Ultrasonic confinement cavitation nanobubble as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an ultrasonic confinement cavitation nano bubble as well as a preparation method and application thereof. The ultrasonic confinement cavitation nanobubble has a core-shell structure, and comprises a core of perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles and a tumor cell membrane shell layer coated on the surface of the core, the inner core in the shell layer is in an oxygen saturation state. Through a multi-dimensional cooperation strategy of TPO (at) CCM nanobubble structure design optimization, bubble generation regulation and control and targeted delivery enhancement, the cavitation effect is remarkably improved, US imaging sensitivity is improved, ROS outbreak is promoted, an efficient tumor killing effect is achieved through cooperative use of the TPO (at) CCM nanobubble and an immune checkpoint inhibitor alphaPD-L1, and the TPO (at) CCM nanobubble can be applied to tumor treatment. The method has great application potential in clinical detection and treatment of cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an ultrasonic confined cavitation nanobubble and a preparation method and application thereof. Background Art

[0002] Cancer, as a disease characterized by malignant tumors, is essentially the uncontrolled proliferation of tumor cells and their continuous invasion of surrounding tissues. The root cause of cancer is usually attributed to gene mutations, which may be triggered by environmental factors, genetic factors, etc., leading to dysregulation of key regulatory mechanisms such as cell proliferation, differentiation and apoptosis, and eventually developing into highly invasive malignant tumors. In addition, cancer cells can escape the attack of the immune system through a variety of mechanisms, including downregulating or changing the expression of surface antigens, releasing immunosuppressive cytokines, upregulating immune checkpoint ligands, and enhancing the expression of anti-apoptotic proteins, which promote the survival and spread of cancer cells in the host. More seriously, cancer cells can escape from the primary tumor and spread to other organs through the blood circulation or lymphatic system to form secondary tumors, which is one of the main causes of death in cancer patients.

[0003] However, existing cancer diagnosis and treatment methods are limited by their strong invasiveness, lack of targeting, and high heterogeneity of the tumor microenvironment, making it difficult for current cancer detection and treatment technologies to meet increasingly complex clinical needs. Specifically, in terms of detection, existing imaging technologies have low sensitivity to early tumors, and traditional contrast agents are limited by their particle size and cannot penetrate deep into tumor tissue for high-resolution imaging, resulting in the inability to evaluate the efficacy of treatment in real time, causing a disconnect between diagnosis and treatment. In terms of treatment, surgical resection may cause damage to normal tissue and tumor residues, while radiotherapy and chemotherapy face challenges such as systemic toxicity, drug resistance, and limited killing effects on hypoxic tumor cells. Therefore, optimizing cancer diagnostic technology and developing more accurate and effective treatments have become important tasks that need to be urgently addressed in the field of global medical research.

[0004] In recent years, ultrasound (US) technology has become a widely used tool for cancer diagnosis and treatment due to its controllability, non-invasiveness and high tissue penetration. In the field of US imaging, contrast-enhanced US imaging (CEUS) significantly enhances the US backscatter signal intensity through the stable cavitation behavior of bubbles, thereby effectively distinguishing target tissue from background tissue. In the field of treatment, the emerging sonodynamic therapy (SDT) uses the inertial cavitation of bubbles under US irradiation to activate sonosensitizers, causing them to produce cytotoxic reactive oxygen species (ROS), thereby achieving efficient tumor clearance. However, the insufficient cavitation effect caused by exogenous US energy and its dependence on oxygen greatly limit the improvement of US imaging contrast and the efficiency of ROS generation, seriously affecting the diagnosis and treatment of cancer. Summary of the invention

[0005] The object of the present invention is to provide an ultrasound-confined cavitation nanobubble and its preparation method and application, so as to overcome the deficiencies of the prior art, optimize the US cavitation effect and tumor microenvironment through a multi-dimensional collaborative strategy, and provide important support for realizing efficient and precise cancer diagnosis and treatment integration.

[0006] To achieve the above object, the technical solution of the present invention is as follows: In the first aspect, the present invention provides an ultrasound-confined cavitation nanobubble with a core-shell structure, including a core of perfluorhexyl bromide-loaded mesoporous titanium dioxide nanoparticles and a tumor cell membrane shell layer coated on the surface of the core; The core in the shell layer is in an oxygen-saturated state.

[0007] In some other embodiments, the mesoporous titanium dioxide nanoparticles are spherical, with a particle size of 200-250 nm and a pore size of 2-20 nm; The ratio of perfluorhexyl bromide to mesoporous titanium dioxide nanoparticles is (10-15) μL:1 mg.

[0008] In some other embodiments, the tumor cell membrane is a breast cancer cell membrane.

[0009] In the second aspect, the present invention provides a preparation method of the ultrasound-confined cavitation nanobubble described in the first aspect, including the following steps: (1) Ultrasonically mix mesoporous titanium dioxide nanoparticles with perfluorhexyl bromide to obtain perfluorhexyl bromide-loaded mesoporous titanium dioxide nanoparticles; (2) Ultrasonically mix tumor cell membrane vesicles with perfluorhexyl bromide-loaded mesoporous titanium dioxide nanoparticles, and extrude to obtain tumor cell membrane-coated perfluorhexyl bromide-loaded mesoporous titanium dioxide nanoparticles; (3) Introduce oxygen into the suspension of tumor cell membrane-coated perfluorhexyl bromide-loaded mesoporous titanium dioxide nanoparticles until oxygen saturation is reached to obtain the ultrasound-confined cavitation nanobubble.

[0010] In some other embodiments, in step (1), the preparation method of the mesoporous titanium dioxide nanoparticles includes the following steps: Mix absolute ethanol, PVP aqueous solution, hydrochloric acid, and TiF 4 solution by stirring at room temperature, carry out the reaction, cool to room temperature after the reaction ends, and obtain mesoporous titanium dioxide nanoparticles after centrifugation, washing, and freeze-drying.

[0011] In some other embodiments, the mixing volume ratio of the absolute ethanol, PVP aqueous solution, hydrochloric acid, and TiF 4 solution is (55-56):(5-10):(0.5-1):(3-6); The concentration of the PVP aqueous solution is 9 - 10 mg / mL; The concentration of the hydrochloric acid is 0.08 - 10.12 mol / L; The concentration of the TiF 4 solution is 35 - 45 mmol / L; The temperature of the reaction is 175 - 185 °C, and the reaction time is 2 - 5 h; The solvent used for washing is water or ethanol.

[0012] In some other embodiments, in step (1), the ratio of the perfluorohexyl bromide to the mesoporous titanium dioxide nanoparticles is (10 - 15) μL:1 mg; The ultrasonic mixing is carried out in ice water for 10 - 15 minutes; In step (2), the tumor cell membrane vesicles are breast cancer cell membrane vesicles; The breast cancer cell membrane vesicles are obtained by extruding a breast cancer cell membrane fragment suspension through a polycarbonate membrane; The pore size of the polycarbonate membrane is 400 - 800 nm, and the number of extrusion times is greater than 10 times; The solvent of the breast cancer cell membrane fragment suspension is a phosphate buffer solution with a concentration of 450 - 550 μg / mL; The preparation method of the breast cancer cell membrane fragments includes the following steps: Digest the cultured breast cancer cells with trypsin, obtain a mouse breast cancer cell precipitate after one centrifugation, and obtain breast cancer cell membrane fragments after washing, resuspension, ice bath, freeze-thaw, and secondary centrifugation.

[0013] In some other embodiments, in step (2), the mass ratio of the perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles to the tumor cell membrane is 1:(1 - 3); The extrusion is carried out using a 400 nm polycarbonate membrane for at least 10 times.

[0014] In some other embodiments, the culture medium for culturing breast cancer cells is RPMI - 1640 medium containing 10% fetal bovine serum; the culture conditions are: temperature is 37 °C, 5% CO 2 ; The rotation speed of the first centrifugation is 770 - 850 rpm, and the time is 3 - 6 minutes; The washing is carried out by washing the cell precipitate with PBS 1 - 2 times; The resuspension is to resuspend in a hypotonic lysis buffer containing a membrane protein extraction reagent and PMSF; The time of the ice bath is 10 - 20 minutes; The freeze-thaw process is to freeze the cell suspension with liquid nitrogen and then thaw it at room temperature; The secondary centrifugation is to centrifuge at a speed of 650 - 750 g for 10 - 15 minutes at 4 °C, collect the supernatant, and then centrifuge at a speed of 13000 - 15000 g for 30 - 40 minutes.

[0015] In a third aspect, the present invention provides the application of the ultrasonic confinement cavitation nanobubbles described in the first aspect in the preparation of drugs for treating tumors and ultrasonic imaging.

[0016] The inventive concept adopted by the present invention: How to effectively enhance the ultrasonic cavitation effect and reshape the tumor microenvironment has become an important direction to break through the existing technical barriers. In the regulation of the US cavitation effect, by reducing the cavitation energy threshold and increasing the number of cavitation nuclei, the utilization rate of ultrasonic energy can be significantly improved, and at the same time, the explosive generation of ROS can be promoted; combined with the regulation of the hypoxic microenvironment and targeted delivery technology, it is possible to enhance oxygen supply and enhance tumor-specific killing. In addition, the stable cavitation behavior of bubbles during cavitation can significantly enhance the US imaging contrast, providing a new idea for real-time monitoring under US imaging guidance. Therefore, optimizing the US cavitation effect and tumor microenvironment through a multi-dimensional collaborative strategy will provide important support for realizing efficient and precise cancer diagnosis and treatment integration.

[0017] The principle of using the ultrasonic confinement cavitation nanobubbles (named TPO@CCM) of the present invention for the detection and treatment of primary tumors and metastatic tumors is as Figure 1 shown, where (a) is the synthesis schematic diagram of TPO@CCM, (b) is the ultrasonic imaging detection of TPO@CCM for primary tumors and metastatic tumors, and (c) is the treatment of primary tumors and inhibition of tumor metastasis by TPO@CCM combined with αPD-L1. Figure 1 In (a), perfluorhexyl bromide (PFHB) is loaded inside titanium dioxide nanoparticles (MTO) with a special mesoporous structure to make PFHB-loaded MTO nanomaterials (TP), and oxygen is introduced into it to achieve oxygen saturation, and finally, it is biomimetically coated with tumor cell membranes (CCM) to make nanobubble materials (TPO@CCM).

[0018] Figure 1 In (b) and Figure 1In (c), as a typical representative of inorganic sonosensitizers, MTO can generate various ROS through US activation. Its special hollow-confined mesoporous structure can not only serve as an efficient drug carrier but also endow the material with a high specific surface area and excellent gas adsorption capacity, providing more nucleation sites for reducing the cavitation threshold. The loaded PFHB, as a perfluorocarbon compound, has a high dissolved oxygen capacity and US-responsive liquid-gas phase transition characteristics. It can vaporize into bubbles under US stimulation while releasing pre-loaded oxygen, which can not only relieve the tumor hypoxic microenvironment and break the oxygen dependence limitation of SDT but also provide more cavitation nuclei for enhancing the cavitation effect. In addition, the coating of CCM endows the material with homologous targeting ability, and by utilizing the homing effect of membrane surface adhesion molecules and source tumor cells, it significantly improves the active enrichment efficiency of nanobubbles at the tumor site.

[0019] Advantages of the present invention: Through a multi-dimensional collaborative strategy of optimizing the structure design of TPO@CCM nanobubbles, regulating bubble generation, and enhancing targeted delivery, the present invention significantly improves the cavitation effect, not only improving the US imaging sensitivity but also promoting the burst of ROS. When used in combination with the immune checkpoint inhibitor αPD-L1, it achieves an efficient tumor killing effect and has great application potential in the clinical detection and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0021] Figure 1 It is a schematic diagram of the ultrasonic-confined cavitation nanobubble TPO@CCM for the detection and treatment of breast cancer in Example 1 of the present invention. Among them, (a) is the schematic diagram of the synthesis of TPO@CCM, (b) is the ultrasonic imaging detection of TPO@CCM for primary tumors and metastatic tumors, and (c) is the treatment of primary tumors and inhibition of tumor metastasis by TPO@CCM combined with αPD-L1; Figure 2 It is the transmission electron microscope images of MTO, TP, and TP@CCM in Example 1 of the present invention. Among them, (a) is MTO, (b) is TP, and (c) is TP@CCM; Figure 3 It is the hydrated particle size distribution diagram and Zeta potential diagram in Example 1 of the present invention. Among them, (a) is the hydrated particle size distribution diagram and (b) is the Zeta potential diagram; Figure 4 It is the SDS-PAGE protein spectrum of TP, CCM, and TP@CCM in Example 1 of the present invention; Figure 5 It is the oxygen release performance of TPO under US excitation in Example 1 of the present invention; Figure 6 Oxygen release performance of TPO@CCM under US excitation in Example 1 of the present invention; Figure 7 In vitro US imaging diagrams and gray value quantification of different materials and in vitro US imaging diagrams and gray value quantification of different concentrations of TPO in Example 1 of the present invention, where (a) in vitro US imaging diagrams and gray value quantification of different materials, (b) in vitro US imaging diagrams and gray value quantification of different concentrations of TPO; Figure 8 Bright-field photos of cavitation clusters generated by different materials under US excitation in Example 1 of the present invention; Figure 9 Degradation of MB and DPBF by different materials under US excitation in Example 1 of the present invention, where (a) MB, (b) DPBF; Figure 10 Survival rates of 4T1 cells after different treatments in Example 1 of the present invention; Figure 11 HIF-1α immunofluorescence staining images of 4T1 cells after different treatments in Example 1 of the present invention; Figure 12 ROS fluorescence staining images and quantification of 4T1 cells after different treatments in Example 1 of the present invention; Figure 13 Flow cytometry quantitative analysis of the maturation of DC cells after different treatments in Example 1 of the present invention, where CD80 and CD86 are the names of protein molecules expressed on the cells; Figure 14 In vitro US imaging diagrams and gray value quantification of primary tumors in different treatment groups in Example 1 of the present invention, where (a) US imaging diagrams, (b) gray value quantification; Figure 15 Typical tumor-like tissues in the livers of mice and in vitro US imaging diagrams of the livers in the sagittal and transverse planes of different treatment groups in Example 1 of the present invention, where (a) tumor-like tissues, (b) in vitro US imaging diagrams of the livers; Figure 16 Changes in tumor volumes of mice in each group in Example 1 of the present invention; Figure 17 Tumor masses and corresponding visualization photos taken out after 16 days of treatment of mice in each group in Example 1 of the present invention, where (a) tumor masses, (b) visualization photos; Figure 18 Flow cytometry analysis of the contents of DC cells and T cells in the tumor tissues of mice in each group in Example 1 of the present invention, where CD4, CD8, CD80, and CD86 are the names of protein molecules expressed on the cells; Figure 19Visualization photos of the lungs of mice in each group and the corresponding H&E staining results after 30 days of treatment in Example 1 of the present invention, where (a) visualization photos and (b) corresponding H&E staining results; Among them, Figures 16 - 19 I:PBS, II:TPO@CCM, III:αPD-L1, IV:TPO@CCM + US, V:TPO@CCM + US + αPD-L1 in Detailed implementation manner

[0022] Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. Specific conditions are not indicated in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. Components not indicated by the manufacturer are all conventional products available commercially.

[0023] Example 1 Preparation of nanobubble material 1. Main experimental materials: Titanium tetrafluoride (TiF 4 , 99%) was purchased from Sigma-Aldrich. Polyvinylpyrrolidone (PVP, Mw = 40,000) was provided by Shanghai Yuanye Bio-Technology Co., Ltd. Hydrochloric acid (HCl) was purchased from Yantai Far East Fine Chemical Co., Ltd. Perfluorohexyl bromide (PFHB) was purchased from Sain Chemical Technology Co., Ltd. Absolute ethanol (C 2 H 5 OH) was supplied by Sinopharm Chemical Reagent Co., Ltd. Sulfur hexafluoride microbubbles for injection (SonoVue) were sourced from Bracco Suisse SA, Switzerland. Methylene blue (MB) and 1,3-diphenylisobenzofuran (DPBF) were purchased from Shanghai Macklin Biochemical Co., Ltd. Hypoxia-inducible factor-1α (HIF-1α) antibody was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. Reactive oxygen species detection kit (DCFH-DA) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. FITC anti-mouse CD11c antibody, Alexa Fluor @RThe anti-mouse CD80 antibody, PE-Cy7 anti-mouse CD86 antibody, PE anti-mouse CD3 antibody, PE-Cy7 anti-mouse CD4 antibody, and APC anti-mouse CD8a antibody were all purchased from Shenzhen Dakewei Biotechnology Co., Ltd. The αPD-L1 antibody was purchased from Bioxcell Co., Ltd. Phosphate-buffered saline (PBS), 4',6-diamidino-2-phenylindole (DAPI), and RPMI-1640 cell culture medium were all provided by Wuhan Sevier Biotechnology Co., Ltd. The Cell Counting Kit-8 (CCK-8) was supplied by Shanghai Titan Technology Co., Ltd. All experiments were performed using deionized (DI) water (Millipore Milli-Q grade, 18.2 MΩ).

[0024] 2. Preparation of nanobubble materials: (1)Preparation of mesoporous titanium dioxide nanomaterials (MTO): First, 55.2 mL of absolute ethanol, 8 mL of PVP aqueous solution (9.75 mg / mL), 500 μL of hydrochloric acid (0.1 M), and 5 mL of TiF 4 solution (40 mM) were successively added to the reaction vessel and stirred at room temperature for 1 hour. Then the above mixture was added to a high-pressure reaction kettle and reacted at a constant temperature of 180 °C for 3 hours. After the reaction solution was cooled to room temperature, it was centrifuged and washed three times with water or ethanol. Finally, the purified solution was dried with a freeze dryer, and both the solution and the freeze-dried powder were stored at 4 °C to obtain mesoporous titanium dioxide nanomaterials, labeled as MTO.

[0025] (2)Preparation of PFHB-loaded MTO nanomaterials (TP): 3 mg of MTO was placed in a 50 mL centrifuge tube equipped with a rubber stopper. After evacuating the air in the centrifuge tube with a vacuum pump, 30 μL of PFHB was quickly injected into the centrifuge tube and sonicated in ice water for 10 minutes to obtain PFHB-loaded MTO nanomaterials, labeled as TP. Subsequently, it was dispersed in PBS (1 mL) for standby.

[0026] (3)Extraction of mouse breast cancer cell membranes (CCM): Mouse breast cancer (4T1) cells were cultured in a culture dish using RPMI-1640 medium containing 10% fetal bovine serum (FBS) (37 °C, 5% CO 2). When the 4T1 cancer cells grew for 36 hours until they reached 80 - 90% of the culture dish, trypsin was used for cell digestion. Subsequently, the cells were centrifuged at 800 rpm for 5 minutes, and the precipitated cells were collected. Then, the cell precipitate was washed 1 - 2 times with PBS and resuspended in a hypotonic lysis buffer containing a membrane protein extraction reagent and PMSF, and placed on ice for 15 minutes. After the ice bath, the method of repeated freezing and thawing was adopted. The cell suspension was frozen with liquid nitrogen and then thawed at room temperature, and this process was repeated twice. The frozen and thawed suspension was centrifuged at 4 °C at a speed of 700 relative centrifugal force (g) for 12 minutes, the supernatant was collected and centrifuged again at a speed of 14000 relative centrifugal force (g) for 40 minutes to obtain a precipitate of mouse breast cancer cell membrane (labeled as CCM) fragments, and the CCM fragment precipitate was resuspended in PBS for later use. (4) Preparation of CCM - coated TP nanomaterials (TP@CCM): First, the obtained CCM fragment suspension (resuspended in PBS) was extruded through polycarbonate membranes with pore sizes of 800 nm and 400 nm at least 10 times to obtain CCM vesicles. Then, the obtained CCM vesicles were mixed with TP (at a mass ratio of 2:1) and sonicated for 5 min. The mixed solution was co - extruded through a 400 nm polycarbonate membrane at least 10 times to obtain CCM - coated TP nanomaterials, labeled as TP@CCM.

[0027] (5) Preparation of oxygen - saturated TP nanomaterials (TPO) and oxygen - saturated TP@CCM nanobubble materials (TPO@CCM): Oxygen was introduced into the suspensions of the obtained TP and TP@CCM 2 until oxygen saturation (until no more oxygen could be charged), and oxygen - saturated TP nanomaterials and oxygen - saturated TP@CCM nanobubble materials could be obtained respectively, which were labeled as TPO and TPO@CCM nanobubbles.

[0028] Performance Characterization I. Characterization of Nanobubble Materials 1. Morphology Characterization: The MTO, TP, and TP@CCM powders were placed in deionized water (DW) to prepare stock solutions with a concentration of 1 mg / mL. Then, each stock solution was diluted 50 times and transferred to 1.5 mL centrifuge tubes, and placed in an ultrasonic cleaning instrument and sonicated for 20 min. Then, 10 μL of the uniformly dispersed diluted solution was taken and dropped onto a copper grid, and dried overnight in an electronic moisture - proof box. Transmission electron microscopy (TEM) was used to take pictures to observe the morphology of the nanoparticles. Figure 2 are the transmission electron micrographs of MTO, TP, and TP@CCM, where (a) MTO, (b) TP, (c) TP@CCM.

[0029] As Figure 2As shown in (a) therein, the prepared MTO presents a typical spherical morphology with an average size of about 220 nm and has a typical mesoporous structure, demonstrating the successful synthesis of MTO. The morphology and size of TP are as shown in Figure 2 in (b) therein. There is no obvious change compared with MTO. The only difference is that its pore structure becomes blurred, indicating that PFHB is loaded into the MTO cavity. Figure 2 In (c) therein, the morphology of TP@CCM is shown. A coating with a cell membrane morphology can be clearly observed on the surface of TP, demonstrating that CCM is successfully wrapped on the surface of TP to obtain TP@CCM.

[0030] 2. Characterization of hydrodynamic diameter and zeta potential: MTO, TP and TP@CCM powders are placed in deionized water to prepare a stock solution with a concentration of 1 mg / mL. Then each stock solution is diluted 10 times, and 3 mL of the diluted liquid is taken into a 5 mL centrifuge tube and sonicated for 20 min. A nanoparticle size and zeta potential analyzer is used to measure the hydrodynamic diameter and zeta potential of the nanoparticles, and the measurement is repeated three times. Figure 3 are the hydrodynamic diameter distribution diagram and zeta potential diagram, where (a) is the hydrodynamic diameter distribution diagram and (b) is the zeta potential diagram.

[0031] As shown in Figure 3 (a) therein, the hydrodynamic diameters of MTO, TP and TP@CCM gradually increase, demonstrating the successful preparation of TP@CCM. Figure 3 (b) therein shows the changes in the zeta potentials of MTO, TP and TP@CCM. Compared with MTO, the potential of TP increases significantly, and the potential of TP@CCM shows an inversion, exhibiting the same potential characteristics as pure CCM, indicating the successful synthesis of the nanoparticles.

[0032] 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment: TP, CCM and TP@CCM are subjected to SDS-PAGE to analyze the protein composition on their surfaces and verify whether TP@CCM retains the protein profile characteristics of CCM. If TP@CCM shows the same protein band pattern as CCM, it indicates that CCM has been successfully coated on the surface of TP. Figure 4 is the SDS-PAGE protein profile of TP, CCM and TP@CCM.

[0033] As can be seen from Figure 4 therein, there is no protein on the surface of pure TP, while after coating CCM on the surface of TP, the protein profile of the obtained TP@CCM is almost the same as that of pure CCM, once again demonstrating the successful loading of CCM.

[0034] 4. Oxygen release experiment of TPO: The experiment was divided into six groups: deoxygenated water (DOW), DOW + US, oxygen-saturated MTO (MTO@O 2 ), MTO@O 2 +US, TPO, and TPO + US. First, N 2 was introduced into deionized water to prepare deoxygenated water, and each sample was dispersed into the deoxygenated water so that the final working concentration of the sample was 250 μg / mL. Then, a dissolved oxygen meter was used to record the oxygen concentration in each aqueous solution. After 180 s of recording, an ultrasound (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle) was applied to each experimental group that required ultrasonic treatment for 420 s, and the oxygen concentration was continuously recorded during the ultrasound. After the ultrasound ended, the oxygen concentration in each aqueous solution was continuously recorded until the end of the experiment. Figure 5 shows the oxygen release performance of TPO under US excitation.

[0035] From Figure 5 , it can be seen that when US was applied to the system, the oxygen concentration in each experimental group increased, but the oxygen concentration in the TPO group showed the most intense explosive increase, indicating that TPO is very sensitive to external US stimuli and has excellent US responsiveness, and can release a large amount of O 2 . 5. Oxygen release experiment of TPO@CCM: The experimental procedure was the same as that in part 4, but at this time, only the differences among the three groups of DOW, TPO + US, and TPO@CCM + US were compared to study whether the presence of CCM would affect the release of oxygen. Figure 6 shows the oxygen release performance of TPO@CCM under US excitation.

[0036] From Figure 6 , it can be seen that during the US irradiation, the dissolved oxygen concentration released by TPO@CCM was only slightly lower than that of TPO, and the difference was negligible. This result indicates that CCM can effectively coat the surface of TPO without significantly affecting the loading and release behavior of O 2 , confirming the potential application value of TPO@CCM in in vivo tumor therapy. II. In vitro experiments of TPO: 1. In vitro US imaging experiment: The in vitro US imaging performance of TPO was tested using a self-made agar model. First, agar, glycerol, and distilled water (mass ratio 3:11:86) were uniformly mixed and heated until the agar was completely dissolved. Then, the obtained hot mixture was poured into a shaping vessel and left to set at room temperature. A silicone tube was placed on the surface of the set agar, and the hot mixture was poured again to cover the silicone tube, and it was left to set at room temperature again so that the silicone tube was embedded in the agar model. Next, different samples (PBS, MTO, TP, TPO, SonoVue) with the same concentration (250 μg / mL) were injected into the silicone tube, where PBS was used as a negative control and the clinical contrast agent SonoVue was used as a positive control; or different concentrations of TPO (0, 0.125, 0.25, 0.5, 1 mg / mL) were injected. An ultrasonic imaging system was used to image the prepared agar model (system parameters remained unchanged during the entire imaging acquisition process) to evaluate the in vitro US imaging performance of different samples and different concentrations of TPO. The above experiments were repeated three times. Figure 7 In vitro US imaging diagrams and gray value quantification for different materials and in vitro US imaging diagrams and gray value quantification for different concentrations of TPO, where (a) In vitro US imaging diagrams and gray value quantification for different materials, (b) In vitro US imaging diagrams and gray value quantification for different concentrations of TPO.

[0037] Given the good oxygen loading and release ability of TPO, the in vitro US imaging performance of TPO was evaluated using a self-made agar model embedded with a silicone tube. As Figure 7 shown in (a) of [reference], the gray value of the PBS group was set to 1, and the ratio of each group relative to the PBS group was examined. Compared with the PBS group, the gray value of the TPO group increased significantly, and its value was similar to that of the clinical contrast agent SonoVue. However, there was not much difference in the gray values of the MTO and TP groups compared with the PBS group. This proves that the application effect of MTO and TP in US imaging is limited, while TPO can significantly improve the contrast and resolution of US imaging by virtue of its excellent oxygen loading ability and controllable release characteristics. We also explored the relationship between the gray value of US imaging and the concentration of TPO. As Figure 7 shown in (b) of [reference], the gray value of the group with a concentration of 0 was set to 1, and the ratio of each group relative to the group with a concentration of 0 was examined. As the concentration of TPO increased, the gray value of US imaging also increased. This indicates that a high concentration of TPO can generate a large number of oxygen microbubbles, significantly enhancing the scattering effect of the material on ultrasonic waves, thereby greatly improving its US imaging ability. 2. Cavitation bubble generation experiment: Prepare suspensions of different samples (MTO, TP, TPO) with the same concentration (250 μg / mL) and deionized water as the control group for standby. Then, drop 20 μL of the liquid on the glass slide according to the grouping, and cover it with a coverslip. Then, perform ultrasound on the liquid through the glass slide (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle, 30 s), and immediately observe and take pictures using a confocal microscope after ultrasound.

[0038] The effect of SDT depends on the ability of the photosensitizer and the intensity of the cavitation effect. When the photosensitizer is determined, enhancing the cavitation intensity is the most direct and effective method to improve the SDT effect. There are two methods to enhance the cavitation intensity, namely reducing the cavitation threshold and increasing the number of cavitation nuclei. MTO can generate various ROS under US stimulation as a photosensitizer, and its mesoporous structure means a large specific surface area, which provides more nucleation sites for reducing the cavitation threshold. The introduction of PFHB and O 2 increases the number of cavitation nuclei and improves the possibility of cavitation bubble generation. To further determine whether the cavitation intensity of the system is enhanced, we evaluated the number of cavitation bubbles generated in different material solutions under the same US conditions. Figure 8 Figure 9 shows the bright-field photos of cavitation clusters generated by different materials under US excitation. As Figure 8 shown, the number of cavitation bubbles in the TPO group is the largest, and the number of cavitation bubbles in the TP, MTO, and DW groups decreases in turn. Such a difference indicates that the mesoporous structure of MTO, the vaporization of PFHB under US, and the introduction of O 2 can all improve the cavitation intensity and are beneficial to the improvement of SDT efficiency. 3. ROS generation experiment: (1) Evaluate the ability of TPO NPs to generate hydroxyl radicals (·OH) by detecting the degradation degree of MB. First, uniformly mix 50 μL of MB solution (1 mM) with 1.55 mL of deionized water, and then add 50 μL of deionized water (control group) or samples with a concentration of 250 μg / mL (MTO, TP, TPO) to the mixture and mix well. Irradiate the mixture with US, detect the absorbance every 0.5 min, and quantify the MB degradation degree according to the change in absorbance at 664 nm.

[0039] (2) Evaluate the generation of singlet oxygen ( 1 O 2The ability of ( ). First, 50 μL of DPBF solution (5 mM) was uniformly mixed with 1.55 mL of deionized water. Then, 50 μL of deionized water (control group) or samples with a concentration of 250 μg / mL (MTO, TP, TPO) were added to the mixed solution and mixed evenly. The mixed solution was irradiated with US, and the absorbance was measured every 1 min. The degradation degree of DPBF was quantified based on the change in absorbance at 398 nm. The above experiments were repeated three times. Figure 9 Degradation of MB and DPBF by different materials under US excitation, where (a) MB and (b) DPBF.

[0040] The SDT performance of TPO was evaluated by detecting the generation of ROS. First, we detected the generation of ·OH through the degradation experiment of MB. The concentration at the 0th minute was set as 1, and the ratio of the concentration of each minute in each group to the concentration at the 0th minute was investigated. As Figure 9 shown in (a) of, under the same US conditions, the degradation of MB in the TPO group was the most, while the degradation of MB in the TP and MTO groups decreased in turn. This indicates that TPO has the strongest ·OH generation ability. At the same time, the generation of 1 O 2 was also detected through the degradation experiment of DPBF. As Figure 9 shown in (b) of, the degradation of DPBF in each experimental group was similar to that of MB, and TPO showed the strongest 1 O 2 generation ability.

[0041] 4. In vitro anti-cancer experiment: First, 4T1 cells (about 5000 per well) were seeded in a 96-well plate and cultured under hypoxia overnight. Then, the old medium was removed, and fresh medium containing PBS, MTO, TP, or TPO (material concentration was 100 μg / mL) was added, and the cells were continuously cultured under hypoxia for 8 h. Then, the ultrasound group was treated with ultrasound for 1 min (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle), and the cells were continuously cultured under hypoxia for 12 h. After removing the old medium, the cells were washed twice with PBS, and CCK-8 reagent was added and incubated for 1 - 4 h. Finally, the absorbance at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the survival rate of 4T1 cells was calculated using the absorbance value. The above experiments were repeated three times.

[0042] Based on the high SDT performance of TPO, we used CCK-8 reagent to evaluate the cell viability of each group after different treatments, taking 4T1 breast cancer cells cultured under hypoxia as an example. Figure 10 Survival rate of 4T1 cells after different treatments. As Figure 10As shown, compared with other experimental groups, the 4T1 cell viability in the TPO + US group was the lowest. This indicates that under US stimulation, TPO has the strongest killing ability against 4T1 cells.

[0043] 5. In vitro anti-cancer mechanism exploration experiment: (1) Detection of cell hypoxia alleviation: (i) Seed 4T1 cells into confocal dishes (about 10 5 cells per dish); (ii) Culture under hypoxia until the cells grow to about 80%; (iii) Remove the old medium and add fresh medium containing PBS, MTO, TP or TPO (material concentration is 100 μg / mL for all); (iv) Continue hypoxic culture for 8 h; (v) Sonicate the sonication group for 1 min (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle); (vi) Continue hypoxic culture for 12 h; (vii) Remove the old medium and wash with PBS 2 - 3 times, then add 4% paraformaldehyde to fix the cells for 30 min; (viii) Remove paraformaldehyde and wash with PBS 3 times, each time for at least 3 min, then add PBS containing 0.5% Triton X-100 to permeabilize the cells for 15 min; (ix) Remove the permeabilization solution and wash with PBS, then add PBS containing 5% bovine serum albumin to block the cells for 1 h; (x) Remove the blocking solution, add diluted anti-HIF-1α antibody (diluted at a ratio of 1:300), and incubate overnight at 4 °C; (xi) Remove the primary antibody and wash with PBS 2 - 3 times, add diluted AlexaFluor 488-conjugated goat anti-mouse IgG secondary antibody, and incubate for 1 h at room temperature in the dark; (xii) Remove the secondary antibody and wash with PBS, add DAPI staining solution, and continue to incubate for 5 min at room temperature in the dark; (xiii) Aspirate the DAPI staining solution and wash with PBS, observe and take pictures through a confocal microscope. The above experiment was repeated three times.

[0044] To clarify the in vitro anti-cancer mechanism of TPO, we first evaluated the hypoxia status of cells in each group using hypoxia-inducible factor HIF-1α immunofluorescence staining. Figure 11 Immunofluorescence staining images of HIF-1α in 4T1 cells after different treatments.

[0045] As Figure 11As shown, the PBS group, TPO group, US group, MTO + US group, and TP + US group all showed strong green immunofluorescence, indicating significant HIF-1α accumulation in these groups, suggesting that the 4T1 cells were in an overall hypoxic state. In contrast, the immunofluorescence signal of the TPO + US group was very weak, indicating a significant reduction in the HIF-1α expression level in the cells of this group and an effective alleviation of the hypoxic state.

[0046] (2)Measurement of intracellular ROS content: (i)–(vi) were the same as those in the “Detection of Alleviation of Cell Hypoxia” section. (vii) Remove the old medium and wash with PBS, and stain the cells according to the instructions of the reactive oxygen species detection kit. (viii) Wash away the excess staining solution with PBS, place it under a confocal microscope for observation and photography. The above experiments were repeated three times.

[0047] The ROS level in 4T1 cells was detected using the DCFH-DA probe. The DCFH-DA probe can be oxidized by intracellular ROS to become 2′,7′-dichlorofluorescein with green fluorescence, and its fluorescence intensity is positively correlated with the ROS level. Figure 12 Figure shows the ROS fluorescence staining images and their quantitative results of 4T1 cells after different treatments. Among them, since the absolute value of fluorescence measurement is affected by various factors such as the instrument and laser intensity, it is more convenient to represent it with a relative value for data unification and comparison. The unit of the relative value is usually arbitrary unit, abbreviated as a.u. in English, which has no practical significance in fluorescence intensity.

[0048] As Figure 12 shown, under US excitation, the cells treated with TPO showed stronger green fluorescence than the cells in other groups, indicating a very high ROS level in the cells of this group. This high level of ROS will cause strong oxidative damage to 4T1 cells, leading to cell death, further revealing the potential mechanism by which TPO exerts its highly efficient anti-cancer effect by inducing ROS generation under US excitation.

[0049] 6. Detection of the maturity of induced dendritic cells (DCs): First, seed 4T1 cells (about 2×10 5 cells per well) into a six-well plate and culture them hypoxically overnight. Remove the old medium, and place the 4T1 cells in a medium containing PBS or TPO and continue hypoxic culture for 8 h. Then, sonicate the sonication group for 1 min (1.0 MHz, 1.5 W / cm 2, 50% duty cycle) and continue culturing for 12 h. Aspirate the supernatant in each culture dish and transfer it to a culture dish with DC 2.0 cells, and continue culturing DC cells for 24 h. Then remove the DC cell supernatant, wash the cells with PBS, and centrifuge at 4 °C and 500 r for 5 min to obtain cell pellets. Add anti-CD11c-FITC, anti-CD86-PE-Cy7, and anti-CD80-Alexa Fluor @R 647 antibodies diluted with 5% FBS to the DC cell pellets and incubate them in the dark at room temperature for 45 min. Finally, centrifuge the cells and wash them with PBS, and immediately use a flow cytometer for detection after resuspension. The above experiment was repeated three times.

[0050] Given that cancer cell debris can induce the maturation of DC cells as tumor-associated antigens, we used flow cytometry to detect the effect of the supernatant of 4T1 cell culture medium under different treatment conditions on the maturation of DC cells. Figure 13 For quantitative analysis of the maturation of DC cells after different treatments by flow cytometry, where CD80 and CD86 are the names of protein molecules expressed on the cells. The value in Q2 (double positive) represents the expression levels of CD86 and CD80 molecules. The higher the value, the more CD86 and CD80 molecules are expressed, indicating a higher degree of DC cell maturation. Q4 (double negative) is the opposite.

[0051] As Figure 13 shown, in the absence of US stimulation, the supernatant of 4T1 cell culture medium treated with PBS and TPO failed to induce the maturation of DC cells. Under US stimulation, the supernatant of the PBS group could only induce the maturation of a small number of DC cells, while the supernatant of the TPO group significantly promoted the maturation of a large number of DC cells. This result reveals that TPO can promote the maturation of DC cells by inducing the process of immunogenic cell death (ICD) under US stimulation, providing an important experimental basis for the potential application of TPO in anti-tumor immunotherapy.

[0052] III. In vivo experiments of TPO@CCM: 1. Establishment of a mouse breast cancer model: Mice (BALB / c, 6 weeks old, female) were purchased from Shandong Pengyue Laboratory Animal Technology Co., Ltd. and allowed to acclimatize in the laboratory for 1 week. All animal experiments were carried out in accordance with the protocols approved by the Experimental Animal Center of Shandong University. First, collect the cultured 4T1 cells by digestion and centrifugation, resuspend them with PBS, and place them on ice for later use. Then remove the hair on the right posterior side of the mouse's back and subcutaneously inject the 4T1 cell suspension (about 10 7 cells). When the tumor grows to 80 - 100 mm 3 in size (about 1 week of growth), the subsequent experiments can be carried out.

[0053] 2. Tumor US imaging experiments: (1) In-situ tumor US imaging: First, 6 days after subcutaneous injection of 4T1 cells (this day was defined as day 1), the tumor-bearing mice were randomly divided into 4 groups. Then, PBS, MTO, TPO, and TPO@CCM (the material concentration was 15 mg / kg for all) were injected into the mice via the tail vein respectively. After 12 h, a US imaging device was used to image the primary tumor sites of the mice. The above experiment was repeated three times.

[0054] (2) Metastatic tumor US imaging: The experimental method was the same as that in "In-situ tumor US imaging", except that the experiment was carried out 44 days after subcutaneous injection of 4T1 cells, and the US imaging site became the liver of the mice. The above experiment was repeated three times.

[0055] Based on the excellent performance of TPO in in-vitro US imaging, we evaluated the US imaging performance of TPO@CCM with tumor targeting ability for tumors in different parts of the body at different time points to monitor the growth and metastasis of tumors in real time. We first systematically evaluated its US imaging performance in primary tumors. Figure 14 US imaging diagrams and gray value quantification for primary tumors of different treatment groups, where (a) US imaging diagram, (b) gray value quantification.

[0056] As Figure 14 shown in (a) of, the gray value of the TPO group increased significantly, while the change in the MTO group was negligible compared with the PBS group, indicating that TPO maintained effective US imaging performance for primary tumors. It is worth noting that TPO@CCM produced the strongest harmonic ultrasound signal at the tumor site, which may be due to the homotypic recognition of CCM for homologous cancer cell lines promoting the specific aggregation of TPO@CCM in tumor tissues, further confirming the great potential of TPO@CCM as an ultrasound contrast agent. To quantitatively analyze this phenomenon, we used Image J software to analyze the gray values of the tumor regions marked by red circles in the figure. As Figure 14 shown in (b) of, consistent with the above discussion results, the gray value of the TPO@CCM group was the highest, indicating that its excellent oxygen-carrying ability and tumor targeting performance significantly improved the US imaging effect.

[0057] To further explore the application potential of TPO@CCM in the detection of small tumors, we evaluated its US imaging performance in metastatic tumors. Given that the lung is one of the main target organs for breast cancer metastasis and should have been the preferred research site, but due to the characteristics of gas-containing tissues in the lung leading to a large attenuation of ultrasound energy and it being difficult to obtain ideal imaging effects, we chose the liver as the representative research site for metastatic tumors. Figure 15Typical tumor-like tissues in the mouse liver and US images of the liver in the sagittal and cross-sectional planes of different treatment groups, where (a) tumor-like tissues and (b) US images of the liver.

[0058] As Figure 15 Shown in (a) of [reference], typical tumor-like tissues were observed in the mouse liver on day 43, confirming the occurrence of liver metastasis. Subsequently, we performed US imaging analysis on the livers of mice in different treatment groups. Figure 15 The results shown in (b) of [reference] indicated that the difference in gray values between liver tumors and normal liver tissues in the TPO group and the TPO@CCM group was significantly higher than that in the PBS group and the MTO group. This finding confirmed the applicability of TPO@CCM in the detection of micrometastatic tumors, providing an important basis for the early diagnosis of tumors and the formulation of clinical treatment strategies.

[0059] 3. Treatment of breast cancer model mice: Five days after subcutaneous injection of 4T1 cells (this day was defined as day 0), the tumor-bearing mice were randomly divided into 5 groups, namely PBS, TPO@CCM, αPD-L1, TPO@CCM + US, and TPO@CCM + αPD-L1 + US groups. TPO@CCM (15 mg / kg) was delivered via the tail vein on days 0, 2, and 4, and the αPD-L1 antibody (100 μg / mouse) was delivered via the tail vein on days 1, 3, and 5. Ultrasound (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle, 10 min) was continuously administered from day 1 to day 5. The tumor volume of the mice was recorded every 2 days (tumor volume V = a×b 2 / 2, where a and b are the length and width of the tumor, respectively), and the recording continued for 16 days. After 16 days, the mice were euthanized, and their blood, solid tumors, and lungs were collected for detection. The number of mice in each group was ≥5, and the experiment was repeated more than three times.

[0060] Figure 16 Shows the changes in tumor volume of mice in each group. As Figure 16As shown, the tumor growth rate in the TPO@CCM group was comparable to that in the PBS group, indicating that TPO@CCM alone could not effectively inhibit tumor growth. Meanwhile, single injection of αPD-L1 also failed to significantly inhibit tumor progression, suggesting that immune checkpoint blockade therapy (ICB) had limited efficacy against refractory immune "cold" 4T1 tumors. In contrast, the TPO@CCM + US group significantly inhibited tumor growth by enhancing ultrasound cavitation effects and alleviating the tumor hypoxic microenvironment, verifying the excellent efficacy of SDT. However, the tumors did not completely regress, suggesting that tumor cells might have an inherent immune evasion mechanism. Notably, the TPO@CCM + US + αPD-L1 group showed the most significant tumor growth inhibition effect among all experimental groups, which benefited from the remarkable advantages of the combined treatment of SDT and immunotherapy, providing a new approach to overcoming tumor immune escape and achieving complete tumor elimination. Sixteen days after treatment, the mice were euthanized and tumor tissues were extracted for photographing and weighing. Figure 17 The tumor masses and corresponding visualized photos of the mice in each group 16 days after treatment are shown, where (a) is the tumor mass and (b) is the visualized photo. As Figure 17 shown in (a) therein, the average tumor masses in the PBS group and the TPO@CCM group were the largest, and the tumor masses in the αPD-L1 group, the TPO@CCM + US group, and the TPO@CCM + αPD-L1 + US group decreased in turn, which was highly consistent with the tumor volume growth trend. In addition, Figure 17 (b) therein shows the ex vivo tumor photos of different treatment groups. Among them, the tumor volume in the TPO@CCM + αPD-L1 + US group was the smallest, and the tumor of one mouse completely regressed, further confirming the synergistic treatment effect of TPO@CCM combined with αPD-L1 under ultrasound irradiation.

[0061] To deeply explore the key mechanisms of action of sonoimmunotherapy, we systematically evaluated key immune indicators such as DC cell maturation and T cell infiltration in the mice. Figure 18 The contents of DC cells and T cells in the tumor tissues of the mice in each group were analyzed by flow cytometry. Among them, CD4, CD8, CD80, and CD86 are all the names of protein molecules expressed on the cells. The larger the value in Q2 in the first row, the higher the expression levels of CD80 and CD86, and the higher the DC cell maturity; the opposite is true for Q4. In the second row, Q1 represents the expression level of CD8, and Q3 represents the expression level of CD4. The higher these two values, the higher the T cell maturity.

[0062] As Figure 18As shown, flow cytometry was used to analyze the composition of immune cells in the tumor microenvironment of mice in each group. The results showed that the proportion of DC cells in the tumor cell microenvironment of the TPO@CCM + US group and the TPO@CCM + US + αPD-L1 group was significantly increased. Notably, the TPO@CCM + US + αPD-L1 treatment group showed the highest proportion of DC maturation, which confirmed that the synergistic effect of SDT and αPD-L1 could effectively promote the release of tumor-associated antigens, thereby activating DC maturation and triggering a systemic anti-tumor immune response. Further analysis showed that the infiltration ratios of CD4 + and CD8 + T cells in the TPO@CCM + US + αPD-L1 treatment group reached the highest levels, indicating that SDT elicited a strong adaptive anti-tumor immune response, and αPD-L1 alleviated tumor immunosuppression by blocking the PD-1 / PD-L1 immune checkpoint pathway, further enhancing this response.

[0063] In addition, we collected the lungs of the surviving mice on day 30 and systematically analyzed the lung metastasis of mice in each group by H&E staining. Figure 19 are the visualized photos of the lungs of mice in each group and the corresponding H&E staining results 30 days after treatment, where (a) visualized photos and (b) corresponding H&E staining results. From Figure 19 in (a) and Figure 19 in (b), it can be seen that obvious small transparent vesicles were visible in the lungs of mice in the PBS group, TPO@CCM group and αPD-L1 group, suggesting the occurrence of tumor metastasis. The H&E staining results further confirmed the above observations, and significant lung metastasis nodules (the nodules are marked with red circles) were shown in these groups. It should be emphasized that almost no metastatic nodules were seen in the lungs of mice in the TPO@CCM + US + αPD-L1 group, in sharp contrast to other groups. All of the above results indicate that cavitation-enhanced SDT combined with αPD-L1-mediated ICB therapy achieved the dual treatment goals of tumor ablation and metastasis inhibition by effectively alleviating the immunosuppressive microenvironment and promoting the production of a large amount of ROS, opening up a new treatment path for clinical tumor treatment.

[0064] The above description is only the preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An ultrasonic confined cavitation nanobubble, characterized in that: It has a core-shell structure, including an inner core of mesoporous titanium dioxide nanoparticles loaded with perfluorohexyl bromide and a tumor cell membrane shell layer coated on the surface of the inner core; The inner core in the shell is in an oxygen saturated state.

2. The ultrasonic confined cavitation nanobubble according to claim 1, characterized in that: The mesoporous titanium dioxide nanoparticles are spherical, with a particle size of 200-250 nm and a pore size of 2-20 nm; The ratio of perfluorohexyl bromide to mesoporous titanium dioxide nanoparticles is (10-15) μL:1 mg.

3. The ultrasonic confined cavitation nanobubble according to claim 1, characterized in that: The tumor cell membrane is a breast cancer cell membrane; The mass ratio of the perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles to the tumor cell membrane is 1:(1-3).

4. A method for preparing ultrasonic confined cavitation nanobubbles according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) ultrasonically mixing mesoporous titanium dioxide nanoparticles and perfluorohexyl bromide to prepare perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles; (2) Ultrasonic mixing of tumor cell membrane vesicles and perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles is performed, and the mixture is extruded to obtain tumor cell membrane-coated perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles; (3) Oxygen is introduced into a suspension of mesoporous titanium dioxide nanoparticles loaded with perfluorohexyl bromide coated with tumor cell membranes until oxygen saturation occurs, thereby obtaining ultrasonic confined cavitation nanobubbles.

5. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 4, characterized in that: In step (1), the method for preparing mesoporous titanium dioxide nanoparticles comprises the following steps: Anhydrous ethanol, PVP aqueous solution, hydrochloric acid and TiF4 solution are stirred and mixed at room temperature, and then reacted. After the reaction is completed, the mixture is cooled to room temperature, and mesoporous titanium dioxide nanoparticles are obtained after centrifugation, washing and freeze-drying.

6. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 5, characterized in that: The mixing volume ratio of the anhydrous ethanol, PVP aqueous solution, hydrochloric acid and TiF4 solution is (55-56): (5-10): (0.5-1): (3-6); The concentration of the PVP aqueous solution is 9-10 mg / mL; The concentration of the hydrochloric acid is 0.08-10.12 mol / L; The concentration of the TiF4 solution is 35-45 mmol / L; The reaction temperature is 175-185 ° C, and the reaction time is 2-5 h; The solvent used for the washing is water or ethanol.

7. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 4, characterized in that: In step (1), the ratio of perfluorohexyl bromide to mesoporous titanium dioxide nanoparticles is (10-15) μL:1 mg; The ultrasonic mixing is performed in ice water for 10-15 minutes; In step (2), the tumor cell membrane vesicles are breast cancer cell membrane vesicles; The breast cancer cell membrane vesicles are obtained by extruding a breast cancer cell membrane fragment suspension through a polycarbonate membrane; The pore size of the polycarbonate membrane is 400-800 nm, and the number of extrusions is greater than 10 times; The solvent of the breast cancer cell membrane fragment suspension is PBS, and the concentration is 450-550μg / mL; The method for preparing breast cancer cell membrane fragments comprises the following steps: The cultured breast cancer cells were digested with trypsin, and the breast cancer cell pellets were obtained after one centrifugation. The breast cancer cell membrane fragments were obtained after washing, resuspending, ice bathing, freezing and thawing, and secondary centrifugation.

8. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 4, characterized in that: In step (2), the mass ratio of the perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles to the tumor cell membrane is 1:(1-3); The extrusion was performed at least 10 times using a 400 nm polycarbonate film.

9. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 7, characterized in that: The breast cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum; the culture conditions were: temperature 37 °C, 5% CO2; The centrifugation speed is 770-850 rpm and the time is 3-6 minutes; The washing comprises washing the cell pellet 1-2 times with PBS; The resuspending is resuspending in a hypotonic lysis buffer containing a membrane protein extraction reagent and PMSF; The ice bath time is 10-20 minutes; The freeze-thaw method is to freeze the cell suspension using liquid nitrogen and then thaw it at room temperature; The secondary centrifugation is performed at 650-750 g for 10-15 minutes at 4 °C, after which the supernatant is collected and centrifuged again at 13000-15000 g for 30-40 minutes.

10. Use of the ultrasonic confined cavitation nanobubble according to any one of claims 1 to 3 in the preparation of drugs for treating tumors and in ultrasonic imaging.

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