Manganese selenium nanoparticles loaded with doxorubicin, preparation method thereof, and application thereof

Abstract

The present invention discloses doxorubicin-loaded manganese-selenium nanoparticles, their preparation method, and applications. The preparation method comprises the following steps: adding potassium permanganate and sodium selenite during the culturing process of Chlorella vulgaris, and synthesizing biological manganese-selenium nanoparticles using the redox system in the Chlorella vulgaris. The microbial cell walls are then disrupted by centrifugation and ultrasonication, and protein molecules deposited on the surface of the nanoparticles are hydrolyzed by protease. Finally, nanomanganese-selenium particles are obtained by step-by-step centrifugation. These particles are then shaken with a DOX solution at room temperature and centrifuged to obtain doxorubicin-loaded manganese-selenium nanoparticles (MnSe@DOX nanoparticles). The preparation method of the present invention is simple and efficient, with very mild synthesis conditions, no pollution to the environment, low cost, and suitable for mass production. The nanoparticles produced by the present invention have excellent anti-tumor effects, converting immunosuppressive "cold" tumors into immunoreactive "hot" tumors, thereby improving the response to ICIs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to doxorubicin-loaded manganese-selenium nanoparticles, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, the treatment for triple-negative breast cancer, which is highly malignant, easily metastatic, and lacks specific targets, mainly relies on surgical resection and chemotherapy and radiotherapy. However, due to the lack of targeting, chemotherapy drugs can cause significant damage to normal cells while killing tumor cells, resulting in significant side effects.

[0003] Doxorubicin is a commonly used first-line chemotherapy drug for breast cancer. Besides directly damaging cells as a chemotherapeutic agent, it can also induce immunogenic cell death and promote the transfer of tumor-associated antigens to dendritic cells, thereby activating dendritic cell maturation and the infiltration of T cells and memory T cells into tumor tissue, transforming immunosuppressive "cold" tumors into immunoreactive "hot" tumors. However, doxorubicin has dose-limiting cardiotoxicity.

[0004] In addition, from an immunological perspective, breast cancer is a "cold tumor" with a low tumor mutation load and a relatively low level of T lymphocyte infiltration. However, triple-negative breast cancer has a relatively richer number of infiltrating lymphocytes than other subtypes of breast cancer, providing a very good immune microenvironment basis for the application of immune checkpoint inhibitors (ICIs). However, a large number of clinical trial results have shown that the single-agent activity of ICIs in metastatic triple-negative breast cancer is not high, and its safety needs to be improved. Therefore, how to design new treatment strategies to provide patients with triple-negative breast cancer with targeted, efficient, and low-toxic treatment options, and to transform immunosuppressive "cold" tumors into immune-responsive "hot" tumors to enhance the anti-tumor effect of ICIs, is a key issue that needs to be urgently addressed in clinical and basic research on breast cancer, and has potential clinical application value. Summary of the Invention

[0005] In order to address the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a manganese-selenium nanoparticle loaded with doxorubicin, a preparation method and application thereof, so as to solve the problem that the single-drug activity of existing ICIs in metastatic triple-negative breast cancer is not high and the safety needs to be improved.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] A method for preparing doxorubicin-loaded manganese-selenium nanoparticles comprises the following steps:

[0008] (1) Preparation of biological manganese-selenium nanoparticles: Cultivate Chlorella, add quercetin solution to the culture system, continue culturing, then add potassium permanganate solution and sodium selenite solution to the culture system, culture for 5-8 days, wait until the culture solution turns brown, collect the Chlorella bodies by centrifugation, crush, sonicate, add trypsin to hydrolyze, and centrifuge again to obtain the product;

[0009] (2) Preparation of doxorubicin-loaded manganese-selenium nanoparticles: taking doxorubicin solution and the biological manganese-selenium nanoparticles prepared in step (1), mixing, fixing the volume, shaking, centrifuging, collecting the precipitate, washing, and preparing.

[0010] Furthermore, the concentration of the quercetin solution added in step (1) is 0.1-0.3 mg / mL.

[0011] Furthermore, the concentration of the potassium permanganate solution added in step (1) is 20-30 μg / mL.

[0012] Furthermore, the concentration of the sodium selenite solution added in step (1) is 0.1-0.15 mg / mL.

[0013] Furthermore, the culture temperature in step (1) is 20-30°C.

[0014] Furthermore, in step (1), the centrifugal speed is 5000-8000 r / min and the time is 5-10 min.

[0015] Furthermore, the crushing time in step (1) is 10-60 min.

[0016] Furthermore, the enzymatic hydrolysis time in step (1) is 2-8 hours.

[0017] Furthermore, in step (1), the centrifugation speed is 5000-12000 r / min and the time is 5-10 min.

[0018] Furthermore, in step (2), the concentration of the doxorubicin solution is 4-6 mg / mL, and the mass ratio of the biological manganese selenium nanoparticles to doxorubicin is 1:1-3.

[0019] Furthermore, in step (2), the shaking temperature is 20-25° C. and the shaking time is 20-30 h.

[0020] Furthermore, in step (2), the centrifugal speed is 12000-15000 r / min, and the time is 5-12 min.

[0021] Manganese selenium nanoparticles loaded with doxorubicin are prepared by the above preparation method.

[0022] Application of the above-mentioned doxorubicin-loaded manganese-selenium nanoparticles in the preparation of drugs for treating tumors.

[0023] The present invention has the following beneficial effects:

[0024] (1) The manganese selenium nanoparticles loaded with doxorubicin prepared by the present invention can be used in tumor treatment. The main advantage is that the nanoparticles reach the tumor site through the EPR effect. Due to the weakly acidic microenvironment of the tumor site, the nanoplatform collapses, thereby releasing the chemotherapy drug doxorubicin Mn 2+ On the one hand, it reduces the systemic toxicity of chemotherapy drugs. On the other hand, in addition to directly damaging cells as a chemotherapy drug, doxorubicin can also cause immunogenic cell death. 2+ It can effectively activate the cGAS-STING pathway, promoting the tumor-killing ability of cytotoxic T cells and NK cells. Therefore, this bio-nanoplatform not only overcomes the poor delivery and severe side effects of commonly used chemotherapeutic drugs in clinical practice, but also significantly enhances the infiltration of cytotoxic immune cells at the tumor site, improving the anti-tumor effect of immune checkpoint inhibitors, and providing new strategies and ideas for the clinical treatment of breast cancer patients. The implementation of this project will help to develop new approaches for cancer treatment.

[0025] (2) The preparation method of the present invention has very mild conditions, is environmentally friendly, low cost, and is suitable for mass production. In addition, the synthesized nanoparticles have good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a TEM image of biological manganese-selenium nanoparticles in Experimental Example 1;

[0027] Figure 2 This is a diagram of the hydrated particle size of biological manganese-selenium nanoparticles in Experimental Example 1;

[0028] Figure 3 This is the XRD pattern of biological manganese selenium nanoparticles in Experimental Example 1;

[0029] Figure 4 This is the XPS spectrum of biological manganese-selenium nanoparticles in Experimental Example 1;

[0030] Figure 5 This is the Mn 2p XPS scan of the biological manganese-selenium nanoparticles in Experimental Example 1;

[0031] Figure 6 This is the Se 3d XPS scan of the biological manganese selenium nanoparticles in Experimental Example 1;

[0032] Figure 7 This is the UV-visible absorption spectrum of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs in Experimental Example 2, where the y-axis is MnSe, DOX, and MnSe@DOX from top to bottom;

[0033] Figure 8 This is the Fourier transform infrared spectrum of the doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs of Experimental Example 2;

[0034] Figure 9 This is the Zeta potential measurement diagram of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs in Experimental Example 2;

[0035] Figure 10 This is the DOX loading rate diagram of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs in Experimental Example 2;

[0036] Figure 11 The release of DOX from doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs under different pH conditions in Experimental Example 2;

[0037] Figure 12 MnSe@DOX NPs loaded with doxorubicin under different pH conditions 2+ release of

[0038] Figure 13 The survival rate of 4T1 cells after treatment with different materials (DOX, MnSe and MnSe@DOX) at different concentrations for 24 hours in Experimental Example 3;

[0039] Figure 14 The experimental results of the anti-tumor effect of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs in animals in Experimental Example 4, where (a) is the tumor growth curve of mice in different treatment groups, (b) is the weight of the tumor in mice in different treatment groups, and (c) is the change in the weight of mice during different treatment periods;

[0040] Figure 15 The experimental results of Experimental Example 4 on the effect of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs on the infiltration and activation of immune cells in tumor tissues, where (a) is the CD8 + (b) The ratio of CD4 T cells in the tumors of mice in different treatment groups. + (c) The proportion of T cells, (d) The proportion of mature DC cells in the tumors of mice in different treatment groups. DETAILED DESCRIPTION

[0041] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0042] Example 1:

[0043] A method for preparing doxorubicin-loaded manganese selenium nanoparticles (MnSe@DOX NPs) comprises the following steps:

[0044] (1) Synthesis of biological manganese-selenium nanoparticles: First, Chlorella vulgaris was cultured in BG11 medium (pH = 8.0, 200 mL) and placed in an artificial light incubator at 25°C. After 24 hours of culture, a 0.2 mg / mL quercetin solution was added to the Chlorella culture system to promote the growth of Chlorella. The culture was continued for another 2 days. A 25 μg / mL KMnO4 solution and a 0.12 mg / mL sodium selenite solution were further added to the culture system. The culture was then continued at 25°C for one week under light. When the solution turned brown, indicating that the biological manganese-selenium nanoparticles had been successfully synthesized in the Chlorella, the Chlorella bodies were collected by centrifugation.

[0045] (2) Isolation and purification of biological manganese-selenium nanoparticles: After the Chlorella culture is completed, the bacterial liquid is centrifuged at 8000 r / min for 10 minutes, the supernatant culture medium is discarded, and the precipitate is collected. The Chlorella cells are treated with a cell ultrasonic disruptor for 40 minutes to rupture the cells and release the intracellular manganese-selenium nanoparticles. After ultrasonic treatment of the cells, trypsin is added to hydrolyze the protein molecules deposited on the surface of the manganese-selenium nanoparticles for 5 hours, followed by step-by-step centrifugation at 10000 r / min to obtain the biological manganese-selenium nanoparticles, which are ultrasonically dispersed in ultrapure water and stored at 4°C.

[0046] (3) Doxorubicin (DOX) loading: 1.2 mL of 5 mg / mL doxorubicin solution and 3 mg of bio-manganese selenium nanoparticles were placed in a 15 mL centrifuge tube and diluted to 6 mL with deionized water. Shake at room temperature for 24 hours. After shaking, centrifuge at 14,000 rpm / min for 10 minutes to collect the precipitate, which is doxorubicin-loaded manganese selenium nanoparticles (MnSe@DOX NPs). The precipitate was then washed with deionized water to completely remove the free doxorubicin. The precipitate was redispersed with 3 mL of deionized water to obtain a MnSe@DOX NPs solution. The solution was stored at 4°C.

[0047] Example 2:

[0048] A method for preparing doxorubicin-loaded manganese selenium nanoparticles (MnSe@DOX NPs) comprises the following steps:

[0049] (1) Synthesis of biological manganese-selenium nanoparticles: First, Chlorella vulgaris was cultured in BG11 medium (pH = 8.0, 200 mL) and placed in an artificial light incubator at 25°C. After 24 hours of culture, a 0.1 mg / mL quercetin solution was added to the Chlorella culture system to promote the growth of Chlorella. The culture was continued for another 2 days. A 20 μg / mL KMnO4 solution and a 0.10 mg / mL sodium selenite solution were further added to the culture system. The culture was then continued at 25°C for one week under light. When the solution turned brown, indicating that the biological manganese-selenium nanoparticles had been successfully synthesized in the Chlorella, the Chlorella bodies were collected by centrifugation.

[0050] (2) Isolation and purification of biological manganese-selenium nanoparticles: After the Chlorella culture is completed, the bacterial liquid is centrifuged at 8000 r / min for 10 minutes, the supernatant culture medium is discarded, and the precipitate is collected. The Chlorella cells are treated with a cell ultrasonic disruptor for 40 minutes to rupture the cells and release the intracellular manganese-selenium nanoparticles. After ultrasonic treatment of the cells, trypsin is added to hydrolyze the protein molecules deposited on the surface of the manganese-selenium nanoparticles for 5 hours, followed by step-by-step centrifugation at 10000 r / min to obtain the biological manganese-selenium nanoparticles, which are ultrasonically dispersed in ultrapure water and stored at 4°C.

[0051] (3) Doxorubicin (DOX) loading: 1.2 mL of 4 mg / mL doxorubicin solution and 3 mg of bio-manganese selenium nanoparticles were placed in a 15 mL centrifuge tube and diluted to 6 mL with deionized water. Shake at room temperature for 24 hours. After shaking, centrifuge at 14,000 rpm / min for 10 minutes to collect the precipitate, which is doxorubicin-loaded manganese selenium nanoparticles (MnSe@DOX NPs). The precipitate was then washed with deionized water to completely remove the free doxorubicin. The precipitate was then redispersed with 3 mL of deionized water to obtain a MnSe@DOX NPs solution. The solution was stored at 4°C.

[0052] Example 3:

[0053] A method for preparing doxorubicin-loaded manganese selenium nanoparticles (MnSe@DOX NPs) comprises the following steps:

[0054] (1) Synthesis of biological manganese-selenium nanoparticles: First, Chlorella vulgaris was cultured in BG11 medium (pH = 8.0, 200 mL) and placed in an artificial light incubator at 25°C. After culturing for 24 hours, a 0.3 mg / mL quercetin solution was added to the Chlorella culture system to promote the growth of Chlorella. The culture was continued for another 2 days. A 30 μg / mL KMnO4 solution and a 0.15 mg / mL sodium selenite solution were further added to the culture system. The culture was then continued at 25°C for one week under light. When the solution turned brown, indicating that the biological manganese-selenium nanoparticles had been successfully synthesized in the Chlorella, the Chlorella bodies were collected by centrifugation.

[0055] (2) Isolation and purification of biological manganese selenium nanoparticles (MnSe): After the culturing of Chlorella, the bacterial liquid was centrifuged at 8000 r / min for 10 min, the supernatant of the culture medium was discarded, and the precipitate was collected. The Chlorella cells were treated with a cell ultrasonic disruptor for 40 min to break the cells and release the intracellular manganese selenium nanoparticles. After ultrasonic treatment of the cells, trypsin was added to hydrolyze the protein molecules deposited on the surface of the manganese selenium nanoparticles for 5 h, followed by step-by-step centrifugation at 10000 r / min to obtain biological manganese selenium nanoparticles, which were ultrasonically dispersed in ultrapure water and stored at 4°C.

[0056] (3) Doxorubicin (DOX) loading: 1.2 mL of 6 mg / mL doxorubicin solution and 3 mg of bio-manganese selenium nanoparticles were placed in a 15 mL centrifuge tube and diluted to 6 mL with deionized water. Shake at room temperature for 24 hours. After shaking, centrifuge at 14,000 rpm / min for 10 minutes to collect the precipitate, which is doxorubicin-loaded manganese selenium nanoparticles (MnSe@DOX NPs). The precipitate was then washed with deionized water to completely remove free doxorubicin. The precipitate was redispersed with 3 mL of deionized water to obtain a MnSe@DOX NPs solution. The solution was stored at 4°C.

[0057] Experimental Example 1: Characterization of Biological Manganese Selenium Nanoparticles

[0058] The biological manganese selenium nanoparticles prepared in Example 1 were subjected to transmission electron microscopy, XRD and XPS measurements. The experimental results are as follows: Figures 1-6 shown.

[0059] Figure 1 Transmission electron microscopy (TEM) results show that the MnSe nanoparticles are about 100 nm in size, with uniform morphology and good dispersion. Figure 2 The particle size distribution results showed that the particle size of the nanomaterials did not change significantly. Figure 3 The XRD pattern of the rice particles matched that of the standard card PDF#27-0311, proving that the synthesized nanoparticles were MnSe. Figure 4The XPS scan of MnSe particles further proves the presence of Se. Perhaps due to the low content of Mn, no Mn peak is shown in the XPS scan. Figure 5 The Mn 2p XPS scan shows the peak of Mn. Figure 6 In order to use XPS Peak software to perform peak fitting on Se 3d peak, the results showed that Se 3d 5 、Se 3d 3 The binding energy is between 54.6 and 57.5 eV reported in the literature. Therefore, the Se in the MnSe nanoparticles is elemental selenium.

[0060] Experimental Example 2: Characterization of Doxorubicin-loaded MnSe@DOX NPs

[0061] The doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs prepared in Example 1 were subjected to UV-visible absorption spectroscopy, Fourier transform infrared spectroscopy and Zeta potential measurements. The experimental results are as follows: Figure 7 , Figure 8 and Figure 9 shown.

[0062] Figure 7 UV-visible absorption spectrum and Figure 8 The Fourier transform infrared spectrum of the sample shows the typical peaks of doxorubicin. Figure 9 The Zeta point measurement results showed that after loading doxorubicin, the Zeta potential of MnSe@DOX changed from -37.1±0.5mV to -20.83±1.0mV. This is because the surface potential of doxorubicin is positive, which increases the surface potential of MnSe@DOX nanoparticles. The above results indicate that doxorubicin has been successfully loaded onto manganese selenium nanoparticles.

[0063] To determine whether MnSe@DOX nanoparticles can release the chemotherapeutic drug DOX in the tumor microenvironment, we examined the release of DOX under different pH conditions and the effect of Mn in MnSe on the release of DOX. 2+ The release of Figure 10 , Figure 11 and Figure 12 As shown, in the weakly acidic tumor microenvironment (pH = 4.5), DOX and Mn 2+ The release of DOX was significantly increased, indicating that the doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs prepared in the present invention have a strong response ability to the tumor microenvironment.

[0064] Experimental Example 3: Evaluation of the anti-tumor effect of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs in vitro

[0065] The manganese selenium nanoparticles MnSe@DOX NPs loaded with doxorubicin and biological manganese selenium nanoparticles MnSe prepared in Example 1 were used as experimental materials, and 2×10 5 4T1 cells were grown on the wall. After the cells attached, they were treated with DOX, MnSe, and MnSe@DOX for 24 h, respectively. The culture medium was discarded, the cells were washed three times with PBS, and fresh culture medium containing 10% CCK8 was added to incubate for 1 h. Finally, the absorbance at 450 nm was measured using a microplate reader.

[0066] The experimental results are as follows Figure 13 As shown in Figure 3, the survival rate of 4T1 cells treated with different treatments decreased with increasing concentrations. More importantly, compared with the DOX and MnSe groups alone, the doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX exhibited stronger cytotoxicity.

[0067] Experimental Example 4: Antitumor Effect of Doxorubicin-Loaded MnSe Nanoparticles MnSe@DOX NPs in Animals

[0068] The doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs and biological manganese selenium nanoparticles MnSe prepared in Example 1 were used as experimental materials to construct a 4T1 subcutaneous tumor-bearing mouse model, and PBS, DOX, MnSe and MnSe@DOX were injected into the tail vein respectively.

[0069] The experimental results are as follows Figure 14 As shown, the results showed that the tumor growth curve of mice in the experimental groups injected with DOX, MnSe and MnSe@DOX was inhibited compared with the control group injected with PBS, and the tumor mass was relatively low. The results showed that the DOX and MnSe material groups alone could partially inhibit tumor growth after treatment. Compared with the DOX and MnSe groups alone, manganese selenium nanoparticles MnSe@DOX loaded with doxorubicin showed stronger anti-tumor effects.

[0070] Experimental Example 5: Effects of doxorubicin-loaded manganese selenium nanoparticles MnSe@DOX NPs on immune cell infiltration and activation in tumor tissue

[0071] The tumor tissues of mice obtained after the treatment in Experiment 4 were used to analyze the infiltration of immune cells in the tumor tissues by flow cytometry. Tumor tissue digestion solution was prepared: DMEM / F12 medium supplemented with 0.0125% Deoxyribonuclease I, 0.05% collagenase type 3 and 0.0125% Neutral protease. First, the tumor tissue was cut into 1 mm 3The cells were then digested in 3 mL of tumor tissue digestion solution at 37°C for 30 min and filtered through a 70 μm filter to obtain a single cell suspension. The red blood cells were then removed using red blood cell lysis buffer, resuspended in PBS, and resuspended in PBS containing 2% FBS. The cells were counted and then added to a flow cytometer at a rate of 1 × 10 6 cells / 100 μL.

[0072] (1) T cell analysis: anti-CD45Ab-PerCP-Cy5.5, anti-CD4Ab-APC, anti-CD8Ab-PE-Cy7.

[0073] (2) Infiltration of natural killer cells (NK cells): anti-CD45Ab-FITC, anti-CD3Ab-APC, anti-CD49bAb-PE.

[0074] (3) Infiltration of dendritic cells (DCs): anti-CD11cAb-PerCP-Cy5.5, anti-CD80Ab-APC, anti-CD86Ab-PE-Cy7.

[0075] Add 1 μL of the corresponding flow cytometry antibody to each tube and stain for 30 minutes at 4°C or on ice in the dark. After staining, add 1 mL of PBS to each tube and centrifuge at 1000 rpm at 4°C for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in 1 mL of PBS before analysis.

[0076] The experimental results are as follows Figure 15 As shown, compared with the control group and the groups treated with DOX or MnSe alone, CD80 + CD86 + The proportion of DC cells and CD4 + T cells, CD8 + The infiltration of T cells and NK cells increased significantly. Taken together, these results indicate that MnSe@DOX can induce the maturation of dendritic cells, increase the recruitment of cytotoxic T cells and NK cells, and enhance the killing of tumor cells by immune effector cells.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing doxorubicin-loaded manganese-selenium nanoparticles, characterized in that: The following steps are involved: (1) Preparation of biological manganese-selenium nanoparticles: Cultivate Chlorella, add quercetin solution to the culture system, continue culturing, then add potassium permanganate solution and sodium selenite solution to the culture system, culture for 5-8 days, wait until the culture solution turns brown, collect the Chlorella bodies by centrifugation, crush, sonicate, add trypsin to hydrolyze, and centrifuge again to obtain the product; (2) Preparation of doxorubicin-loaded manganese-selenium nanoparticles: mixing the doxorubicin solution and the biological manganese-selenium nanoparticles prepared in step (1), adjusting the volume, shaking, centrifuging, collecting the precipitate, washing, and preparing the product; Wherein, the concentration of the potassium permanganate solution added in step (1) is 20-30 μg / mL; the concentration of the sodium selenite solution added is 0.1-0.15 mg / mL; The concentration of the doxorubicin solution in step (2) is 4-6 mg / mL, and the mass ratio of the biological manganese selenium nanoparticles to the doxorubicin is 1:1-3.

2. The method for preparing doxorubicin-loaded manganese-selenium nanoparticles according to claim 1, characterized in that: The concentration of the quercetin solution added in step (1) is 0.1-0.3 mg / mL.

3. The method for preparing doxorubicin-loaded manganese-selenium nanoparticles according to claim 1, characterized in that: The culture temperature in step (1) is 20-30°C.

4. The method for preparing doxorubicin-loaded manganese-selenium nanoparticles according to claim 1, characterized in that: In step (1), the centrifugal speed is 5000-8000 r / min, and the time is 5-10 min; the crushing time is 10-60 min; the enzymatic hydrolysis time is 2-8 h, and the temperature is 20-40°C; the re-centrifugation speed is 5000-12000 r / min, and the time is 5-10 min.

5. The method for preparing doxorubicin-loaded manganese-selenium nanoparticles according to claim 1, characterized in that: In step (2), the shaking temperature is 20-25°C and the time is 20-30 h; the centrifugal speed is 12000-15000 r / min and the time is 5-12 min.

6. Doxorubicin-loaded manganese-selenium nanoparticles prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the doxorubicin-loaded manganese-selenium nanoparticles according to claim 6 in the preparation of a drug for treating triple-negative breast cancer.

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