Platinum boron phosphorus ternary nanoparticle and preparation method and application thereof

Abstract

The application belongs to the technical field of tumor drugs, and particularly relates to platinum-boron-phosphorus ternary nanoparticles as well as a preparation method and application thereof. The platinum-boron-phosphorus ternary nanoparticles have a particle size of 450nm-550nm and are composed of three elements of platinum, boron and phosphorus. Compared with PtNPs alone, the ternary nanoparticles Pt-B-PNPs have stronger catalytic capacity, i.e. the efficiency of catalyzing H2O2 to decompose to generate O2 is significantly improved, and have lower toxicity. Under the stimulation of ultrasonic waves (US), the ternary nanoparticles can play a synergistic effect with a sonosensitizer chlorin E6 to generate a large amount of reactive oxygen species (ROS), and then effectively kill tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of tumor drug technology, specifically relating to a platinum-boron-phosphorus ternary nanoparticle, its preparation method, and its application. Background Technology

[0002] Sonodynamic therapy (SDT) is a modern and novel form of cancer treatment designed to replace traditional tumor ablation methods and address the shortcomings of photodynamic therapy (PDT). Traditional tumor ablation methods, in addition to killing tumor tissue, can also damage adjacent tissues and organs. Furthermore, chemotherapy drugs, besides leading to tumor resistance, can cause toxic damage to the patient's major organs. Unlike the two traditional tumor treatment methods mentioned above, the principle of SDT is roughly the same as that of PDT. First, the enhanced permeability and retention effect (EPR) of solid tumors is used to enrich the acoustic sensitizer at the tumor site. Then, the tumor tissue is locally irradiated with ultrasound of a specific intensity to effectively activate the acoustic sensitizer. Oxygen (O2) in the solid tumor tissue can accept the energy transferred by the acoustic sensitizer in the excited state to form highly cytotoxic reactive oxygen species (ROS), which effectively kill tumor cells. The three components used in SDT treatment, ultrasound (US), acoustic sensitizer, and O2, are non-toxic and have no toxic effect on normal organisms.

[0003] However, in order to obtain more oxygen and nutrients than normal tissues during the growth process, solid tumor tissues often secrete a series of growth factors related to tumor angiogenesis (such as vascular endothelial growth factor); and when the tumor tissue grows to 150-200 micrometers in size, it becomes highly dependent on the supply of nutrients and oxygen from the tumor blood vessels. Therefore, the secretion of factors that promote angiogenesis increases. However, the tumor blood vessels formed at this time are very different from normal blood vessels in terms of structure and morphology, mainly in the following aspects: (1) The overall vascular structure is disordered, the shape is swollen, there are more cystic vessels, and the characteristics of multiple branches; (2) The structure of the blood vessel wall in the tumor tissue is abnormal, and the number of fissures increases; (3) The morphology of endothelial cells is abnormal, and they grow into the lumen; (4) The morphology of pericytes is abnormal, and their function is insufficient. The above characteristics will lead to an increase in ineffective circulation and local blood leakage, the interstitial hydraulic pressure will continue to rise, the hypoxic state will be further aggravated, and ultimately the formation of the malignant tumor microenvironment (TME) will occur. Therefore, although SDT has a series of advantages, the production of ROS, a key molecule for its cytotoxic effect, requires a large amount of O2. Thus, the hypoxic state of TME and the resulting tumor cell hypoxia further limit the application of SDT in tumor treatment. Simultaneously, SDT itself consumes O2 during treatment, which further exacerbates the hypoxia at the tumor site, thereby further inhibiting the therapeutic effect of SDT.

[0004] In existing technologies, dihydroporphyrin E6 (ce6) can be used as a photosensitizer, and Pt NPs as a catalyst to increase the ROS content within solid tumors. Although Pt NPs catalysts can increase the rate of ROS generation from dihydroporphyrin to some extent, the increase in rate and the overall ROS content are relatively low, resulting in poor SDT treatment efficacy. Therefore, it is necessary to develop a catalyst that can increase the rate of ROS generation from dihydroporphyrin. Summary of the Invention

[0005] To address the above problems, one of the objectives of this invention is to provide a ternary nanoparticle, Pt-BP NPs, which is based on improved Pt NPs. This nanoparticle can efficiently catalyze the decomposition of H2O2 in solid tumor cells to generate O2, thereby alleviating the hypoxia of tumor cells. Furthermore, it enhances the ability of Ce6@PLGA NPs to generate ROS after ultrasound stimulation, thus enhancing the therapeutic effect of SDT.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: The present invention provides a platinum-boron-phosphorus ternary nanoparticle with a particle size of 450nm-550nm, which is composed of platinum, boron and phosphorus.

[0007] Another aspect of the present invention provides a method for preparing the above-mentioned platinum-boron-phosphorus ternary nanoparticles, which includes: (1) adding polyethylene glycol aqueous solution to polystyrene microsphere aqueous solution and adjusting the pH to 7-9 to obtain a mixture; (2) adding H2PtCl4 solution to the mixture, letting it stand, adding NH3·H2O, H3PO3 and NaBH4 and reacting in an environment with pH of 3-5 and temperature of 100℃-120℃, washing, and centrifuging to obtain platinum-boron-phosphorus ternary nanoparticles.

[0008] In another aspect, the present invention provides a composition for treating solid tumors, comprising the above-mentioned platinum-boron-phosphorus ternary nanoparticles and a photosensitizer for photodynamic therapy and / or sonodynamic therapy.

[0009] In another aspect, the present invention provides the use of the above-mentioned platinum-boron-phosphorus ternary nanoparticles in the preparation of agents for photodynamic therapy and / or sonodynamic therapy of solid tumors.

[0010] The beneficial effects of the present invention include: (1) The ternary nanoparticles Pt-BP NPs provided by the present invention have stronger catalytic ability than Pt NPs alone, that is, the efficiency of catalyzing the decomposition of H2O2 to produce O2 is significantly improved; and they have lower toxicity; under the stimulation of ultrasound (US), they can work synergistically with the acoustic sensitizer dihydroporphyrin E6 to produce a large amount of reactive oxygen species (ROS), thereby effectively killing tumor cells.

[0011] (2) The SDT cell killing effect of Pt-BP NPs provided by the present invention can not only significantly induce cell apoptosis, but also effectively trigger cell necrosis signaling pathway. Attached Figure Description

[0012] Figure 1 The effect of different molar ratios of H3PO3 and NaBH4 on the particle size of Pt-BP NPs; Figure 2 A) Characterization of Pt-BP NPs; B) Transmission electron microscopy (TEM) image of Pt-BP NPs; C) Particle size distribution of Pt-BP NPs; D) Zeta potential of Pt-BP NPs; EDS elemental analysis of Pt-BP NPs.

[0013] Figure 3 XPS spectra analysis of Pt-BP NPs; A represents Pt element; B represents B element; C represents the XPS spectrum of P element; Figure 4Characterization of Ce6@PLGA NPs; A is a transmission electron microscope image of Ce6@PLGA NPs; B is a particle size distribution map of Ce6@PLGA NPs; C is a Zeta potential map of Ce6@PLGA NPs; D is a drug release curve of Ce6@PLGA NPs (10 μg / mL). Figure 5 Characterization of Pt NPs; A is a transmission electron microscope image of Pt NPs; B is a comparison of the catalytic performance of Pt NPs and commercial Pt NPs (10 μg / mL). Figure 6 The values ​​are: A) Catalytic current of Pt-BP NPs under different environmental conditions; B) Catalytic current of Pt-BP NPs under different pH conditions; C) Catalytic current of Pt-BP NPs after 1 month of storage; D) Catalytic current of Pt-BP NPs under different intensities of ultrasonic stimulation; E) Catalytic current of Pt-BP NPs at the same concentration in H2O2 solutions of different concentrations (10 μg / mL⁻¹). Figure 7 The oxygen production capacity of Pt-BP NPs was observed. Figure 8 SOSG results of Ce6@PLGA NPs under different experimental conditions: A shows the SOSG fluorescence intensity analysis results of Ce6@PLGA NPs (10 μg / mL) under different US irradiation times; B shows the SOSG fluorescence intensity analysis results of Pt NPs and Pt-BP NPs (10 μg / mL) alone under different US irradiation times; C shows the SOSG fluorescence intensity analysis results of the reaction of Pt and Pt-BP NPs with Ce6@PLGA NPs (10 μg / mL) in H2O2 aqueous solution. Figure 9 The study observed the hemolytic effects of Pt NPs, Pt-BP NPs, and Ce6@PLGA NPs; I: positive control group; II: Ce6@PLGA NPs group; III: Pt NPs group; IV: Pt-BP NPs group; V: Pt+Ce6@PLGA NPs group; VI: Ce6@PLGA+Pt-BP NPs group; VII: negative control group. Figure 10The study investigated the uptake of Pt NPs, Pt-B-PNPs, and Ce6@PLGA NPs by SKOV3 ovarian cancer cells. A) CLSM was used to evaluate the uptake of different NP types after incubation for 1 h and 4 h: I. Pt NPs; II. Pt-B-PNPs; III. Ce6@PLGA NPs. B) A fluorescence microplate reader was used to evaluate the uptake of Pt NPs after incubation for 1 h and 4 h. C) A fluorescence microplate reader was used to evaluate the uptake of Pt-B-PNPs after incubation for 1 h and 4 h. D) A fluorescence microplate reader was used to evaluate the uptake of Ce6@PLGA NPs after incubation for 1 h and 4 h. Figure 11 The results show the analysis of different types of NPs in different cancer cells; A represents SKOV3 ovarian cancer cells; B represents the CCK-8 toxicity analysis results in A2780 ovarian cancer cells. Figure 12 A compares the catalytic performance of Pt-BP NPs and Pt NPs in SKOV3 cells; A shows the analysis results of H2O2 consumption and O2 production efficiency of Pt NPs and Pt-B-PNPs; B shows the expression of HIF-1α in the SKOV3 cell line. Figure 13 Analysis of apoptosis and necrosis signaling pathways in ovarian cancer cells after SDT treatment; A: Detection and analysis of apoptosis signaling pathway; B: Detection and analysis of necrosis signaling pathway. Figure 14 To detect the ROS content in ovarian cancer cells after SDT treatment; A represents the amount of intracellular ROS produced under different experimental conditions; B represents the detection of intracellular ROS content by a fluorescence microplate reader; I: Control group; II: US treatment group; III: US+Pt NPs group; IV: US+Pt-BP NPs group; V: US+Ce6@PLGA NPs group; VI: US+Ce6@PLGA+Pt NPs group; VII: US+Ce6@PLGA+Pt-BP NPs group; Figure 15 The results show the cell death of ovarian cancer cells after SDT treatment; A: Cell death observation under confocal microscopy; B: Cell death detection results by CCK-8 assay; C: Cell death detection results by flow cytometry. I: Control group; II: US treatment group; III: US + Pt NPs group; IV: US + Pt-BP NPs group; V: US + Ce6@PLGA NPs group; VI: US + Ce6@PLGA + Pt NPs group; VII: US + Ce6@PLGA + Pt-BP NPs group. Figure 16Fluorescence imaging analysis of drugs at tumor sites; A shows the metabolism of different types of NPs at the tumor site after orthotopic injection; B shows the aggregation of major organs after orthotopic injection of Pt-BP NPs; I: Pt NPs group; II: Pt-BP NPs group; III: Ce6@PLGA NPs group; IV: Ce6@PLGA+Pt NPs group; V: Ce6@PLGA+Pt-BP NPs group; Figure 17 Figure 1 shows the changes in physiological indicators of mice during the 16-day treatment period; Figure 2 shows the changes in tumor volume of mice; Figure 3 shows the changes in body weight of mice. I: Control group; II: US treatment group; III: US+Pt NPs group; IV: US+Pt-BP NPs group; V: US+Ce6@PLGA NPs group; VI: US+Ce6@PLGA+Pt NPs group; VII: US+Ce6@PLGA+Pt-BP NPs group (n=5). Figure 18 The in vivo antitumor effects of Pt-BP NPs and Ce6@PLGA NPs are shown in Figure A. A photograph of tumor-bearing mice after 16 days of treatment; B. HE staining of tumors from dissected tumor-bearing mice after 16 days of treatment; C. TUNEL staining of tumors from dissected tumor-bearing mice after 16 days of treatment; D. A photograph of tumors from dissected tumor-bearing mice after 16 days of treatment. I: Control group; II: US group; III: US+Pt NPs group; IV: US+Pt-BP NPs group; V: US+Ce6@PLGA NPs. Figure 19 H&E staining analysis of major organs; Figure 20 For the analysis of blood biochemical indicators. Detailed Implementation

[0014] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0015] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0016] This invention provides a platinum-boron-phosphorus ternary nanoparticle with a particle size of 450mm-550mm, such as 460mm, 480mm, 500mm, 520mm, or 540mm; it is composed of platinum, boron, and phosphorus. It should be noted that the molar ratio of B to P can be (1-3):(1-3), such as 1:2, 2:1, 1:3, or 3:1; furthermore, the molar ratio of Pt, B, and P can be 0.005:(1-3):(1-3). It should also be noted that different molar ratios do not significantly affect the particle size of the prepared boron-phosphorus ternary nanoparticles.

[0017] These platinum-boron-phosphorus ternary nanoparticles (containing boron and phosphorus, both beneficial trace elements with good biocompatibility) exhibit stronger catalytic activity than nanoparticles prepared solely from platinum, showing a significantly improved efficiency in catalyzing the decomposition of H₂O₂ to produce O₂. They also possess lower toxicity. Under ultrasound (US) stimulation, they synergistically interact with the sonosensitive agent dihydroporphyrin E6 to generate a large amount of reactive oxygen species (ROS), effectively killing tumor cells. Furthermore, Pt-BP NPs demonstrate good biostability, do not cause erythrocyte rupture, and do not require an additional carrier to perform their catalytic function. This avoids the increased costs and poor stability associated with complex synthesis in future industrial applications, while also preventing potential drug toxicity and financial burdens on patients caused by the use of excessive carrier materials.

[0018] Furthermore, after effective accumulation at the tumor site, nanomedicines are metabolized by the tumor tissue and participate in the body's circulation. To avoid damage to major organs and the vascular system, the size of the nanomedicines is crucial. Currently, the size of nanomedicines used in vivo is mainly distributed between 0 and 700 nm (excluding 0). The platinum-boron-phosphorus ternary nanoparticles in this invention fall within this range and meet the requirements.

[0019] Another embodiment of the present invention provides a method for preparing the above-mentioned platinum-boron-phosphorus ternary nanoparticles, which includes: (1) adding polyethylene glycol aqueous solution to polystyrene microsphere aqueous solution and adjusting pH to 7-9 to obtain a mixture; (2) adding H2PtCl4 solution to the mixture, letting it stand, adding NH3·H2O, H3PO3 and NaBH4 and reacting in an environment with pH of 3-5 and temperature of 100℃-120℃, washing, and centrifuging to obtain platinum-boron-phosphorus ternary nanoparticles.

[0020] In this invention, a nano-alloy preparation approach is used to reduce Pt NPs via a hydrothermal method using PS as a reaction template and NaBH4 and H3PO3 as co-catalysts. NaBH4 and H3PO3, in addition to acting as catalysts, also serve as sources of B and P elements, thus jointly forming Pt-BP NPs. Furthermore, this synthesis method is green, simple, and widely applicable.

[0021] In some specific embodiments, in the above-mentioned method for preparing platinum-boron-phosphorus ternary nanoparticles, the pH is adjusted to 8 in step (1); and / or the reaction is carried out in an environment with a pH of 4 in step (2).

[0022] Another embodiment of the present invention provides a composition for treating solid tumors, comprising the above-described platinum-boron-phosphorus ternary nanoparticles and a photosensitizer for photodynamic therapy and / or sonodynamic therapy. The above-described platinum-boron-phosphorus ternary nanoparticles can be combined with a photosensitizer for photodynamic therapy to generate a large amount of reactive oxygen species to kill tumors, or they can be combined with a photosensitizer suitable for sonodynamic therapy (ultrasound) to generate a large amount of reactive oxygen species to kill tumors.

[0023] In some specific embodiments, the photosensitizers suitable for photodynamic therapy and / or sonodynamic therapy are in nanoscale form. In these embodiments, the excellent film-forming properties of PLGA can be utilized to encapsulate Ce6 via ultrasonic emulsification to form Ce6@PLGA nanospheres, thereby improving the water solubility of Ce6. Besides nanoscale forms, other forms that can participate in in vivo circulation are also possible.

[0024] In some specific embodiments, the photosensitizer for the aforementioned photodynamic therapy and / or sonodynamic therapy is preferably dihydroporphyrin E6. Ce6, as a classic photosensitizer molecule, is widely used in PDT. However, due to the limited tissue penetration depth of PDT, its therapeutic effect on deep tumors is often poor. In Ce6@PLGA NPs, the presence of the sonosensitive molecule Ce6 allows for some inhibition of tumor growth when external ultrasound stimulation is applied. However, due to the hypoxic environment of the tumor microenvironment, its ability to inhibit tumor growth is weak. Since Pt NPs have a weak ability to catalyze the decomposition of intracellular H2O2, when Pt NPs + Ce6@PLGA NPs are used in combination, the tumor growth inhibition effect under external ultrasound stimulation is not significantly different from that of Ce6@PLGA NPs alone. Pt-BP NPs, due to their excellent ability to catalyze the decomposition of intracellular H2O2 to produce O2, produce the greatest anti-tumor effect when used in combination with Ce6@PLGA NPs.

[0025] Another embodiment of the present invention provides the application of the above-mentioned platinum-boron-phosphorus ternary nanoparticles in the preparation of agents for photodynamic therapy and / or sonodynamic therapy of solid tumors.

[0026] In some specific embodiments, solid tumors include, but are not limited to: breast fibroids, uterine fibroids, breast cancer, liver cancer, lung cancer, brain tumors, schwannomas, soft tissue sarcomas, osteosarcomas, gastric cancer, colon cancer, kidney cancer, bladder cancer, ovarian cancer, prostate cancer, lymphoma, and myeloma.

[0027] In some specific embodiments, the solid tumor can be ovarian cancer.

[0028] In some specific embodiments, applications include the use of platinum-boron-phosphorus ternary nanoparticles in the preparation of drugs for treating ovarian cancer by promoting the expression of C-Caspase3,8,9 and C-PARP.

[0029] This invention, through in-depth analysis of the ovarian cancer cell death mechanism induced by Pt-BP NPs combined with Ce6@PLGANPs and SDT, reveals for the first time in this study that the mechanism inducing ovarian cancer cell death, in addition to traditional apoptosis, also triggers the cell necrosis signaling pathway. Caspase-8 and 9 are two other key molecules in the apoptosis mechanism network and can form a positive feedback effect with caspase 3. PARP is a multifunctional protein post-translational modification enzyme present in most eukaryotic cells, playing an important role in maintaining cell homeostasis. When cells undergo apoptosis, caspase 3 is activated, thereby cleaving PARP to form inactive C-PARP. Therefore, an increase in C-PARP content is also a marker of the onset of apoptosis.

[0030] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0031] In this embodiment of the invention, the main instruments and equipment used include: Malvern laser particle size analyzer (ZEN3600) from Malvern Panalytical, X-ray electron spectrometer (ESCALAB250Xi) from Thermofisher, USA, transmission electron microscope (S-4800) from Hitachi, Japan, dissolved oxygen analyzer (JPBJ-608) from INESAS Scientific, Shanghai, low-speed centrifuge (TD4N) from Hunan Xiangyi Co., Ltd., optical microscope (E100) from Nikon, Japan, confocal microscope (Ti2-E) from Nikon, Japan, fluorescence microplate reader from BMGLABTECH, Germany, flow cytometer (Cytoflex S) from Beckman Coulter, USA, 660nm laser emitter from Xi'an Zhongchuan Optoelectronics Co., Ltd., gel imaging system (Doc™ XR) from Bio-Rad, Shanghai, and small animal in vivo fluorescence imaging system from Viber Lourmat, France.

[0032] In this embodiment of the invention, the main reagents used include: PS nanospheres from Suzhou Zhiyi Technology Co., Ltd., PtNPs from Sigma-Aldrich (USA), chloroplatinic acid (H2PtCl4) from Sigma-Aldrich (USA), sodium borohydride (NaBH4) from Nantong Hongzhi Chemical Co., Ltd., phosphorous acid (H3PO3) from Nantong Hongzhi Chemical Co., Ltd., dihydroporphyrin e6 (Ce6) from Shanghai Maclean Co., Ltd., 30wt% hydrogen peroxide aqueous solution (H2O2) from Sigma-Aldrich (USA), singlet oxygen assay kit (SOSG) from Thermofisher (USA), McCoy's 5A complete medium from Wuhan Pronosai Co., Ltd., A2780 complete medium from Wuhan Pronosai Co., Ltd., CCK-8 assay kit from Shanghai Beyotime Co., Ltd., and Ru(DPP)3Cl2 from Sigma-Aldrich (USA). H2DCFHDA comes from Sigma-Aldich, Inc. in the United States.

[0033] In this embodiment of the invention, the experimental animals used were 4-6 week old SPF-grade female nude mice provided by Chongqing Enswell Co., Ltd. Animal husbandry and related experimental procedures all complied with the "Regulations for the Management of Experimental Animals" approved by the Animal Ethics Committee of Chongqing Medical University.

[0034] In this embodiment of the invention, both the SKOV3 ovarian cancer cell line and the A2780 ovarian cancer cell line were provided by Wuhan Pronosai Co., Ltd.

[0035] I. Preparation of Pt-BP NPs, Pt NPs and Ce6@PLGA NPs Example 1: Preparation of Pt-BP NPs After carefully cleaning the weighing spoon and glass rod with ultrapure water and anhydrous ethanol in an ultrasonic cleaner, wipe away excess water with filter paper. Accurately weigh 20 mg of PS (polystyrene microspheres) using an electronic balance and dissolve it in 6 mL of ultrapure water. Stir the liquid in the beaker evenly with a glass rod. After the solution stabilizes, place the beaker on a magnetic stirrer and stir at a rate of 400 rpm / min. During the stirring process, add freshly prepared polyethylene glycol (PEG) aqueous solution dropwise to the beaker. The pH of the solution was then adjusted to 8.0 using sodium hydroxide and concentrated hydrochloric acid. After the solution stabilized, 0.5 mL of 20 mL H2PtCl4 was added to the solution, ensuring that the pipette was suspended in the air and that the drops were spaced apart to avoid continuous addition and the formation of a liquid stream. After the solution was allowed to stand at room temperature for 10 min, 0.4 mL of freshly prepared 10 wt% NH3·H2O and appropriate amounts of H3PO3 and NaBH4 were added. The solution was then incubated in a 110°C water bath for 10 min, followed by the addition of toluene solution to adjust the pH to 4, and the reaction was continued for 3 h. Finally, the solution was washed with ultrapure water, ethanol, and ultrapure water in that order, and then centrifuged at 10000 rpm to obtain the final product.

[0036] Different Pt-BP NPs were prepared by setting the molar ratio of NaBH4 to H3PO3 to 1:0, 0:1, 1:2, 2:1, 1:3 and 3:1 respectively, according to the preparation method described above.

[0037] Example 2 Preparation of Pt NPs The weighing spoon was carefully cleaned in an ultrasonic cleaner with ultrapure water and anhydrous ethanol, and excess water was wiped off with filter paper. 10 mg of H2PtCl4 was accurately weighed using an electronic balance and dissolved in 10 mL of ultrapure water to prepare a 5% H2PtCl4 aqueous solution. Then, 200 μL of H2PtCl4 was accurately pipetted into 10 mL of ultrapure water, and 10 mL of NaBH4 solution (0.28 mg / mL) was added to the mixed solution. The mixture was placed on a magnetic stirrer and reacted at room temperature for 1 h (400 rpm / min). The black reaction solution was then centrifuged in a high-speed centrifuge (12000 rpm / min). Finally, the solution was washed three times with ultrapure water and ethanol to obtain PtNPs.

[0038] Example 3 Preparation of sound-sensitizing agent Ce6@PLGA NPs Ce6@PLGA NPs were prepared using an ultrasonic emulsification strategy, with the following steps: First, a weighing spoon was carefully cleaned in an ultrasonic cleaner with ultrapure water and anhydrous ethanol, and excess water was wiped away with filter paper. 50 mg of PLGA powder and 2 mg of Ce6 powder were accurately weighed using an electronic balance and dissolved in 2 mL of dichloromethane to prepare a suspension. Then, 8 mL of 4% polyvinyl alcohol solution was accurately pipetted into the above suspension, and ultrasonic emulsification was performed for 5 min using an ultrasonic emulsion apparatus to obtain a black emulsion. Next, 10 mL of 2% isopropanol was added to the solution, and the mixture was stirred at room temperature for 4 hours to evaporate excess dichloromethane. Finally, the product was washed with ultrapure water at 4°C to obtain the final product.

[0039] II. Characterization of Pt-BP NPs, Pt NPs and Ce6@PLGA NPs Example 4 Characterization of Pt-BP NPs (1) Pt-BP NPs particle size characterization prepared from precursors with different molar ratios 1.5 mL of Pt-BP NPs solutions prepared with different molar ratios of H3PO3 and NABH4 in Example 1 were taken and their particle size distribution was analyzed using a Malvern particle size analyzer. The results are as follows: Figure 1 As shown, the horizontal axis represents the diameter of the synthesized NPs, and the vertical axis represents the distribution intensity of NPs with a specific particle size. When the molar ratio of NaBH4 to H3PO3 is 1:0, that is, NaBH4 is used alone as a reducing agent, the measured NP particle size distribution intensity is 0, indicating that no NPs are formed. When the molar ratio of NaBH4 to H3PO3 is 0:1, the measured NP particle size distribution intensity is also 0, consistent with the previous results, indicating that no NPs are formed. This indirectly proves that using two reducing agents together is crucial for the formation of Pt-BP NPs.

[0040] Furthermore, when the molar ratio of NaBH4 to H3PO3 was 1:2, the particle size distribution of the synthesized Pt-BP NPs was mainly around 500 nm; when the molar ratios were 2:1, 1:3, and 3:1, the particle size distribution of the prepared Pt-BP NPs was also mainly around 500 nm. This indicates that the molar ratio of NaBH4 to H3PO3 has little effect on the particle size. For subsequent verification, a molar ratio of NaBH4 to H3PO3 of 1:2 was selected.

[0041] (2) Characterization of Pt-BP NPs prepared from precursors with a molar ratio of 1:2 (a) Particle size characterization 1.5 mL of the Pt-BP NPs solution prepared in Example 1 with a molar ratio of 1:2 was taken and its zeta potential was determined using a Malvern particle size analyzer. The morphology of the Pt-BP NPs was observed using transmission electron microscopy (TEM), and elemental distribution analysis was performed using X-ray energy dispersive spectroscopy. The results are as follows: Figure 2 As shown, the synthesized Pt-BP NPs are generally spherical with a relatively uniform morphology and a particle size of about 500 nm. This result is consistent with the particle size analyzer results (see...). Figure 2 A and Figure 2 B).

[0042] (b) Surface potential characterization In addition, the Pt-BP NPs in (a) were placed in a Malvern laser particle size analyzer for further observation of their surface potential, and the results are as follows. Figure 2 As shown in Figure C, the horizontal axis represents the electric potential intensity, and the vertical axis represents the number of particles. The results show that the surface potential of Pt-BP NPs is concentrated around -21.33mV. The NPs surface is negatively charged, and the plasma proteins in blood vessels are also negatively charged. According to the principle of like charges repelling each other, Pt-BP NPs will not bind to the blood vessel wall or the main proteins in the plasma, thereby reducing the possibility of vascular embolism.

[0043] (c) Element composition, i.e., valence state In addition, the elemental composition of Pt-BP NPs in (a) was observed using Energy Dispersive Spectroscopy (EDS), and the results are as follows: Figure 2 As shown in Figure D, Pt-BP NPs simultaneously contain Pt, B, and P elements. The valence states of these elements were observed using X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 3 As shown.

[0044] The above experimental results fully demonstrate the successful synthesis of Pt-BP NPs.

[0045] Example 5 Characterization of Ce6@PLGA NPs 10 mg of Ce6@PLGA NPs lyophilized powder was dispersed in PBS and its particle size distribution and zeta potential were detected by Malvern particle size analyzer; its morphology was observed by TEM.

[0046] The morphological characteristics of Ce6@PLGA NPs are as follows: Figure 4As shown, the synthesized Ce6@PLGA NPs are generally spherical and have a relatively uniform morphology. PLGA forms a transparent thin film that coats the Ce6 particles, with a particle size of around 120 nm. This result is consistent with the particle size analyzer results. Figure 4 A and Figure 4 B), and the particle size distribution range is relatively narrow, further proving that the synthesized Ce6@PLGANPs have good uniformity.

[0047] The surface charge detection results of Ce6@PLGANPs are as follows: Figure 4 As shown in Figure C, Ce6@PLGANPs carry a negative charge on their surface, with an absolute value of 6.03±0.2mV, indicating that Ce6@PLGA NPs have good potential for in vivo applications.

[0048] The release of Ce6 from Ce6@PLGA NPs under US stimulation was also measured, and the results are as follows: Figure 4 As shown in Figure D, the horizontal axis represents the time after US irradiation, and the vertical axis represents the drug release efficiency, which is calculated as: Drug release efficiency = Absorbance value of supernatant / Absorbance value of original solution * 100%. The Ce6 release rate is highest within the first two hours after US irradiation, gradually slowing down over time, reaching 80% drug release efficiency after 10 hours.

[0049] Example 6 Characterization of Pt NPs In this embodiment of the invention, the catalytic performance testing method includes: pouring 0.3 μm diameter aluminum oxide powder onto deer hide and diluting it with ultrapure water; then placing a glassy carbon electrode on the deer hide, polishing it clean, and placing it in a clean beaker; then cleaning it in an ultrasonic cleaner for 3 minutes each in the order of ultrapure water, anhydrous ethanol, and ultrapure water; then placing the electrode upside down on a clean foam board and drying it in a dryer at 37°C.

[0050] Take 0.5 μL of Pt NPs aqueous solution (10 mg / mL) -1 As for the observation using TEM after drying on a clean glass slide, the results are as follows: Figure 5 As shown in Figure A, the prepared Pt NPs are irregularly spherical with a particle size of approximately 120 nm.

[0051] In addition, to verify the catalytic performance of Pt NPs, the catalytic performance of Pt NPs of the same concentration and commercial Pt NPs (Sigma-Aldrich, USA) in H2O2 aqueous solutions of different concentrations was compared. The results are as follows: Figure 5 As shown in B, the prepared Pt NPs exhibit catalytic performance consistent with commercial Pt NPs and can be used for further research.

[0052] III. Performance Testing of Pt-BP NPs Example 7: Observation of catalytic stability and comparison of catalytic ability of Pt-BP NPs under different environments Electrochemical sensor technology is a detection method that converts the biochemical signal of a substance to be detected into an electrical signal using a specially designed sensor. It is widely studied due to its high sensitivity, wide detection range, and low cost. Since H2O2 undergoes electron transfer during a redox reaction, this invention uses a conventional glassy carbon electrode to construct a three-electrode detection system to detect the number of electrons generated by Pt NPs and Pt-BP NPs in the catalytic decomposition of H2O2 to produce O2, and records the results using time-current curves. The results are as follows: Figure 6 As shown in Figure E (where the larger values ​​on the vertical axis represent Pt-BP NPs and the smaller values ​​represent Pt NPs), the horizontal axis represents the reaction time and the vertical axis represents the change in current intensity. The larger the absolute value, the greater the amount of electrons transferred.

[0053] In this embodiment of the invention, the glassy carbon electrode was treated according to the following method before the following experiments were conducted: Alumina powder with a diameter of 0.3 μm was poured onto deerskin and diluted with ultrapure water. The glassy carbon electrode was then placed on the deerskin, polished clean, and placed in a clean beaker. It was then cleaned in an ultrasonic cleaner for 3 minutes each time with ultrapure water, anhydrous ethanol, and then ultrapure water in that order. The electrode was then placed upside down on a clean foam board and dried in a dryer at 37°C.

[0054] (1) Observation of pH stability of Pt-BP NPs Prepare aqueous solutions with pH values ​​of 1, 2, 3, 4, 5, 6, and 7.4 using sodium hydroxide and concentrated hydrochloric acid under a pH meter. Add 10 μL of Pt-BP NPs aqueous solution to a clean glassy carbon electrode and dry it for later use. Connect a saturated calomel electrode, a platinum wire electrode, and a working electrode with added Pt-BP NPs to form a three-electrode detection system. Insert the working electrode into the aqueous solutions with pH values ​​of 1, 2, 3, 4, 5, 6, and 7.4 respectively, and add 30 wt% H₂O₂ solution when the reaction is stable. Detect and record the current curves using an electrochemical workstation.

[0055] The results are as follows Figure 6As shown in Figure A, the results indicate the changes in the catalytic ability of Pt-BP NPs to decompose H2O2 under different pH reaction conditions. Since H2O2 is easily decomposed under alkaline conditions and the tumor microenvironment is generally acidic, the changes in the catalytic performance of Pt-BP NPs under different acidic conditions were mainly simulated. When the pH values ​​were 7.4, 6, 5, 4, 3, 2 and 1, the catalytic current value increased from 540 mA to 580 mA. The increase in the absolute value of the current may be due to the increased decomposition rate of H2O2 under acidic conditions, which led to a slight increase in the absolute value of the catalytic current.

[0056] (2) Observation of the time stability of Pt-BP NPs Each glassy carbon electrode was then dried again by adding 10 μL of LPt-BP NPs aqueous solution. The glassy carbon electrodes were then placed at room temperature for 30 days, and their catalytic current curves were measured using an electrochemical workstation on days 0, 5, 10, 15, 20, 25, and 30. The measurement method involved first connecting a saturated calomel electrode, a platinum wire electrode, and a working electrode to form a three-electrode detection system. The prepared working electrode was placed in PBS detection solution, and after the electrode stabilized, 20 μL of 30 wt% H₂O₂ solution (1.6 mol L⁻¹) was added. The current curves were then detected and recorded using an electrochemical workstation.

[0057] The results are as follows Figure 6 As shown in Figure B, the results show that the current value of the working electrode changes little within 30 days, further demonstrating that the prepared Pt-BP NPs have good time stability.

[0058] (3) Observation of temperature stability of Pt-BP NPs Because changes in the surface properties of nanomaterials can trigger a heat-generating reaction when stimulated by US, thus altering the ambient temperature, we simultaneously observed the heat generation of Pt-BP NPs under different ambient temperatures. 10 μL of Pt-BP NPs solution was added to each glassy carbon electrode. The PBS detection solution was heated to 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C. A three-electrode detection system was formed by connecting a saturated calomel electrode, a platinum wire electrode, and a working electrode. The working electrode was placed in the heated PBS detection solution, and after the electrode stabilized, 20 μL of 30 wt% H₂O₂ solution (1.6 mol L⁻¹) was added. The current curves were detected and recorded using an electrochemical workstation.

[0059] The results are as follows Figure 6As shown in Figure C, when the temperature is set to 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, the change in catalytic current at the working electrode is small, further indicating that the catalytic activity of Pt-BP NPs is not significantly affected by ambient temperature.

[0060] (4) Observation of the ultrasonic stability of Pt-BP NPs Each glassy carbon electrode was then dried again by adding 10 μL of LPt-BP NPs aqueous solution. The dried electrodes were then irradiated with ultrasonic waves of 0 W, 0.5 W, 1 W, 1.5 W, and 2 W for 180 s, and the catalytic current curves were then tested.

[0061] The results are as follows Figure 6 As shown in Figure D, the results indicate that the catalytic activity of Pt-BP NPs did not change significantly as the ultrasonic intensity varied from 0 W to 2 W, suggesting that the prepared Pt-BP NPs have good ultrasonic stability.

[0062] (5) Comparison of catalytic abilities of Pt-BP NPs and Pt NPs Each glassy carbon electrode was dried again after being dried with 10 μL of Pt-BP NPs and Pt NPs aqueous solution. Then, a saturated calomel electrode, a platinum wire electrode, and a working electrode were connected to form a three-electrode detection system. The working electrode was placed in a heated PBS detection solution and waited for the electrode to stabilize before adding 20 μL of H2O2 solution (1.6 mol L⁻¹). The current curve was detected and recorded using an electrochemical workstation.

[0063] The results are as follows Figure 6 As shown in Figure E (where the larger values ​​on the ordinate represent Pt-BP NPs and the smaller values ​​represent Pt NPs), the results indicate that the absolute values ​​of the catalytic currents of both Pt-BP NPs and Pt NPs increase with increasing H2O2 concentration. However, the absolute values ​​of the catalytic currents of Pt-B-PNPs are greater than those of Pt NPs at the same concentration, further demonstrating that Pt-B-PNPs exhibit superior catalytic performance for H2O2 decomposition compared to Pt NPs alone. Since Pt-B NPs and Pt-P NPs cannot be synthesized (see Figure E),... Figure 1 Therefore, this study did not conduct a comparative experiment on the catalytic properties of the two types of nanoparticles mentioned above.

[0064] Example 8: Observation of the in vitro oxygen production capacity of Pt-BP NPs To calibrate the dissolved oxygen meter, insert its probe into a zero-oxygen solution. Before actual testing, immerse the probe in the zero-oxygen solution and do not remove it prematurely. Prepare 5 ml of different test solutions (ultrapure water, containing 20 μL of 1M H2O2 aqueous solution, containing 20 μL of LPt NPs and 20 μL of 1M H2O2 aqueous solution, containing 20 μL of LPt-BP NPs and 20 μL of 1M H2O2 aqueous solution). Place the beakers containing the different test solutions on a magnetic stirrer and stir evenly at 350 rpm / min. Insert the dissolved oxygen meter probe into the beaker, being careful not to let it touch the beaker wall. Record the dissolved oxygen reading every 5 seconds for a total of 15 minutes.

[0065] The results are as follows Figure 7 As shown, the horizontal axis represents the dissolved oxygen meter detection time, and the vertical axis represents the O2 concentration. The results show that in the presence of only ultrapure water (H2O), no O2 is generated, as indicated by the flat linear graph. With the addition of H2O2, trace amounts of O2 are detected due to the redox reaction of H2O2 itself. When Pt NPs are added to the system, the O2 production rate increases in the first 100 seconds because Pt NPs can catalyze the decomposition of H2O2 to produce O2. However, due to the limited catalytic performance of Pt NPs on H2O2 decomposition, the O2 production rate slows down after the first 100 seconds and stabilizes after 300 seconds. When the Pt NPs are replaced with newly synthesized Pt-BP NPs, the O2 production rate increases significantly and stabilizes around 500 seconds. Analysis of the blue and purple curves in the figure shows that although Pt NPs can catalyze the decomposition of H2O2 to produce O2, the O2 production rate is low. This further indicates that Pt-BP NPs can significantly improve the catalytic efficiency of Pt NPs, thereby catalyzing the decomposition of more H2O2 to produce O2 in the same amount of time.

[0066] Example 9: Observation of ROS generation capability of Ce6@PLGA NPs 100 μg of SOSG powder was added to 33 μL of methanol-water solution to prepare a 5 mM stock solution. An appropriate amount of the stock solution was diluted to prepare a 5 μM working solution. Ce6@PLGA NPs were added to the 5 μM working solution to prepare a detection solution with a final concentration of 10 μg / mL⁻¹, and the solution was shielded from light with aluminum foil. After ultrasonic irradiation, the solution was aspirated into cuvettes at different time points, and the fluorescence intensity was measured using a fluorescence spectrophotometer. Pt-BP NPs and Pt NPs were separately added to the working solution to prepare a final concentration of 10 μg / mL. -1The detection solution was prepared and shielded from light with aluminum foil. After ultrasonic irradiation, the solution was aspirated into cuvettes at different time points, and the fluorescence intensity was measured using a fluorescence spectrophotometer. Ce6@PLGA NPs, PtNPs, and Pt-BP NPs were aspirated into a 5 μM working solution to prepare a final concentration of 10 μg / mL. -1 The detection solution was prepared by adding 20 μL of H₂O₂ aqueous solutions with concentrations of 0 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.25 mM, and 1.5 mM to the detection solution and shielding it from light with aluminum foil. After ultrasonic irradiation, the solution was aspirated into a cuvette at the reaction endpoint and the fluorescence intensity was measured using a fluorescence spectrophotometer (1 W, 180 s).

[0067] The results are as follows Figure 8 As shown in Figure A, the horizontal axis represents the duration of US wave irradiation, and the vertical axis represents the relative fluorescence intensity. A larger value indicates... 1 The more O2 produced, the higher the fluorescence intensity. When only Ce6@PLGA NPs are present in the reaction system, the fluorescence intensity increases with the duration of US irradiation, indicating that Ce6@PLGA NPs can effectively produce O2 under US irradiation. 1 O2; in Figure 8 In B, the horizontal and vertical coordinates are illustrated in the same way. Figure 8 A. When only Pt NPs and Pt-BP NPs are present in the system, no effective fluorescence intensity is observed regardless of the reaction time. This indicates that US wave irradiation cannot induce SDT reaction in isolated Pt NPs or isolated Pt-BP NPs. 1 O2; in Figure 8 In Figure C, the horizontal axis represents different concentrations of H2O2 in the reaction system, and the vertical axis represents the relative fluorescence intensity. As can be seen from the figure, when Ce6@PLGA NPs+Pt NPs are present in the reaction system, the relative fluorescence intensity increases continuously with the increase of US wave irradiation time, but it is still lower than the relative fluorescence intensity produced by Ce6@PLGA NPs+Pt-B-PNPs in the reaction system under the same US wave irradiation time. This result further illustrates that compared with Pt NPs alone, Pt-BP NPs have a stronger ability to catalyze the decomposition of H2O2 to produce O2, and can synergistically interact with Ce6@PLGA NPs.

[0068] Example 10 Observation of hemolytic effects of Pt-BP NPs, Ce6@PLGA NPs and Pt NPs This invention primarily observes whether the synthesized NPs can induce hemolysis with erythrocytes. Specifically, two healthy female nude mice were anesthetized intraperitoneally, and their eyeballs were quickly removed, with fresh blood collected in EP tubes containing anticoagulant. The blood was diluted 20-fold with PBS and centrifuged at 4°C for 5 minutes (2000 rpm / min). After washing the tubes five times with PBS, the erythrocyte precipitate at the bottom of the tubes was prepared into a erythrocyte suspension. Experimental groups were then formed: erythrocytes + 200 μL chloroform (positive control), and erythrocytes + 50 μL Pt NPs (experimental group, 10 μg / mL). -1 ), red blood cells + 50uL Pt-BP NPs (experimental group, 10ugmL) -1 ), Red blood cells + 50uL Ce6@PLGA NPs (Experimental group, 10ug mL) -1 Red blood cells + 50 μL Pt NPs + 50 μL Ce6@PLGA NPs (experimental group, 10 μg / mL) -1 Red blood cells + 50 μL Pt-BP NPs + 50 μL Ce6@PLGA NPs; the above solution was incubated at 37°C for 4 h and then observed and photographed.

[0069] The results are as follows Figure 9 As shown, from left to right, the groups are: I: Positive control group; II: Ce6@PLGA NPs group; III: Pt NPs group; IV: Pt-BP NPs group; V: Pt+Ce6@PLGA NPs group; VI: Ce6@PLGA+Pt-BP NPs group; VII: Negative control group. In the positive control group, the solution was turbid red, indicating significant hemolysis of red blood cells. However, in the Ce6@PLGA NPs group, Pt NPs group, Pt-BP NPs group, Pt+Ce6@PLGA NPs group, Ce6@PLGA+Pt-B-PNPs group, and the negative control group, red blood cells mainly accumulated at the bottom of the EP tube, and no hemolysis occurred, further demonstrating the good biocompatibility of the synthesized NPs.

[0070] IV. Application of Pt-BP NPs and Ce6@PLGA NPs in SDT Treatment of Ovarian Cancer Cell Models Example 11 Observation of cellular uptake of Pt NPs, Pt-BP NPs and Ce6@PLGA NPs (1) NPs fluorescent labeling After carefully cleaning the weighing spoon and glass rod with ultrapure water and anhydrous ethanol in an ultrasonic cleaner, excess water was wiped off with filter paper. 2 mg of lyophilized Pt NPs powder and 50 mg of PLGA powder were accurately weighed using an electronic balance and mixed into a 50 mL centrifuge tube. The tube was then wrapped with aluminum foil, and 2 mL of dichloromethane and 4 mL of 8% PVA solution were added. The mixture was first ultrasonically vibrated for 3 min in an ultrasonic cleaner, followed by ultrasonic emulsification using a sonic vibrator (5 min, vibrate for 5 s, pause for 5 s) to obtain a black emulsion. 15 mL of 2% isopropanol was added to the solution to stabilize the newly synthesized NPs. The centrifuge tube was then placed in a fume hood and stirred for 4 hours with a magnetic stirrer to evaporate any unreacted dichloromethane. The mixture was then centrifuged at 4°C (8000 rpm / min), washed, and the final product, Pt@PLGA NPs, was obtained. The preparation method for Pt-BP@PLGA NPs was the same as for Pt@PLGA NPs. After preparing DIO working solution and incubating Pt@PLGA NPs at 37°C for 15 min, the Pt@PLGA NPs were washed three times by centrifugation using sterile serum-free medium (Pt@PLGA NPs: 10 μg / mL). The fluorescent labeling method for Pt-BP NPs was the same as that for Pt NPs. The fluorescent labeling method for Ce6@PLGA NPs was the same as that for Pt NPs, but it was not necessary to re-encapsulate PLGA. In addition, only the DIO staining solution needed to be replaced with DIL.

[0071] The results are as follows Figure 10 As shown in Figure A, Pt NPs and Pt-BP NPs were labeled with red fluorescence, while Ce6@PLGA NPs were labeled with green fluorescence. NPs were distributed intracellularly, primarily in the cytoplasm, with no significant uptake observed in the nucleus. Initially, after 1 hour of reaction, uptake of NPs by tumor cells was almost unobservable; only tumor cell nuclei stained blue by nuclear dyes were clearly visible under confocal microscopy. However, with increasing reaction time, uptake of all three types of NPs by tumor cells significantly increased, a result consistent with subsequent fluorescence microplate reader assays.

[0072] (2) Observation of cell uptake of Pt NPs, Pt-BP NPs and Ce6@PLGA NPs After removing the SKOV3 cell line from the cell culture incubator, cell growth was observed under an optical microscope. Cell culture flasks in the logarithmic growth phase were removed, old culture medium was discarded, and the cells were washed with PBS. Cells were digested with trypsin using a sterile pipette tip, resuspended in complete culture medium, and the resuspended solution was transferred to a centrifuge tube for centrifugation. After centrifugation, the old culture medium was discarded, and 1 mL of fresh complete culture medium was added to the centrifuge tube to resuspend the cells. The cell resuspended solution was then seeded into 6-well plates and cultured for 72 h (ensuring that the culture medium volume in each well reaches 2 mL). The old culture medium was discarded, and the cells were washed three times repeatedly with sterile PBS. Fresh culture medium (SKOV3: McCoy's 5A; A2780: DMEM) was then added to the well plates. The experimental groups were as follows: Control group (PBS), Experimental group 1 (Pt NPs), Experimental group 2 (Pt-BP NPs), and Experimental group 3 (Ce6@PLGA NPs). 10 μg / mL of Pt NPs, Pt-BP NPs, and Ce6@PLGA NPs were added to 6-well plates, and the cells were cultured for 1 h and 4 h, respectively. After culture, the old culture medium was discarded, and the cells were washed three times with sterile PBS. 1 mL of trypsin digest was added to each well, and the cells were centrifuged and collected after digestion. The cells were resuspended in sterile PBS, and the fluorescence intensity was detected using a fluorescence microplate reader. To more directly observe the NP uptake behavior of cells, the CLSM technique was used to observe the NP uptake process. First, confocal culture dishes were sterilized with UV light. Then, SKOV3 cell lines in logarithmic growth phase were seeded into the culture dishes and incubated in a cell culture incubator for 72 h (37°C, 5% CO2). After the incubation period, 10 μg / mL of Pt NPs, Pt-BP NPs, and Ce6@PLGA NPs were added to the culture dishes, and the cells were cultured for another 1 h and 4 h, respectively. Finally, after discarding the old culture medium and washing the cells three times with sterile PBS, the confocal dish was placed under CLSM for observation.

[0073] The results are as follows Figure 10 As shown in Figure B, the fluorescence intensity signals of the three NPs were very weak in the first 1 hour of the reaction, indicating that only a very small amount of NPs were taken up by the cells. Subsequently, as the reaction time increased, the uptake of the three NPs by the cells significantly increased after 4 hours. Therefore, we used 4 hours as the reaction time for subsequent NPs to enter the cells. These experimental results confirm that all three prepared NPs can be effectively taken up by SKOV3 cells.

[0074] Example 12 Cytotoxicity observation of Pt NPs, Pt-BP NPs and Ce6@PLGA NPs SKOV3 and A2780 cell lines were seeded into 96-well plates, ensuring a cell density of 5 × 10⁶ cells per well.3 The well plates were then placed in a cell culture incubator and cultured for 24 hours (37°C, 5% CO2). NPs were added to the well plates at different concentrations. After culturing, the old culture medium was discarded, and the cells were washed three times with sterile PBS. mcCoy'5A containing 10% CCK-8 reagent and DMEM medium were added, and the cells were cultured for another 2 hours. The absorbance of each well in the 96-well plate was then measured at 450 nm using a multi-mode microplate reader. Cell viability in the experimental group was calculated as: (Experimental group absorbance / Control group absorbance) × 100%.

[0075] The results are as follows Figure 11 A and Figure 11 As shown in B (where, Figure 11 In A, the vertical axis values ​​for each concentration group, from left to right, are Ce6@PLGA, Pt, and Pt-BP, respectively. Figure 11 In Figure B, the ordinate values ​​for each concentration group (from left to right: Ce6@PLGA, Pt, and Pt-BP) represent the molar concentration of NPs, and the ordinate represents cell viability. In the SKOV3 tumor cell line, we found that as the concentration of Ce6@PLGA NPs increased, the cell viability remained essentially at 100%. This indicates that Ce6@PLGA NPs have low toxicity and good biocompatibility. However, with increasing concentrations of Pt NPs and Pt-BP NPs, the survival rates of SKOV3 and A2780 cells significantly decreased. When the NP concentrations reached Pt NPs = 25 μg / mL and Pt-BP NPs = 200 μg / mL, cell survival rates dropped below 50%. This indicates that in the SKOV3 tumor cell line, Pt NPs = 25 μg / mL and Pt-B-PNPs = Pt-BP NPs = 200 μg / mL represent the IC50 values ​​of the two nanomedicines, respectively. In both the SKOV3 and A2780 cell lines, the IC50 value of Pt-BP NPs was 8 times that of Pt NPs. These experimental results further demonstrate that the toxicity of Pt-BP NPs is significantly lower than that of Pt NPs.

[0076] Example 13: Detection of H2O2 consumption, O2 production, and HIF-1α factor expression in ovarian cancer cells. H2DCFHDA can be oxidized to DCF by H2O2 and emits green fluorescence, with the fluorescence intensity increasing with increasing H2O2 concentration; Ru(DPP)3Cl2 emits red fluorescence, but the fluorescence intensity decreases with increasing O2 content. This invention utilizes the fluorescence properties of these two probes to detect changes in intracellular H2O2 and O2 content. First, SKOV3 cells in the logarithmic growth phase were... 3Cells were seeded at a density of 10 cells / well in 12-well plates, and then the plates were placed in a cell culture incubator (37°C, 5% CO2) for 72 h. After the culture, the old medium was replaced with fresh medium. Pt NPs and Pt-BP NPs (10 μg / mL) were added to the experimental group, and the same volume of physiological saline was added to the control group. After 4 h, the old medium was discarded and the cells were washed three times with sterile PBS before adding fresh medium. Then, an appropriate amount of H2DCFHDA and Ru(DPP)3Cl2 probe were added to the well plate and incubated in the cell culture incubator in the dark for 20 min. After the incubation, the H2DCFHDA and Ru(DPP)3Cl2 probes that had not entered the cells were washed away with sterile PBS, and the cells were observed using CLSM.

[0077] The results are as follows Figure 12 As shown, in Figure 12 In diagram A, the red color represents the oxygen probe, which emits red fluorescence, but the fluorescence intensity decreases as the oxygen concentration in the environment increases. When Pt NPs are added, the red fluorescence intensity does not change significantly compared to the control group, indicating that Pt NPs only produce trace amounts of O2. With the addition of the same concentration of Pt-BP NPs, the red fluorescence intensity within tumor cells decreases significantly, indicating that the addition of Pt-BP NPs can significantly improve the hypoxic environment within tumor cells. Meanwhile, the green fluorescence represents the H2O2 probe. Unlike the O2 probe, it does not produce fluorescence itself, but its fluorescence intensity increases continuously with the increase of H2O2 in the environment. Compared with the control group, the addition of Pt NPs did not reduce the fluorescence intensity of the green fluorescent probe, further indicating that Pt NPs have a relatively low efficiency in catalyzing the decomposition of H2O2. However, when Pt-BP NPs of the same concentration and SKOV3 tumor cells were co-incubated for 4 hours, the fluorescence intensity of the green fluorescent probe inside the tumor cells decreased significantly. This also proves that Pt-BP NPs can also exert a strong ability to catalyze the decomposition of H2O2 inside tumor cells, and the catalytic efficiency is significantly higher than that of Pt NPs of the same concentration.

[0078] To further investigate the catalytic activity of NPs, this embodiment of the invention used Western blotting (WB) to detect the expression of HIF-1α in SKOV3 and A2780 cell lines. (HIF-1 is a heterodimer composed of two subunits, HIF-1α and HIF-1β (molecular weight: HIF-1α = 120 KD, HIF-1β = 91~94 KD). HIF-1β functions as a structural protein within the cell, and its expression level is unaffected by external factors. HIF-1α is the active subunit of HIF-1, and its expression level is affected by changes in intracellular O2 content. HIF-1α expression increases when cells are in a hypoxic state and decreases when the hypoxic environment is alleviated.) First, cells in the logarithmic growth phase were transferred to 6-well plates and cultured for 72 hours. Then, Pt NPs and Pt-BP NPs (10 μg / mL) were added to the experimental group, while the same volume of physiological saline was added to the control group and cultured for 4 hours. After 4 hours, the cells were removed from the cell culture incubator, the old culture medium was discarded, and the cells were washed three times with sterile PBS to remove Pt NPs and Pt-BP NPs that had not entered the cells. Then, 300 μL of IRA cell lysis buffer was added to each well, and the plate was placed on ice for lysis. After thawing for 5 minutes, the cells were scraped into EP tubes, sonicated twice, and vortexed five times. Finally, the cells were centrifuged at 12,000 rpm / min at 4°C for 20 minutes. Then, the EP tubes were placed in a water bath at 95°C for 6 minutes. After the lysis buffer cooled to room temperature, its protein content was determined (BCA method). 30 μg of protein was added to each well and separated by electrophoresis on a 10% SDS-PAGE gel. After electrophoresis, the gel was cut according to the molecular weights of GAPDH and HIF-α, and then transferred to a PVDF membrane and electroporated under constant current (250 mA, 2 h). The PVDF membrane was then blocked with skim milk at room temperature for 80 min and incubated overnight at 4°C with HIF-1α primary antibody dilution buffer. After washing with 1×TBST, the PVDF membrane was soaked in secondary antibody dilution buffer and incubated again at 37°C for 1 h. Finally, the PVDF membrane was treated with chemiluminescence staining solution and observed using a chemiluminescence imaging system.

[0079] The results are as follows Figure 12As shown in Figure B, in the SKOV3 cell line, after treating with the same concentrations of Ce6@PLGA NPs, Pt NPs, and Pt-BP NPs for the same duration, the expression level of HIF-1α in the Pt-BP NPs group significantly decreased. This indicates that the hypoxic environment inside the tumor cells was effectively improved, further demonstrating that Pt-BP NPs have a stronger ability to catalyze the decomposition of H2O2 to produce O2 compared to Pt NPs. This conclusion was also verified in the A2780 ovarian cancer cell line.

[0080] Example 14 Analysis of apoptosis and necrosis signaling pathways in ovarian cancer cells after SDT treatment First, SKOV3 cell lines were seeded in 6-well plates at a density of 5 × 10⁶ cells / well. 3 Cells were added to each well. 10 μg / mL of Ce6@PLGA NPs and Pt-BP NPs were then added to each well, and the cells were incubated for 4 hours. After incubation, the cells were washed three times with sterile PBS and irradiated with 1W US light for 180 seconds. Western blotting was then used to detect apoptosis and necrosis-related signaling pathway molecules at different time gradients (time = 0, 0.5, 1, and 2 hours). In simpler terms, 300 μL of IRIPA cell lysis buffer was added to each well, and the plate was placed on ice for 5 minutes. Cells were then scraped into EP tubes, sonicated twice, and vortexed five times. Finally, the cells were centrifuged at 12000 rpm / min at 4°C for 20 minutes. The EP tubes were then placed in a water bath at 95°C for 6 minutes. After the lysis buffer cooled to room temperature, the protein content was measured (BCA method). 30 μg of protein was added to each well and separated by electrophoresis on a 10% SDS-PAGE gel. After electrophoresis, the gel was cut according to the molecular weight of ACTIN and different target molecules, and then transferred to a PVDF membrane and electroporated at a constant current (250 mA, 2 h). The PVDF membrane was then blocked with skim milk at room temperature for 80 min, followed by overnight incubation at 4°C with the corresponding primary antibody dilution buffer for each target molecule. After washing with 1×TBST, the PVDF membrane was immersed in secondary antibody dilution buffer and incubated again at 37°C for 1 h. Finally, the PVDF membrane was treated with chemiluminescence imaging solution and observed using a chemiluminescence imaging system.

[0081] The results are as follows Figure 13As shown in Figure A, 0.5 h after low-frequency ultrasound irradiation, the expression levels of C-Caspase3, 8, 9, and C-PARP significantly increased, and their expression levels further increased with the extension of reaction time. This indicates that SKOV3 cells can effectively undergo apoptosis after US irradiation. Further examination of MAPK signaling pathway protein expression revealed that the phosphorylation levels of P38 and JNK significantly increased 0.5 h after SDT treatment, roughly coinciding with the aforementioned time of apoptosis initiation. This suggests that the activation of P38 and JNK protein kinases may be an early event in SDT-induced apoptosis of SKOV3 tumor cells. Simultaneously, the phosphorylation levels of C-jun and ATF-2, and the expression level of MKK4 protein in the JNK signaling pathway significantly increased; ERK phosphorylation also increased, but its expression level began to decrease after 1 h of SDT treatment, indicating that the ERK signaling pathway was also activated, but to a lower degree.

[0082] Similarly, in this embodiment of the invention, the occurrence of cell necrosis in SKOV3 cells that had taken up Pt-BP NPs and Ce6@PLGA NPs after US treatment was analyzed, and the results are as follows: Figure 13 As shown in Figure B, the expression levels of cell necrosis signaling proteins gradually increased with increasing US irradiation time. These results indicate that SKOV3 cells that have taken up Pt-BP NPs and Ce6@PLGA NPs can not only undergo apoptosis but also effectively undergo cell necrosis after US treatment.

[0083] Example 15: Detection of ROS content in ovarian cancer cells after SDT treatment First, SKOV3 cells were seeded into confocal culture dishes and incubated in a cell culture incubator for 48 hours (37°C, 5% CO2). Then, the old culture medium was discarded, and the dish was rinsed three times with sterile PBS. Since the cells in the culture dish are not very firmly attached, the sterile pipette tip should be used from the edge of the confocal culture dish during rinsing, and the movements should be as gentle as possible to prevent cell detachment. (If cell detachment is severe in this step, poly-L-lysine can be added to the confocal culture dish beforehand for cell fixation). After rinsing, fresh complete culture medium was added to the culture dishes, and experimental groups were formed. The groups were as follows: I: Negative control group; II: Pt NPs alone; III: Ce6@PLGA NPs alone; IV: Pt-B-PNPs alone; V: Ce6@PLGA NPs + US; VI: Ce6@PLGA NPs + Pt NPs + US; VII: Ce6@PLGA NPs + Pt-BP NPs + US. The NP concentration in each experimental group was 10 μg / mL. US irradiation intensity and time were 1 W and 180 s, respectively. After treatment, 15 μM of DCFH-DA fluorescent probe was added to each group and incubated in a cell culture incubator for 15 min, ensuring complete protection from light. After incubation, the culture dishes were washed three times with serum-free mcCoy5'A medium to remove excess DCFH-DA fluorescent probe, handling as gently as possible. Finally, discard the serum-free mcCoy5'A medium and add 500 μL of sterile PBS to each culture dish. Then, place the culture dishes under a CLSM (Clean Microplate Reader) for observation. The detection procedure for the fluorescence microplate reader is roughly the same as that for the CLSM, but the cells need to be seeded in 6-well plates. After the experimental treatment, digest the cells with trypsin, collect the cells in EP tubes, and place them on ice (ice should be prepared in advance). Detect the fluorescence intensity using a fluorescence microplate reader.

[0084] ROS mainly includes singlet oxygen. １ The effects of O2, H2O2, and superoxide radical anions (O2•−) on cells depend primarily on their concentration. The level of ROS within tumor cells and the ROS scavenging system maintain a dynamic balance, allowing tumor cells to grow normally. When the ROS concentration increases and disrupts this balance, it can kill tumor cells.

[0085] The results are as follows Figure 14As shown, in the Control group, the green fluorescence detected was extremely weak because no treatment was performed on the cells. When tumor cells were treated with ultrasound, no obvious green fluorescence was observed, indicating that ultrasound alone could not stimulate ROS production in tumor cells. Next, Pt NPs and Pt-BP NPs were added to the ultrasound treatment, but still no green fluorescence was produced, indicating that neither Pt NPs nor Pt-BP NPs could effectively produce ROS under ultrasound irradiation. Then, Pt NPs and Pt-BP NPs were replaced with PLGA@Ce6 NPs coated with a sonosensitive agent. After ultrasound irradiation, weak green fluorescence was observed in the cells, indicating that tumor cells irradiated with ultrasound could effectively produce ROS. Next, Pt NPs were added to the above experimental group. After ultrasound irradiation, although tumor cells could still produce ROS, the amount of ROS produced was not significantly different from that produced by using PLGA@Ce6 NPs alone, further indicating that Pt NPs had low efficiency in catalyzing the decomposition of H2O2 to produce O2 and could not effectively alleviate the hypoxic environment within tumor cells. Finally, Pt NPs were replaced with Pt-BP. NPs combined with Ce6@PLGA NPs produced ROS under US irradiation. The results showed a large amount of green fluorescence within the cells, indicating sufficient ROS generation. This further demonstrates that Pt-BP NPs significantly enhance the catalytic ability of H2O2 decomposition, effectively alleviating the hypoxic microenvironment within tumor cells. The same experimental results were also verified using a fluorescence microplate reader, as shown in the results below. Figure 14 As shown in B.

[0086] Example 16: Double staining experiment of live and dead cells after SDT treatment First, SKOV3 cells were seeded in confocal culture dishes and cultured in a cell culture incubator for 48 hours (37°C, 5% CO2). Then, the old culture medium was discarded, and the dish was rinsed three times with sterile PBS. Since the cells in the culture dishes are not very firmly attached, the sterile pipette tip should be used from the edge of the confocal culture dish during rinsing, and the movements should be as gentle as possible to prevent cell detachment. After rinsing, fresh complete culture medium was added to the culture dishes, and the experimental groups were determined as follows: I: Negative control group; II: Pt NPs alone; III: Ce6@PLGA NPs alone; IV: Pt-BP NPs alone; V: Ce6@PLGA NPs + US; VI: Ce6@PLGA NPs + Pt NPs + US; VII: Ce6@PLGA NPs + Pt-BP NPs + US. The NP concentration in each experimental group was 10 μg / mL. The US irradiation intensity and duration were 1 W and 180 s, respectively. After treatment, each group was stained with Calcein AM / PI cell double staining kit and incubated at 4°C for 15 min. Light protection was maintained throughout the process. After incubation, the culture dishes were washed three times with serum-free mcCoy5'A medium to remove any Calcein AM / PI dye molecules that had not entered the cells, handling them as gently as possible. Finally, the serum-free mcCoy5'A medium was discarded, and 500 μL of sterile PBS was added to each culture dish. The dishes were then placed under CLSM for observation. Flow cytometry and fluorescence microplate reader detection procedures were largely the same as CLSM, but cells needed to be seeded in 6-well plates. After experimental treatment, cells were digested with trypsin, collected in EP tubes, and placed on ice (ice should be prepared in advance) for detection using flow cytometry and fluorescence microplate reader.

[0087] CLSM observation results as follows Figure 15As shown in Figure A, in the Control group, since no cell treatment was performed, all cells observed in the field of view were green. After ultrasound treatment of the tumor cells, the result was the same as in the Control group; all cells observed in the field of view were still green, indicating that ultrasound stimulation alone could not induce cell death. Next, Pt NPs and Pt-BP NPs were added to the ultrasound treatment, but the cells observed in the field of view were still predominantly green, indicating that neither Pt NPs nor Pt-BP NPs could effectively kill tumor cells under ultrasound irradiation. Then, Pt-BP NPs and Pt-BP NPs were replaced with PLGA@Ce6 NPs coated with the sonosensitive agent Ce6. After US irradiation, red cells appeared in the field of view, indicating that the sonosensitive agent, under the influence of US irradiation, could have a certain killing effect on tumor cells. Finally, Pt NPs were added to the above experimental groups. After US irradiation, although tumor cells also died, the killing efficiency was the same as the previous experimental group. Finally, we replaced Pt NPs with Pt-BP NPs combined with PLGA@Ce6 NPs, which together generated ROS under US irradiation, thereby killing tumor cells. The results showed that the CLSM field was filled with red cells. This result indicates that Pt-BP NPs can effectively enhance the killing effect of SDT on tumor cells. Simultaneously, the same experimental results were observed using a CCK-8 kit and flow cytometry. Figure 15 B and Figure 15 C).

[0088] V. In vivo in situ injection imaging and antitumor activity studies of Pt-BP NPs and Ce6@PLGA NPs In the following examples, the establishment of the SKOV3 ovarian cancer-bearing mouse model included: after resuscitating SKOV3 cells, transferring them to T75 culture flasks at a 1:1 volume ratio for further culture; observing cell growth under an optical microscope after 48 hours; if growth was good, continuing cell passage at a 1:1 volume ratio; replacing old culture medium with fresh culture medium for the first 24 hours before cell collection; resuspending cells in sterile PBS after collection and storing the cell suspension on ice. Then, using healthy nude mice, after disinfection with alcohol, 150 μL of cell suspension was injected subcutaneously near the right hind limb, and the needle was slowly withdrawn while pressing the injection site with a sterile cotton swab to prevent cell fluid leakage; the tumor volume was calculated using the following formula: V = a × b² / 2, where V = tumor volume (mm³), a = longest diameter of the tumor (mm), and b = shortest diameter of the tumor (mm).

[0089] Example 17 In vivo fluorescence imaging of Pt-BP NPs and Ce6@PLGA NPs (1) In vivo tumor fluorescence imaging To observe the accumulation of Pt-BP NPs, Pt NPs, and Ce6@PLGA NPs in subcutaneous tumors of nude mice and their distribution in major organs after in situ injection of nanomedicines into tumor tissue, we first fluorescently labeled the nanomedicines and then injected 25 μL of Pt NPs, Pt-BP NPs, and Ce6@PLGA NPs into the tumors of mice using the in situ injection method. To ensure the best imaging quality, we injected the maximum concentration of nanomedicines into the tumors at a safe dose that would not cause mortality in the mice. During injection, the drugs were administered slowly, and the needle was inserted from the center of the tumor as much as possible to ensure uniform drug distribution; after injection, the needle was slowly withdrawn to prevent backflow or leakage of the drug. After ensuring that no more fluid seepage occurs at the injection site, the mice are numbered and placed in an anesthesia box filled with isoflurane gas for gas anesthesia. Once the mice are in anesthetized state, they are transferred to a small animal in vivo fluorescence imaging device for observation. During the observation process, the mice's condition is monitored at all times. If the mice awaken, they need to be transferred to the anesthesia box for continued anesthesia.

[0090] The results are as follows Figure 16 As shown in Figure A (red fluorescence indicates high drug accumulation and high fluorescence intensity; blue fluorescence indicates low drug accumulation and low fluorescence intensity), in Pt NPs, after 72 hours of in-situ injection of nanomedicine, the red and blue fluorescent regions significantly decreased, indicating that although the drug was metabolized at the tumor site, some drug still remained there (indicating a relatively high drug injection dose and increased drug accumulation at the tumor site). Similar experimental results were observed in the Pt-BP NPs group, the Ce6@PLGA NPs group, and the Pt+Ce6@PLGA NPs group. However, in the Pt-B-P+Ce6@PLGA NPs group, a high drug concentration was observed at the tumor site 72 hours after in situ injection. This phenomenon may be related to the particle size of the Pt-BP NPs. Unlike NPs used for conventional tail vein injection, the Pt-BP NPs synthesized in this invention have a diameter of approximately 500 nm to meet the requirements of in situ injection. This is larger than the particle size of NPs used in ordinary studies. Therefore, during metabolism at the tumor site, the increased particle size may reduce the probability of passing through the tumor capillary gaps, thereby prolonging the accumulation time of Pt-BP NPs at the tumor site. Therefore, to ensure the normal viability of mice during treatment, a gradient-decreasing injection dose was selected during in situ tumor injection.

[0091] (2) Fluorescence imaging of isolated tumors and major organs Since mice themselves contain certain biofluorescence and excretions of varying degrees, in order to avoid interference from these biological factors during in vivo imaging of mice, we injected the same concentration and volume of nanomedicine as those used in tumor in vivo fluorescence imaging, and then removed the tumors and major organs (heart, liver, spleen, lung, and kidney) of the mice for fluorescence observation using a small animal in vivo fluorescence imaging system.

[0092] The results are as follows Figure 16 As shown in Figure B (from left to right: heart, liver, spleen, lung, kidney, tumor), after 72 hours of metabolism, Pt-BP NPs+Ce6@PLGA NPs were distributed to varying degrees in all the aforementioned tissues, with larger accumulations in the liver and lungs. Meanwhile, drug accumulation at the tumor site decreased, and the mice's viability remained normal. Because Pt-BP NPs accumulated in all major organs of the mice, subsequent studies involved H&E staining of pathological sections of the major organs to observe whether they caused pathological damage.

[0093] Example 18: In vivo antitumor activity study of Pt-BP NPs and Ce6@PLGA NPs (1) Observation of SDT treatment in SKOV3 ovarian cancer-bearing mice Thirty-five Nude mice were randomly divided into seven groups (n=5 per group), numbered I-VII: I: control group, II: US irradiation group, III: Pt NPs group, IV: Pt-BP NPs group, V: Ce6@PLGA NPs group, VI: Pt NPs+Ce6@PLGA NPs+US irradiation group, and VII: Pt-BP NPs+Ce6@PLGA NPs+US irradiation group. Subcutaneous tumors in the mice reached 80 mm². 3 Different treatments were administered to mice in different experimental groups. In the control group, 25 μL of physiological saline was injected into the tumor site. All experimental groups received 25 μL of saline at a concentration of 5 mg / kg. Four hours after the in situ injection, the mice were irradiated with ultrasound for 2 weeks and 5 minutes. To ensure normal mouse viability, the mice were administered the drug three times at four-day intervals, and changes in volume and weight were recorded. On day 16, after treatment, live digital photography was performed on mice in each group, and subcutaneous tumors were removed for digital image acquisition.

[0094] The results are as follows Figure 17 A and Figure 17As shown in Figure B, compared with the control group, the tumor volume gradually increased with the increase of mouse feeding time in the US irradiation group, the US irradiation + Pt NPs group, and the US irradiation + Pt-BP NPs group, and there was no significant difference in tumor volume changes, indicating that US irradiation alone, Pt NPs, and Pt-BP NPs alone cannot play a good killing role against ovarian cancer cells. However, in the US irradiation + Ce6@PLGA NPs experimental group, the tumor growth rate was significantly inhibited compared with the control group, but the degree of inhibition was still low and could not achieve the ideal effect. This is because the presence of the hypoxic microenvironment inside the tumor cells reduced the production of ROS, and the O2 content also decreased with the increase of treatment time during SDT treatment, further weakening the killing ability of SDT on tumor cells. When Pt NPs were added to Ce6@PLGA NPs and external ultrasound stimulation was applied, the tumor volume change was similar to that in the ultrasound irradiation + Ce6@PLGA NPs group, indicating that Pt NPs still cannot effectively improve the hypoxic microenvironment of tumor cells in animals.

[0095] (2) Pathological observation of tumor tissue After treatment on day 16, the mice were euthanized, and their major organs (heart, liver, spleen, lung, kidney) and tumor tissues were removed. After rinsing off surface blood with sterile PBS, the tissues were fixed in formalin for 72 hours. Tissue embedding was then performed using the following steps: different tissues were removed from the formalin fixative and rinsed with flowing PBS for 12 hours to remove residual fixative. Then, a gradient dehydration process was performed, with the following sequence: soaking in anhydrous ethanol at concentrations of 70%, 80%, 85%, 90%, and 95% for 50 minutes each; followed by soaking in anhydrous ethanol I and anhydrous ethanol II for 40 minutes each. After the gradient dehydration, a clearing solution I (2:3 volume ratio of anhydrous ethanol to xylene) was prepared, followed by a clearing solution II (1:3 volume ratio of anhydrous ethanol to xylene). The different tissues after gradient dehydration were then soaked in clearing solution I, clearing solution II, and xylene solution for 15 minutes, 10 minutes, and 5 minutes, respectively. During the clearing process, the transparency of the tissue should be observed promptly, and the process should be stopped at appropriate times to prevent tissue fragmentation. After clearing, prepare the appropriate number of wax cups according to the actual amount of tissue and melt the solid paraffin at a temperature controlled within the range of 55-60°C. Immediately afterward, perform tissue paraffin infiltration three times, with each infiltration lasting 0.5h, 1h, and 2h. After tissue paraffin infiltration, use a sharp, non-abrasive blade to trim the paraffin block into a rectangle. Then, set the section thickness to 20μm and further smooth the trimmed paraffin block. Adjust the section thickness to 5-10μm depending on the type of tissue. Place the prepared paraffin slides on clean glass slides and use a spreader set to 43°C-44°C for spreading. Stop spreading when the paraffin slides are completely spread on the spreader. Finally, set the oven temperature to 45°C and place the spread slides in the oven to bake until the moisture evaporates and the paraffin slides are completely adhered to the glass slide. The baking time should be adjusted appropriately according to the different types of tissue wax slides.

[0096] The results showed that, compared with the previous experimental group, the growth rate and volume of mouse tumors were significantly inhibited after Pt-BP NPs+Ce6@PLGA NPs+external ultrasound stimulation. Furthermore, the tumor volume decreased continuously with the extension of treatment time, reaching less than 20 mm at the treatment endpoint, achieving the desired experimental effect. Figure 18 A and Figure 18 D) and the tumor tissue damage is the greatest ( Figure 18 B and Figure 18 C).

[0097] The above demonstrates that Pt-BP NPs can effectively alleviate the hypoxic microenvironment within tumor cells, thereby further enhancing the killing ability of SDT on ovarian cancer cells and achieving the best tumor treatment effect. The mouse weight change curves showed no significant difference in weight change between the experimental groups and the control group as the treatment time increased, further indicating that the prepared Pt NPs, Ce6@PLGA NPs, and Pt-BP NPs have good biocompatibility.

[0098] Example 19: In vivo biosafety observation of Pt-BP NPs and Ce6@PLGA NPs In this embodiment of the invention, to observe the organ toxicity of Pt-BP NPs and Ce6@PLGA NPs during US tumor treatment, we performed H&E staining analysis on the major organs of mice that were orthotopically injected with the above nanomedicines at different time points at the start of treatment. The results are as follows: Figure 19 As shown, from the start point to the end point of treatment, the nanomedicine caused no significant organic damage to the major organs in the body.

[0099] Simultaneously, in this embodiment of the invention, after the completion of treatment, mice in the US-irradiated Pt-BP NPs and Ce6@PLGA NPs experimental groups were anesthetized, and blood samples were collected via tail vein for relevant biochemical index analysis. The results are as follows: Figure 20 As shown, there was no significant difference in biochemical indicators between the experimental group mice and the control group. The above experimental results indicate that the Pt-B-PNPs and Ce6@PLGA NPs prepared in this study have high biosafety.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A platinum-boron-phosphorus ternary nanoparticle, characterized in that, Its particle size is 450nm-550nm, and it is composed of three elements: platinum, boron, and phosphorus. The preparation method of platinum-boron-phosphorus ternary nanoparticles includes: (1) adding polyethylene glycol aqueous solution to polystyrene microsphere aqueous solution and adjusting the pH to 7-9 to obtain a mixture; (2) adding H2PtCl4 solution to the mixture, letting it stand, adding NH3·H2O, H3PO3 and NaBH4, and then reacting in an environment with pH of 3-5 and temperature of 100℃-120℃, washing, and centrifuging to obtain platinum-boron-phosphorus ternary nanoparticles; platinum-boron-phosphorus ternary nanoparticles are used to catalyze the decomposition of H2O2 to produce O2.

2. The platinum-boron-phosphorus ternary nanoparticles according to claim 1, characterized in that, In step (1), the pH is adjusted to 8; and / or in step (2), the reaction is carried out in an environment with a pH of 4.

3. A composition for treating solid tumors, characterized in that, Includes the platinum-boron-phosphorus ternary nanoparticles as described in claim 1 or 2 and photosensitizers for photodynamic therapy and / or sonodynamic therapy.

4. The composition for treating solid tumors according to claim 3, characterized in that, Photosensitizers suitable for photodynamic therapy and / or sonodynamic therapy are in nanoscale form.

5. A composition for treating solid tumors according to claim 3 or 4, characterized in that, The photosensitizer for photodynamic therapy and / or sonodynamic therapy is dihydroporphyrin E6.

6. The use of the platinum-boron-phosphorus ternary nanoparticles according to claim 1 or 2 in the preparation of formulations for photodynamic therapy and / or sonodynamic therapy of solid tumors.

7. The application according to claim 6, characterized in that, Solid tumors include breast fibroids, uterine fibroids, breast cancer, liver cancer, lung cancer, brain tumors, schwannomas, soft tissue sarcomas, osteosarcomas, stomach cancer, colon cancer, kidney cancer, bladder cancer, ovarian cancer, prostate cancer, lymphoma, and myeloma.

8. The application according to claim 6 or 7, characterized in that, The solid tumor is ovarian cancer.

9. The application according to claim 8, characterized in that, Applications include: the use of platinum-boron-phosphorus ternary nanoparticles in the preparation of drugs that treat ovarian cancer by promoting the expression of C-Caspase3,8,9 and C-PARP.

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