A preparation method of a self-loaded oxygen UCST type ruthenium nanodiagnostic and therapeutic agent for triple negative breast cancer targeted synergistic treatment

Abstract

The application discloses a preparation method of a self-loaded oxygen UCST type ruthenium nanodiagnosis and treatment agent for triple negative breast cancer targeted synergistic treatment, wherein the nanodiagnosis and treatment agent takes a poly(acrylic acid-acrylonitrile-butyl hexafluoromethyl methacrylate) temperature-sensitive polymer as a carrier, simultaneously loads a chemotherapeutic drug salinomycin, a ruthenium-based photosensitizer Ru(pdt)3Cl2, a targeting ligand folic acid and an oxygen carrying agent perfluorohexane through a nano co-assembly technology. The system integrates chemotherapy, photodynamic therapy, active targeting and self-oxygen supply functions. The preparation method synthesizes the carrier through a RAFT polymerization and adopts an ultrasonic-assisted co-assembly process, which is simple and controllable. In-vitro experiments show that the nanodiagnosis and treatment agent has excellent cell targeting, active oxygen generation capacity and remarkable synergistic anti-tumor effect, and has a good application prospect in the diagnosis and treatment of triple negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and nanomaterials technology, and specifically relates to a method for preparing a self-oxygen-loaded UCST-type ruthenium nanoparticle therapeutic agent for targeted synergistic therapy of triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) lacks effective targeted therapies because it does not express estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HGF-2). Clinical treatment heavily relies on chemotherapy, but the overall prognosis is poor. The treatment challenges stem primarily from the following factors: First, tumor cells are prone to multidrug resistance (MDR), a complex mechanism involving overactivation of drug efflux pumps (such as P-glycoproteins) and enhanced DNA repair, often leading to chemotherapy failure. Second, the presence of a subset of cancer stem cells (CSCs) is considered a significant cause of tumor recurrence, metastasis, and resistance to traditional therapies. Furthermore, the abnormal vascular network resulting from rapid tumor proliferation leads to severe hypoxia in the tumor microenvironment (TME). This hypoxia not only promotes tumor invasion and metastasis but also severely limits the efficacy of oxygen-dependent therapies such as photodynamic therapy (PDT), which generate cytotoxic reactive oxygen species.

[0003] To address these challenges, nanomedicine delivery systems have been extensively studied. Nanocarriers based on enhanced permeability and retention (EPR) effects can achieve passive targeted accumulation of drugs at tumor sites. Furthermore, stimulus-responsive nanosystems (e.g., pH-sensitive, reduction-sensitive) are designed to release drugs in response to specific signals in the tumor microenvironment, aiming to improve therapeutic specificity. Among various stimulus-responsive materials, thermosensitive polymers have attracted attention because they can be remotely spatiotemporally controlled via external heat sources (such as near-infrared light). Polymers with a high critical solution temperature (UCST) can undergo reversible phase separation at temperatures above their phase transition points, providing another possible mechanism for heat-triggered drug release.

[0004] Meanwhile, combination therapy strategies, especially co-delivery strategies based on nanoplatforms, have become one of the research directions for improving anti-tumor efficacy, aiming to achieve synergy between different mechanisms such as chemotherapy and phototherapy. However, how to efficiently and stably load different therapeutic agents onto the same carrier and ensure their synergistic function still presents challenges in material design and preparation processes. Especially for PDT, overcoming tumor hypoxia is the core of improving its efficacy. Although some studies have explored the introduction of oxygen-carrying materials such as perfluorocarbons into the treatment system, how to effectively integrate them with other functional modules and achieve efficient release and utilization of oxygen at the lesion site still needs further exploration. In terms of photosensitizer development, metal complexes such as ruthenium(II) polypyridine compounds have shown potential as PDT photosensitizers due to their excellent optical properties and modifiability.

[0005] In summary, while existing technologies have made progress in single-treatment strategies or simple combinations for TNBC, developing integrated therapeutic formulations that can systematically address multidrug resistance, eliminate tumor stem cells, and effectively improve the hypoxic tumor microenvironment remains a crucial challenge. An innovative nanotherapeutic platform that integrates active targeting, intelligent controlled release, self-contained oxygen support, and multimodal synergistic therapy is urgently needed and holds great promise for advancing the treatment of TNBC. Summary of the Invention

[0006] In view of the above, it is necessary to provide a multifunctional nanotherapeutic agent that can simultaneously achieve active targeting, self-oxygenated photodynamic therapy, reversal of multidrug resistance, and thermally triggered precise drug release for the efficient synergistic treatment of triple-negative breast cancer.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The purpose of this invention is to propose a self-oxygenated UCST-type nanotherapeutic agent for targeted synergistic therapy of triple-negative breast cancer. The nanotherapeutic agent uses thermosensitive polymer nanoparticles with a high critical solution temperature (UCST) as a carrier. The carrier is loaded with chemotherapeutic drugs, photosensitizers, targeting ligands and liquid perfluorocarbon compounds. The UCST phase transition temperature of the thermosensitive polymer is 42±2℃.

[0009] In this invention, the temperature-sensitive polymer is poly(acrylic acid-acrylonitrile-butyl hexafluoromethacrylate) (P(AA-AN-C7F6)), which is copolymerized by acrylic acid (AA), acrylonitrile (AN) and butyl hexafluoromethacrylate (C7F6) through a reversible addition-fragmentation chain transfer polymerization reaction, wherein the molar ratio of AA, AN and C7F6 is 4:1:1.

[0010] In this invention, the chemotherapeutic drug is thalinomycin or a pharmaceutically acceptable salt thereof; the photosensitizer is a ruthenium(II) polypyridine complex; the targeting ligand is folic acid; and the liquid perfluorocarbon compound is perfluorohexane.

[0011] In this invention, the ruthenium(II) polypyridine complex is Ru(pdt)3Cl2, wherein pdt is 1,10-phenanthroline-5,6-dione.

[0012] In this invention, the mass ratio of the carrier to the chemotherapy drug, photosensitizer, and targeting ligand is 10:1:1:1; the amount of liquid perfluorocarbon compound added is 0.5%-5% of the carrier mass.

[0013] This invention also proposes a method for preparing the aforementioned self-oxygen-carrying UCST-type nanotherapeutic agent, comprising the following steps:

[0014] S1. Preparation of thermosensitive polymer carrier: Acrylic acid, acrylonitrile, butyl hexafluoromethacrylate, RAFT chain transfer agent and initiator are dissolved in an organic solvent, and after deoxygenation, the mixture is reacted at 55-65℃ for 20-28 hours under an inert atmosphere. After the reaction is completed, the mixture is purified to obtain P(AA-AN-C7F6) polymer.

[0015] S2. Co-assembly of nanotherapeutic agents: The P(AA-AN-C7F6) polymer obtained in step S1, the chemotherapeutic drug, photosensitizer, and targeting ligand are dissolved in dimethyl sulfoxide to obtain stock solutions of each component; the stock solutions of each component are mixed in proportion, and under ice bath and stirring conditions, deionized water is slowly added to the mixture, followed by the addition of liquid perfluorocarbon compound;

[0016] S3. Ultrasonic induction and post-processing: The mixed system obtained in step S2 is subjected to ultrasonic treatment to induce the formation of nanoparticles. Subsequently, the obtained nano dispersion is purified by dialysis and freeze-dried to obtain the nanotherapeutic agent powder.

[0017] In this invention, further, in step S1, the RAFT chain transfer agent is cyanomethyldodecyl carbonyl trithiocarboxylate, and the initiator is azobisisobutyronitrile.

[0018] In this invention, further, in step S2, the final concentrations of the P(AA-AN-C7F6) polymer, chemotherapeutic drug, photosensitizer, and targeting ligand in the mixture are 1-5 mg / mL, 0.1-0.5 mg / mL, 0.1-0.5 mg / mL, and 0.1-0.5 mg / mL, respectively.

[0019] In this invention, further, in step S3, the conditions for ultrasonic treatment are: power 200-400W, pulse mode, working for 3-5 seconds followed by an interval of 3-5 seconds, and a total ultrasonic time of 2-5 minutes.

[0020] The present invention also proposes the application of the aforementioned self-oxygen-loaded UCST-type nanotherapeutic agent in the preparation of drugs for the diagnosis and / or treatment of triple-negative breast cancer.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects:

[0022] 1. This invention constructs a multifunctional, integrated synergistic therapy platform. Its core lies in integrating a thermosensitive polymer carrier, thalinomycin (a chemotherapy drug capable of reversing multidrug resistance), a ruthenium-based photosensitizer that generates singlet oxygen, a folic acid ligand for active targeting, and perfluorohexane (a highly efficient oxygen-carrying agent) into a single nanoparticle using controllable nano-co-assembly technology. This "four-in-one" design not only simplifies formulation but, more importantly, ensures consistent spatiotemporal delivery of each functional unit, laying the material foundation for the effective synergy of chemotherapy and photodynamic therapy, and the organic combination of active targeting and intelligent drug release at the lesion site.

[0023] 2. This invention achieves intelligent response to the tumor microenvironment and precise controlled drug release. The high critical solution temperature (UCST) of the carrier polymer allows it to remain stable at body temperature, while undergoing a phase transition triggered by mild local hyperthermia in the tumor, rapidly releasing the loaded drug. This heat-triggered drug release mechanism, combined with folic acid-mediated active targeting, enhances the drug's concentration within tumor cells, thereby improving therapeutic efficacy while potentially significantly reducing systemic toxicity to normal tissues, embodying the precision medicine concept of "on-demand treatment."

[0024] 3. This invention effectively addresses the two core challenges of tumor hypoxia and multidrug resistance. The loaded perfluorohexane, acting as a built-in oxygen reservoir, significantly improves the hypoxic microenvironment within the tumor, thereby ensuring the photosensitizer continuously and efficiently generates cytotoxic reactive oxygen species under light irradiation, completely reversing the limited efficacy of traditional photodynamic therapy in hypoxic areas. Simultaneously, the selected thalinomycin not only directly kills tumor cells but has also been shown to inhibit tumor stem cell activity and downregulate P-glycoprotein expression, thus possessing dual potential in reversing multidrug resistance and preventing tumor recurrence and metastasis.

[0025] 4. The nanotherapeutic agent of this invention has a simple and controllable preparation process with good translational prospects. From the RAFT-controlled polymerization synthesis of the carrier to the ultrasound-assisted one-step co-assembly, the entire process is characterized by mild conditions, good reproducibility, and uniform and stable nanoparticle properties. Systematic in vitro experiments have verified the system's excellent active targeting ability, significantly enhanced reactive oxygen species generation, and outstanding synergistic cell-killing effect, fully demonstrating the rationality of its integrated design and providing a solid and reliable experimental basis for subsequent in vivo studies and clinical translation. Attached Figure Description

[0026] Figure 1 This is the synthetic route of the thermosensitive polymer P(AA-AN-C7F6) in the embodiments of the present invention.

[0027] Figure 2 Transmission electron microscopy (TEM) images of P(AA-AN-C7F6) polymer nanoparticles and PRSP nanotherapeutic agents.

[0028] Figure 3 The transmittance of an aqueous solution (1 mg / mL) of P(AA-AN-C7F6) polymer as a function of temperature is used to determine its high critical solution temperature (UCST).

[0029] Figure 4 These are confocal microscopy images, showing the effect of folic acid modification on the uptake of PRSP nanotherapeutic agents by 4T1 cells.

[0030] Figure 5 Fluorescent images of reactive oxygen species (ROS) generated in 4T1 cells after laser irradiation in different treatment groups.

[0031] Figure 6 The in vitro cytotoxicity of different treatment groups on 4T1 cells was determined by the MTT assay.

[0032] Figure 7 The images show the live / dead staining fluorescence of 4T1 cells after different treatment regimens. Detailed Implementation

[0033] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] In all embodiments, reagents and instruments not specifically described are commercially available conventional products, and operating methods not specifically described are conventional methods in the art.

[0035] Example 1: Synthesis of thermosensitive polymer P(AA-AN-C7F6)

[0036] Synthetic routes such as Figure 1 As shown. Accurately weigh acrylic acid (AA, 144.12 mg, 2.0 mmol), acrylonitrile (AN, 15.25 mg, 0.5 mmol), butyl hexafluoromethacrylate (C7F6, 120 mg, 0.5 mmol), RAFT chain transfer agent cyanomethyl dodecyl carbonyl trithiocarboxylate (5.6 mg), and initiator azobisisobutyronitrile (AIBN, 3.3 mg), place them in a round-bottom flask, and add 14 mL of anhydrous N,N-dimethylformamide (DMF) to dissolve them.

[0037] The reaction system was subjected to three cycles of "freezing-vacuuming-thawing-nitrogen purging" to completely remove oxygen, and then sealed under a nitrogen atmosphere. The reaction flask was placed in an oil bath at 55°C and the reaction was magnetically stirred for 24 hours.

[0038] After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 and dialyzed in deionized water for 3 days (changing the water every 8 hours) to remove small molecule impurities. The dialysis solution was then freeze-dried to obtain a white flocculent crude product.

[0039] To obtain purified polymer, the crude product was dissolved in deionized water (5 mg / mL), heated to boiling until completely dissolved, allowed to cool at room temperature for 1 hour, and centrifuged at 12000 rpm for 10 minutes. The supernatant was collected and freeze-dried to obtain a white powder of pure product P(AA-AN-C7F6).

[0040] The transmittance of a 1 mg / mL polymer aqueous solution at 500 nm wavelength was monitored as a function of temperature (25℃ to 50℃) using a UV-Vis spectrophotometer at a heating rate of 1℃ / min. The temperature at which 50% transmittance was achieved was defined as the UCST. Results are as follows: Figure 3 As shown, the UCST of the obtained polymer is approximately 42°C.

[0041] Example 2: Preparation of self-loaded oxygen-containing UCST-type nanotherapeutic agents (PRSP)

[0042] This embodiment provides a specific method for preparing the nanotherapeutic agent, but it should not be construed as limiting the scope of the invention.

[0043] (1) Preparation of stock solutions: Weigh 10 mg of P(AA-AN-C7F6) polymer, 1 mg of Ru(pdt)3Cl2, 1 mg of salamicin sodium (Sal-Na) and 1 mg of folic acid (FA). Dissolve the polymer in 2 mL of dimethyl sulfoxide (DMSO) to prepare a 5 mg / mL solution; dissolve the other three components in 0.1 mL of DMSO to prepare a 10 mg / mL solution.

[0044] (2) Co-assembly: Take 2 mL of polymer solution into a beaker, and add 100 µL each of Ru(pdt)3Cl2, Sal-Na and FA solutions in sequence, and vortex to mix. Place the beaker in an ice-water bath, and slowly add 3 mL of deionized water dropwise at a rate of 1 mL / min using a syringe pump while stirring magnetically at 500 rpm. Then, add 40 µL of perfluorohexane (PFH).

[0045] (3) Ultrasonic induction and purification: Immediately place the mixture in an ice bath and treat it with pulsed ultrasound (300 W power, 3 seconds working / 5 seconds intermittent) for 3 minutes until the solution shows a pale blue opalescence. Transfer the obtained nano-dispersion to a MWCO 3500 dialysis bag and dialyze it in deionized water for 3 days (changing the water 3 times a day) to remove organic solvents and free molecules. The dialysate was freeze-dried to obtain a pale blue sponge-like solid, which is the target nano-therapeutic agent, denoted as PRSP. Its morphology was observed by TEM, and the results are as follows. Figure 2 As shown, the PRSP nanoparticles are regularly spherical and uniformly distributed.

[0046] Example 3: Folic acid-mediated active targeted cell uptake experiment

[0047] The mouse triple-negative breast cancer cell line 4T1, which highly expresses the folate receptor, was used as a model. 4T1 cells were cultured at a rate of 5 × 10⁶ cells / year. 4 Inoculated at a density of cells / well in confocal culture dishes and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0048] Three groups were set up: ① control group (no drug added); ② PRSP(-FA) group (prepared according to the method of Example 2, but without folic acid); ③ PRSP(+FA) group (prepared according to the method of Example 2). The concentration of nanoparticles was uniformly adjusted to 40 µg / mL (based on polymer) using serum-free medium. The medium was then replaced, and the corresponding drugs were added, followed by incubation for 4 hours.

[0049] After incubation, the cells were washed three times with PBS. Intracellular fluorescence signals were observed directly under a confocal microscope using the autofluorescence of Ru(pdt)3Cl2 (excitation / emission: 488 nm / >650 nm).

[0050] The results are as follows Figure 4As shown, the fluorescence signal intensity in the PRSP group cells modified with folic acid was significantly stronger, demonstrating that folic acid modification enhanced the targeted uptake efficiency.

[0051] Example 4: Detection of Intracellular Reactive Oxygen Species (ROS) Generation

[0052] To verify the enhancing effect of perfluorohexane (PFH)'s oxygen-carrying capacity on photodynamic therapy, ROS levels were measured. Cell culture was performed as in Example 3.

[0053] Six groups were set up: ① control group; ② free Ru(pdt)3Cl2 group; ③ PRS group (without PFH and FA); ④ PRS + laser group; ⑤ PRSP group (without FA); ⑥ PRSP + laser group. The drug concentration (as Ru) was consistent across all groups. After incubation with the drugs for 4 hours, the mixture was washed with PBS.

[0054] Add serum-free medium containing 10 µM DCFH-DA fluorescent probe and incubate in the dark for 30 minutes. After washing with PBS, the laser group was subjected to a 450 nm laser (power density 293 mW / cm²). 2 Irradiate for 2 minutes. Immediately after irradiation, detect green fluorescence (excitation / emission: 488 nm / 525 nm) under a confocal microscope; its intensity reflects the intracellular ROS level.

[0055] The results are as follows Figure 5 As shown, the PRSP plus laser irradiation group produced the strongest fluorescence signal, indicating that PFH loading can effectively improve hypoxia and enhance photodynamic therapy.

[0056] Example 5: In vitro cytotoxicity assay (MTT method)

[0057] To evaluate the synergistic therapeutic effect of nanotherapeutic agents. 4T1 cells were cultured at 5 × 10⁻⁶ cells / day. 3 One sample per well was inoculated into a 96-well plate and cultured for 24 hours. Grouping was the same as in Example 4.

[0058] After incubation with the drug for 4 hours, the laser group was irradiated (under the same conditions as in Example 4). After irradiation, the culture medium was replaced with fresh complete culture medium, and the culture was continued for 12 hours.

[0059] Add 20 µL of MTT solution (5 mg / mL) to each well, incubate for 4 hours, discard the supernatant, and add 150 µL of DMSO to dissolve the formazan crystals. Measure the absorbance (OD) at 570 nm using a microplate reader and calculate the cell viability (average OD of experimental groups / average OD of control groups × 100%). Each group should have at least 3 replicates, and the experiment should be independently repeated 3 times.

[0060] The results are as follows Figure 6As shown, the PRSP+laser group exhibited the strongest cell growth inhibition and the lowest cell survival rate, demonstrating the synergistic effect between chemotherapy and self-oxygenated enhanced photodynamic therapy.

[0061] Example 6: Live / Dead Cell Staining Assay

[0062] Visually assess cell viability. 4T1 cells were seeded in 24-well plates (5 × 10⁶ cells / well). 3 (each hole), grouping and processing are the same as in Example 5.

[0063] After 6 hours of treatment, the culture medium was discarded, and the cells were washed with PBS. A mixed staining solution of Calcein-AM (2 µM) and propidium iodide (PI, 4 µM) was added, and the cells were incubated in the dark for 20 minutes. After washing again with PBS, the cells were observed and photographed under a fluorescence microscope: live cells showed green fluorescence, and dead cell nuclei showed red fluorescence.

[0064] The results are as follows Figure 7 As shown, compared with the control group, the PRSP plus laser irradiation group had the highest proportion of dead cells, which directly confirms its synergistic killing efficacy.

[0065] Based on the results of the above embodiments, the nanotherapeutic agent provided by this invention achieves active targeting of tumor cells through folic acid modification, enables heat-triggered drug release through the UCST carrier properties, alleviates tumor hypoxia and enhances photodynamic therapy by loading perfluorohexane (PFH), and the loaded thalimycin helps overcome multidrug resistance. In vitro experiments show that this system has good cellular uptake, reactive oxygen species generation, and synergistic antitumor effects.

[0066] The above embodiments only illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A self-oxygenated UCST-type nanotherapeutic agent for targeted synergistic therapy of triple-negative breast cancer, characterized in that, The nanotherapeutic agent uses thermosensitive polymer nanoparticles with a high critical solution temperature (UCST) as a carrier, on which chemotherapy drugs, photosensitizers, targeting ligands and liquid perfluorocarbon compounds are co-loaded; wherein, the UCST phase transition temperature of the thermosensitive polymer is 42±2℃.

2. The nanotherapeutic agent according to claim 1, characterized in that, The temperature-sensitive polymer is poly(acrylic acid-acrylonitrile-butyl hexafluoromethacrylate), which is copolymerized by acrylic acid (AA), acrylonitrile (AN) and butyl hexafluoromethacrylate (C7F6) through a reversible addition-fragmentation chain transfer polymerization reaction, wherein the molar ratio of AA, AN and C7F6 is 4:1:

1.

3. The nanotherapeutic agent according to claim 1, characterized in that, The chemotherapeutic drug is thalinomycin or a pharmaceutically acceptable salt thereof; the photosensitizer is a ruthenium(II) polypyridine complex; the targeting ligand is folic acid; and the liquid perfluorocarbon compound is perfluorohexane.

4. The nanotherapeutic agent according to claim 3, characterized in that, The ruthenium(II) polypyridine complex is Ru(pdt)3Cl2, wherein pdt is 1,10-phenanthroline-5,6-dione.

5. The nanotherapeutic agent according to any one of claims 1-4, characterized in that, The mass ratio of the carrier to the chemotherapy drug, photosensitizer, and targeting ligand is 10:1:1:1; the amount of liquid perfluorocarbon compound added is 0.5%-5% of the carrier mass.

6. A method for preparing a self-oxygen-loaded UCST-type nanotherapeutic agent according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of thermosensitive polymer carrier: Acrylic acid, acrylonitrile, butyl hexafluoromethacrylate, RAFT chain transfer agent and initiator are dissolved in an organic solvent, and after deoxygenation, the mixture is reacted at 55-65℃ for 20-28 hours under an inert atmosphere. After the reaction is completed, the mixture is purified to obtain P(AA-AN-C7F6) polymer. S2. Co-assembly of nanotherapeutic agents: The P(AA-AN-C7F6) polymer obtained in step S1, the chemotherapeutic drug, photosensitizer, and targeting ligand are dissolved in dimethyl sulfoxide to obtain stock solutions of each component; the stock solutions of each component are mixed in proportion, and under ice bath and stirring conditions, deionized water is slowly added to the mixture, followed by the addition of liquid perfluorocarbon compound; S3. Ultrasonic induction and post-processing: The mixed system obtained in step S2 is subjected to ultrasonic treatment to induce the formation of nanoparticles. Subsequently, the obtained nano dispersion is purified by dialysis and freeze-dried to obtain the nanotherapeutic agent powder.

7. The preparation method according to claim 6, characterized in that, In step S1, the RAFT chain transfer agent is cyanomethyl dodecyl carbonyl trithiocarboxylate, and the initiator is azobisisobutyronitrile.

8. The preparation method according to claim 6, characterized in that, In step S2, the final concentrations of the P(AA-AN-C7F6) polymer, chemotherapeutic drug, photosensitizer, and targeting ligand in the mixture are 1-5 mg / mL, 0.1-0.5 mg / mL, 0.1-0.5 mg / mL, and 0.1-0.5 mg / mL, respectively.

9. The preparation method according to claim 6, characterized in that, In step S3, the conditions for ultrasonic treatment are: power 200-400W, pulse mode, working for 3-5 seconds followed by an interval of 3-5 seconds, and a total ultrasonic time of 2-5 minutes.

10. The use of a self-oxygen-loaded UCST nanotherapeutic agent according to any one of claims 1-5 in the preparation of a medicament for the diagnosis and / or treatment of triple-negative breast cancer.

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