in conjunction with embodiments, rather than limiting the present invention. Unless otherwise stated in the context of the present application, the technical terms and abbreviations used in the present application have the conventional meanings known to those skilled in the art; unless otherwise stated, the raw material compounds used in the following embodiments are all commercially available.

[0038] Example 1

[0039] Preparation of BiMn / BSA

[0040] BiMn / BSA nanoparticles are prepared by adding Bi 3+ and Mn 2+Synthesis by a biomineralization method in the presence. First, 250 mg of BSA was dissolved in 8 mL of ultrapure water, and then 1 mL of Bi(NO3)3 (50 mM, containing 2 M HNO3) and 1 mL of MnCl2 (200 mM) solution were gradually added while stirring at room temperature for 30 minutes. Subsequently, the pH value of the solution was adjusted to 12 by adding 2 M NaOH solution, and stirring was continued for 24 hours. The resulting suspension was dialyzed against deionized water through a dialysis membrane (3.4 kDa) for 24 hours to remove excess precursors, and then BiMn / BSA was obtained by freeze-drying.

[0041] Comparative Example 1

[0042] Compared with Example 1, the difference is only that MnCl2 is missing, and the missing molar amount is supplemented with an equimolar amount of Bi(NO3)3. Other operations and parameters are the same as in Example 1. The prepared material is labeled as Bi / BSA nanoparticles.

[0043] Comparative Example 2

[0044] Compared with Example 1, the difference is only that Bi(NO3)3 is missing, and the missing molar amount is supplemented with an equimolar amount of MnCl2. Other operations and parameters are the same as in Example 1. The prepared material is labeled as Mn / BSA nanoparticles.

[0045] Example 2

[0046] Characterization of BiMn / BSA

[0047] Transmission electron microscopy (TEM-EDS, Titan G260-300, FEI, USA) images showed that BiMn / BSA had a uniform plate-like structure with an average diameter of about 110 nm ( Figure 2 A), which was consistent with the results of dynamic light scattering (DLS) measurements ( Figure 2 B). Elemental mapping analysis detected C, O, N, S, Bi, and Mn, confirming BSA-mediated Bi / Mn biomineralization ( Figure 2 C). X-ray photoelectron spectroscopy (XPS, ThermoFisher-VG Scientific, ESCALAB250Xi, USA) analysis determined the composition of Bi and Mn in BiMn / BSA ( Figure 2 D, Figure 2 E, Figure 2 F). In addition, the Bi and Mn contents were measured to be 1.1% and 0.87% respectively by inductively coupled plasma atomic emission spectroscopy (ICP-AES) ( Figure 2 G). To study the colloidal stability, BiMn / BSA was redispersed in PBS or DMEM cell culture medium containing FBS, and it was observed that its hydrodynamic size remained almost unchanged within 48 hoursFigure 2 H). The incorporation of Mn element endows the nanoparticles with peroxidase-like activity, which helps to alleviate tumor hypoxia and enhance the effect of oxygen-dependent radiotherapy. By adding H2O2 to BiMn / BSA and measuring the oxidation using a portable dissolved oxygen instrument (JPBJ-609L, INESA Scientific Instrument Co., Ltd., China), it was observed that oxygen was rapidly generated within 10 minutes ( Figure 2 I). In contrast, the Mn-free nanoparticles (i.e., Bi / BSA) did not show this phenomenon. It can be seen that the catalytic oxidation ability originates from the Mn component, and the oxygen generation rate increases in a concentration-dependent manner with the increase in the concentration of BiMn / BSA ( Figure 2 J).

[0048] Example 3

[0049] Cell viability assay

[0050] First, 4T1 and MDA-MB-231 cells were evenly distributed in 96-well plates at 37 °C, ensuring that each well contained approximately 5000 cells. After the cells grew on the bottom of the wells and covered approximately 60% of the area, the original medium was replaced with a new medium containing different concentrations of Bi / BSA, MnBSA, or BiMn / BSA to set up experimental groups. The cells were incubated in the new medium for another 6 hours and then irradiated with different doses of radiation such as 0.1, 2, 4, 8 Gy. After radiation treatment, the cell viability was measured using a CCK-8 kit, and the absorbance value (OD) was read at a wavelength of 460 nm using a multifunctional microplate reader. The cell viability was evaluated according to the viability calculation formula: cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100. As shown in Figures (3A - 3D), when not irradiated, BiMn / BSA had minimal cytotoxicity to cells, showing good biocompatibility. When exposed to different concentrations of radiation, it could kill tumor cells in a concentration-dependent manner. As Figure 3 A - Figure 3 B shows, BiMn / BSA showed stronger cell damage activity than Bi / BSA at higher concentrations, resulting in a lower IC50 value. This enhanced effect may be attributed to the catalytic oxidation of Bi, which increases the oxygen substrate for radiotherapy ( Figure 3 C, Figure 3 D). The DNA free radicals generated by irradiation react with the in-situ generated oxygen, causing irreparable DNA damage (i.e., DNA double-strand breaks), leading to cell death.

[0051] Example 4

[0052] Cell apoptosis analysis

[0053] 4T1 cells (1×10^5 cells per well) were incubated in a 6-well plate for 6 hours. They were incubated with PBS, Bi / BSA (50 μg / ml), or BiMn / BSA (50 μg / ml) for 6 hours and exposed to different radiation doses, and then the cells were incubated for another 18 hours. After the cells were treated with trypsin, they were washed twice with PBS in a centrifuge tube and collected. According to the instructions of the AnnexinV-FITC / PI apoptosis detection kit provided by Yeasen Co., Ltd. (Shanghai), the cells were co-incubated with 50 μL of Annexin V (10%) and 50 μL of PI (10%) in the dark at room temperature for 15 minutes. 400 μL of binding buffer was added to each sample, and then it was analyzed using a flow cytometer (FACSVerse, BD, USA). The results showed that after radiotherapy, the number of late apoptotic cells increased significantly, especially after binding with BiMn / BSA nanoparticles, and the effect was more significant. The combination of BiMn / BSA and radiotherapy induced apoptosis in more than 90% of the cells.

[0054] Example 5

[0055] Reactive oxygen species (ROS) detection

[0056] (1) 4T1 or MD-MBA-231 cells (5×10^4 cells) were seeded in confocal dishes and incubated overnight. Then, they were treated with PBS, Bi / BSA (50 μg / ml), or BiMn / BSA (50 μg / ml) for 6 hours, followed by irradiation with or without X-rays (4 Gy). After 18 hours of incubation, DCFH-DA (10 μM) was added for staining for 1 hour. The cell nuclei were stained with Hoechst 33342 for 15 minutes, and it was detected using a confocal laser scanning microscope (CLSM). The results showed that compared with the control group, the cells treated with simple Bi / BSA (50 μg / ml) or BiMn / BSA (50 μg / ml) only showed weak fluorescence, while the fluorescence increased significantly after combined radiotherapy ( Figure 3 E).

[0057] (2) Cells were cultured to near confluence at 37 °C under 5% CO2. Then the cells were treated with media containing different concentrations (e.g., 0, 10, 25, 50, 100, 250, 500 μg / mL) of Bi / BSA, MnBSA, or BiMn / BSA. After 6 hours, they were irradiated with 4 Gy. After treatment, they were stained with 1 μM (1:1000) DCFDA dye by incubating at 37 °C for 30 minutes. After staining, the cells were washed twice with PBS and then resuspended in the buffer for flow cytometry analysis. The fluorescence intensity was measured using a flow cytometer at a specific wavelength (e.g., excitation at 488 nm and emission at 525 nm) to evaluate the ROS level. The results were similar to those of the confocal detection. Figure 3F). Both BiMn / BSA can significantly enhance the radiosensitization effect of radiotherapy.

[0058] Example 6

[0059] DNA damage detection

[0060] The cells were treated according to the method in the above endogenous ROS generation experiment. After irradiating with 4 Gy X-rays and incubating for 18 hours, the cells were first fixed with 4% formaldehyde for 10 minutes, and then permeabilized with 0.15% TritonX-100 for 15 minutes. Subsequently, the cells were treated with blocking buffer and incubated with the primary antibody against γ-H2AX and the secondary antibody Alexa Fluor 555-labeled rabbit anti-rabbit IgG, respectively. The cell nuclei were stained with DAPI (1 μg / mL) and detected by confocal laser scanning microscopy (CLSM). The results showed that the Bi nanoparticles-containing group showed a strong radiosensitization effect, and the effect of BiMn / BSA was better ( Figure 3 G).

[0061] Example 7

[0062] Western blotting

[0063] First, 4T1 cells (1×10^5 cells per well) were incubated in a 6-well plate for 6 hours. They were incubated with PBS, Bi / BSA (50 μg / ml) or BiMn / BSA (50 μg / ml) for 6 hours and exposed to different radiation doses, and then the cells were incubated for another 18 hours. Protein samples were collected and separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After separation, the proteins (such as Sting and phosphorylated Sting) were transferred to a nitrocellulose or PVDF membrane. Then, the membrane was treated with a blocking solution (such as skim milk or bovine serum albumin) to prevent non-specific binding of antibodies. Then, specific primary antibodies were added to the membrane to bind to the target proteins. After washing off the excess primary antibodies, secondary antibodies conjugated with enzymes (such as horseradish peroxidase or alkaline phosphatase) were added. Finally, chemiluminescent substrates were added, and the luminescence signal indicated the presence and quantity of the target proteins through the reaction of the enzyme conjugated with the secondary antibody. It was visible that in the Control group and Bi / BSA group, there was no obvious phosphorylation of STING, while obvious phosphorylation was visible in the BiMn / BSA group ( Figure 3 H, Figure 3 I). Collecting tumor tissues treated differently for Western blotting detection also obtained the same trend. Figure 5 E, Figure 5 F), which means that manganese plays a key role in activating the STING pathway both in vivo and in vitro.

[0064] Example 8

[0065] In vivo / in vitro biodistribution study

[0066] To observe the biodistribution of BiMn / BSA, we established a 4T1 subcutaneous tumor model. And, Cy5.5-labeled nanoparticles (BiMn / Cy5.5-BSA) were synthesized by replacing BSA with Cy5.5-BSA. BALB / c mice bearing 4T1 tumors were given BiMn / Cy5.5-BSA by intravenous injection at an equivalent dose of 2 mg / kg of Cy5.5. Fluorescence images were captured at 1, 2, 4, 8, 12, and 24 hours after injection using a live optical imaging system (PerkinElmer, IVIS Lumina, USA). Figure 4 A). Twenty-four hours after injection, the mice bearing 4T1 tumors were euthanized, and the major organs (heart, liver, spleen, lung, kidney) and tumors were collected for ex vivo imaging. Figure 4 B). To determine the penetration of the nanoparticles in the tumors, the tumor tissues were sectioned and examined under a fluorescence microscope. In addition, ICP-OES (Prodigy 7, Leeman Laboratories, Hudson, USA) was used to measure the concentrations of Bi and Mn in the tissues. The major organs and tumors were digested with aqua regia and heated overnight at 70 °C. Then, the suspension was centrifuged to remove the residues and diluted with deionized water. Finally, sample analysis was performed using ICP-OES. Figure 4 C, Figure 4 D) The results showed that BiMn / BSA had good tumor targeting after radiotherapy treatment.

[0067] Example 9

[0068] Magnetic Resonance Imaging

[0069] For in vivo magnetic resonance imaging, the BiMn / BSA solution (50 μg / mL) was intravenously injected into tumor-bearing mice. MR imaging of the tumor region was collected at the eighth hour after injection. In addition, a series of BiMn / BSA solutions (1.0 mL) were imaged using a 3T MR scanner in the range of Mn concentrations from 0 to 100 μg / ml. The results showed that BiMn / BSA had good magnetic resonance sensitization effects and had the potential to become a magnetic resonance imaging contrast agent. Figure 4 F, Figure 4 G).

[0070] Example 10

[0071] In Vivo Antitumor Therapy

[0072] Tumor-bearing BALB / c mice were randomly divided into 6 groups (7 mice in each group): (1) PBS; (2) RT; (3) Bi / BSA; (4) RT + Bi / BSA; (5) BiMn / BSA; (6) RT + BiMn / BSA. Mice received 4 Gy radiotherapy on day 0 and day 4. On day 3, Bi / BSA (50 μg / kg) or BiMn / BSA (50 μg / kg) was injected intravenously. The tumor size and body weight of the mice were recorded every two days. The formula for calculating the tumor volume of the mice was: V = (width^2 × length) / 2. On day 16, blood, tumors, and major organs (heart, liver, spleen, lung, and kidney) were collected for further analysis. As Figure 5 As shown in Figures B - D, it can be seen that RT combined with BiMn / BSA showed the best therapeutic effect, significantly inhibiting tumor growth without significantly affecting the body weight gain of the mice.

[0073] Example 11

[0074] H&E and immunofluorescence staining of tumor tissues

[0075] The obtained tumor tissues were immersed in 4% formaldehyde, then dehydrated with absolute ethanol (56 - 58 °C), and then embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H&E) and (anti-γ-H2AX) staining, and then imaged using a Nikon Ni-E (Nikon, Minato, Japan) microscope. In addition, TUNEL, caspase-3, and HIF-1α detections were also performed according to the standard protocol. A series of images were taken using a fluorescence microscope. The results showed that RT combined with BiMn / BSA was the most effective in inducing apoptosis of tumor cells.

[0076] Example 12

[0077] Blood biochemical and blood routine tests

[0078] Biological safety assessment involved collecting blood samples by cardiac puncture to evaluate the physiological status of the mice. Serum was extracted, and various biomarkers, including AST, ALT, BUN, Cre, and CK-MB, were analyzed using blood biochemical tests. In addition, a blood sample was also collected for blood routine tests to determine the levels of different blood cells, such as red blood cells, white blood cells, and platelets. These tests were performed after different treatments to evaluate their effects on the health of the mice. The results were then compared with the normal reference ranges of each parameter to detect any significant changes. Figure 6 The results showed that RT combined with BiMn / BS showed good biological safety, confirming the clinical application potential of this combination strategy.

[0079] Example 13

[0080] H&E staining of important organs

[0081] The heart, liver, spleen, lungs, and kidneys were collected and fixed with 4% formaldehyde. Sections of the organs were stained with hematoxylin and eosin (H&E) to show the tissue structure and identify morphological changes. Then, H&E-stained sections were imaged using a Nikon Ni-E microscope (Nikon, Minato, Japan) to evaluate each organ for damage. Figure 7 The results showed that there was no non-specific damage to important organs, indicating better prospects for clinical application.

Claims

1. Use of a BiMn / BSA nano-sensitizer in the preparation of a radiotherapy sensitizing drug for treating triple-negative breast cancer, characterized in that, The preparation method of the BiMn / BSA nano-sensitizer is as follows: First, dissolve BSA in water, and then add Bi 3+ source and Mn 2+ source. After mixing evenly, adjust the pH of the system to 10-13, carry out mineralization treatment, then dialyze and freeze-dry to obtain the BiMn / BSA nano-sensitizer.

2. The application according to claim 1, wherein Bi 3+ source, Mn 2+ The molar ratio of the source is 1:3 to 5; Bi 3+ The molar amount of the source and the weight ratio of BSA are 0.1 to 1 mmol / g.

3. The application according to claim 1, characterized in that The described Bi 3+ source is a water-soluble salt that can ionize Bi 3+ .

4. The application according to claim 3, characterized in that The described Bi 3+ source is bismuth nitrate.

5. The application according to claim 1, characterized in that, The Mn described above 2+ source is a water-soluble salt that can ionize Mn 2+ .

6. The application according to claim 5, characterized in that, The described Mn 2+ source is at least one of manganese nitrate and manganese chloride.

7. The application according to claim 1, characterized in that The mineralization treatment time is above 20 h.

8. The application according to claim 7, wherein The mineralization treatment time is 20 - 30 h.

9. The application according to claim 1, wherein It is combined with pharmaceutically acceptable excipients to prepare the radiotherapy sensitizing drug for triple-negative breast cancer.

10. The application according to claim 9, wherein The radiotherapy sensitizing drug for triple-negative breast cancer has a pharmaceutically acceptable dosage form.

11. The application according to claim 10, wherein The radiotherapy sensitizing drug for triple-negative breast cancer is an injection.

12. The application according to any one of claims 1 to 11, characterized in that, It is prepared into a radiotherapy sensitizing drug for triple-negative breast cancer suitable for a radiotherapy dose below 5 Gy.

13. The application according to claim 12, characterized in that, It is prepared into a radiotherapy sensitizing drug for triple-negative breast cancer suitable for a radiotherapy dose of 2 - 4.5 Gy.

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