Ultrasonic confined cavitation nanobubbles and their preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an ultrasonic confined cavitation nanobubble and its preparation method and application. The ultrasonic confined cavitation nanobubble has a core-shell structure, including an inner core of mesoporous titanium dioxide nanoparticles loaded with perfluorohexyl bromide and a tumor cell membrane shell coated on the surface of the inner core; the inner core in the shell is in an oxygen-saturated state. The present invention significantly improves the cavitation effect through a multi-dimensional synergistic strategy of optimizing the design of TPO@CCM nanobubble structure, regulating bubble generation and enhancing targeted delivery, which not only improves US imaging sensitivity, but also promotes the outbreak of ROS, and is used in conjunction with the immune checkpoint inhibitor αPD-L1 to achieve an efficient tumor killing effect, and has great application potential in the clinical detection and treatment of cancer.

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, a disease characterized by malignant tumors, is essentially the uncontrolled proliferation of tumor cells and their persistent invasion of surrounding tissues. The root cause of cancer is often attributed to gene mutations, which can be triggered by environmental and genetic factors, leading to dysregulation of key regulatory mechanisms such as cell proliferation, differentiation, and apoptosis, ultimately developing into highly invasive malignancies. Furthermore, cancer cells can evade the immune system through various mechanisms, including downregulating or altering the expression of surface antigens, releasing immunosuppressive cytokines, upregulating immune checkpoint ligands, and enhancing the expression of anti-apoptotic proteins. These mechanisms promote the survival and spread of cancer cells within the host. More seriously, cancer cells can escape from the primary tumor and spread to other organs through the bloodstream or lymphatic system, forming 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 the increasingly complex clinical needs. Specifically, in terms of detection, existing imaging technologies have low sensitivity for early-stage 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 therapeutic effect in real time during treatment, 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 effectiveness in killing 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 have greatly limited 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 purpose of the present invention is to provide an ultrasonic confined cavitation nanobubble and its preparation method and application, so as to overcome the shortcomings of the existing technology, optimize the US cavitation effect and tumor microenvironment through a multi-dimensional collaborative strategy, and provide important support for the realization of efficient and accurate integrated cancer diagnosis and treatment.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In a first aspect, the present invention provides an ultrasonic confined cavitation nanobubble having a core-shell structure, comprising an inner core of mesoporous titanium dioxide nanoparticles loaded with perfluorohexyl bromide and a tumor cell membrane shell coated on the surface of the inner core;

[0008] The core in the shell is in an oxygen saturated state.

[0009] 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;

[0010] The ratio of perfluorohexyl bromide to mesoporous titanium dioxide nanoparticles is (10-15) μL:1 mg.

[0011] In some other embodiments, the tumor cell membrane is a breast cancer cell membrane.

[0012] In a second aspect, the present invention provides a method for preparing the ultrasonic confined cavitation nanobubbles described in the first aspect, comprising the following steps:

[0013] (1) ultrasonically mixing mesoporous titanium dioxide nanoparticles with perfluorohexyl bromide to prepare perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles;

[0014] (2) Ultrasonic mixing of tumor cell membrane vesicles and perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles was performed, and the mixture was extruded to obtain tumor cell membrane-coated perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles;

[0015] (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 ultrasonically confined cavitation nanobubbles.

[0016] In some other embodiments, in step (1), the method for preparing mesoporous titanium dioxide nanoparticles comprises the following steps:

[0017] Anhydrous ethanol, PVP aqueous solution, hydrochloric acid and TiF4 solution were stirred and mixed at room temperature, and then reacted. After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed and freeze-dried to obtain mesoporous titanium dioxide nanoparticles.

[0018] In some other embodiments, 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);

[0019] The concentration of the PVP aqueous solution is 9-10 mg / mL;

[0020] The concentration of the hydrochloric acid is 0.08-10.12 mol / L;

[0021] The concentration of the TiF4 solution is 35-45 mmol / L;

[0022] The reaction temperature is 175-185 ° C, and the reaction time is 2-5 h;

[0023] The solvent used for the washing is water or ethanol.

[0024] In some other embodiments, in step (1), the ratio of perfluorohexyl bromide to mesoporous titanium dioxide nanoparticles is (10-15) μL:1 mg;

[0025] The ultrasonic mixing is performed in ice water for 10-15 minutes;

[0026] In step (2), the tumor cell membrane vesicles are breast cancer cell membrane vesicles;

[0027] The breast cancer cell membrane vesicles are obtained by extruding a breast cancer cell membrane fragment suspension through a polycarbonate membrane;

[0028] The pore size of the polycarbonate membrane is 400-800 nm, and the number of extrusions is greater than 10;

[0029] The breast cancer cell membrane fragment suspension solvent is phosphate buffer solution, the concentration is 450-550μg / mL;

[0030] The method for preparing breast cancer cell membrane fragments comprises the following steps:

[0031] The cultured breast cancer cells were digested with trypsin, and mouse 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.

[0032] 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);

[0033] The extrusion was performed at least 10 times using a 400 nm polycarbonate film.

[0034] In some other embodiments, the breast cancer cells are cultured in a medium containing RPMI-1640 medium containing 10% fetal bovine serum; the culture conditions are: a temperature of 37°C and 5% CO2;

[0035] The centrifugation speed is 770-850 rpm and the time is 3-6 minutes;

[0036] The washing step is to wash the cell pellet 1-2 times with PBS;

[0037] The resuspending is resuspending in a hypotonic lysis buffer containing a membrane protein extraction reagent and PMSF;

[0038] The ice bath time is 10-20 minutes;

[0039] The freeze-thaw method is to freeze the cell suspension using liquid nitrogen and then thaw it at room temperature;

[0040] The secondary centrifugation was performed at 650-750 g for 10-15 minutes at 4 °C, and the supernatant was collected and centrifuged again at 13000-15000 g for 30-40 minutes.

[0041] In a third aspect, the present invention provides the use of the ultrasonic confined cavitation nanobubbles described in the first aspect in the preparation of drugs for treating tumors and in ultrasonic imaging.

[0042] The inventive concept adopted by the present invention is:

[0043] How to effectively enhance the ultrasonic cavitation effect and reshape the tumor microenvironment has become an important direction for breaking through existing technological barriers. In the regulation of the US cavitation effect, by lowering the cavitation energy threshold and increasing the number of cavitation nuclei, the utilization rate of ultrasonic energy can be significantly improved, while promoting the explosive generation of ROS; combined with hypoxic microenvironment regulation and targeted delivery technology, it is possible to enhance tumor-specific killing while achieving enhanced oxygen supply. In addition, the stable cavitation behavior of bubbles during the cavitation process can significantly enhance US imaging contrast, providing a new idea for real-time monitoring under the guidance of US imaging. Therefore, optimizing the US cavitation effect and tumor microenvironment through a multi-dimensional collaborative strategy will provide important support for the realization of efficient and accurate integrated cancer diagnosis and treatment.

[0044] The present invention uses ultrasound confined cavitation nanobubbles (named TPO@CCM) for the detection and treatment of primary tumors and metastatic tumors. Figure 1 As shown, (a) schematic diagram of the synthesis of TPO@CCM, (b) ultrasound imaging detection of primary tumors and metastatic tumors by TPO@CCM, and (c) TPO@CCM combined with αPD-L1 for the treatment of primary tumors and inhibition of tumor metastasis. Figure 1(a) is a process in which perfluorohexyl bromide (PFHB) is loaded inside titanium dioxide nanoparticles (MTO) with a special mesoporous structure to make PFHB-loaded MTO nanomaterial (TP), oxygen is introduced into the material to achieve oxygen saturation, and finally the material is biomimetically coated with tumor cell membrane (CCM) to make a nanobubble material (TPO@CCM).

[0045] Figure 1 (b) and Figure 1 In (c), MTO, a typical representative of inorganic sonosensitizers, can generate a variety of ROS through US activation. Its unique hollow confined mesoporous structure not only serves as an efficient drug carrier, but also gives the material a high specific surface area and excellent gas adsorption capacity, providing more nucleation sites to lower the cavitation threshold. The loaded PFHB, a perfluorocarbon compound, has high oxygen solubility and US-responsive liquid-gas phase transition properties. It can vaporize into bubbles under US stimulation and release preloaded oxygen simultaneously, which can not only alleviate the tumor hypoxic microenvironment and break the oxygen-dependent limitation of SDT, but also provide more cavitation nuclei to enhance the cavitation effect. In addition, the CCM coating gives the material homologous targeting ability, utilizing the homing effect of membrane surface adhesion molecules and source tumor cells to significantly improve the active enrichment efficiency of nanobubbles at the tumor site.

[0046] Beneficial effects of the present invention:

[0047] The present invention significantly enhances the cavitation effect through a multi-dimensional synergistic strategy of optimizing the TPO@CCM nanobubble structure, regulating bubble generation, and enhancing targeted delivery. This not only improves US imaging sensitivity but also promotes the outbreak of ROS. It also achieves a highly efficient tumor-killing effect when used in conjunction with the immune checkpoint inhibitor αPD-L1, and has great application potential in the clinical detection and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0049] Figure 1 Schematic diagram of the use of ultrasonic confined cavitation nanobubbles TPO@CCM for the detection and treatment of breast cancer in Example 1 of the present invention, including: (a) schematic diagram of the synthesis of TPO@CCM, (b) ultrasonic imaging detection of primary tumors and metastases using TPO@CCM, and (c) TPO@CCM combined with αPD-L1 for the treatment of primary tumors and inhibition of tumor metastasis;

[0050] Figure 2Transmission electron microscopy images of MTO, TP, and TP@CCM in Example 1 of the present invention, where (a) MTO, (b) TP, and (c) TP@CCM;

[0051] Figure 3 The hydrated particle size distribution diagram and the Zeta potential diagram in Example 1 of the present invention, wherein: (a) the hydrated particle size distribution diagram, (b) the Zeta potential diagram;

[0052] Figure 4 This is the SDS-PAGE protein spectrum of TP, CCM and TP@CCM in Example 1 of the present invention;

[0053] Figure 5 is the oxygen release performance of TPO under US excitation in Example 1 of the present invention;

[0054] Figure 6 The oxygen release performance of TPO@CCM under US excitation in Example 1 of the present invention;

[0055] Figure 7 The in vitro US imaging images and grayscale value quantification of different materials and the in vitro US imaging images and grayscale value quantification of TPO at different concentrations in Example 1 of the present invention, wherein (a) in vitro US imaging images and grayscale value quantification of different materials, (b) in vitro US imaging images and grayscale value quantification of TPO at different concentrations;

[0056] Figure 8 Bright field photographs of cavitation clusters generated by different materials under US excitation in Example 1 of the present invention;

[0057] Figure 9 The degradation of MB and DPBF by different materials under US excitation in Example 1 of the present invention, where (a) MB, (b) DPBF;

[0058] Figure 10 is the survival rate of 4T1 cells after different treatments in Example 1 of the present invention;

[0059] Figure 11 These are images of HIF-1α immunofluorescence staining of 4T1 cells after different treatments in Example 1 of the present invention;

[0060] Figure 12 The ROS fluorescence staining images and quantification of 4T1 cells after different treatments in Example 1 of the present invention are shown;

[0061] Figure 13 The flow cytometry analysis of the maturation of DC cells after different treatments in Example 1 of the present invention is as follows, wherein CD80 and CD86 are the names of protein molecules expressed on the cells;

[0062] Figure 14The US imaging images and grayscale value quantification of primary tumors in different treatment groups in Example 1 of the present invention, wherein (a) US imaging image, (b) grayscale value quantification;

[0063] Figure 15 Typical tumor-like tissue in the mouse liver and US imaging images of the liver in sagittal and transverse sections of different treatment groups in Example 1 of the present invention, including (a) tumor-like tissue and (b) liver US imaging;

[0064] Figure 16 The changes in tumor volume of each group of mice in Example 1 of the present invention;

[0065] Figure 17 The tumor masses and corresponding visual photos of mice in each group treated for 16 days in Example 1 of the present invention are shown, where (a) tumor mass and (b) visual photos are shown.

[0066] Figure 18 The flow cytometry analysis of the content of DC cells and T cells in the tumor tissues of each group of mice in Example 1 of the present invention, wherein CD4, CD8, CD80, and CD86 are the names of protein molecules expressed on cells;

[0067] Figure 19 These are the visualized photos and corresponding H&E staining results of the lungs of mice in each group after 30 days of treatment in Example 1 of the present invention, wherein (a) visualized photos, (b) corresponding H&E staining results;

[0068] in, Figure 16-19 Among them, I:PBS, II:TPO@CCM, III:αPD-L1, IV:TPO@CCM + US, V:TPO@CCM + US + αPD-L1. DETAILED DESCRIPTION

[0069] Those skilled in the art will appreciate that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the examples were performed under conventional conditions or manufacturer's recommended conditions. Components used without manufacturer's indication are commercially available conventional products.

[0070] Example 1

[0071] Preparation of nanobubble materials

[0072] 1. Main experimental materials:

[0073] Titanium tetrafluoride (TiF4, 99%) was purchased from Sigma-Aldrich. Polyvinylpyrrolidone (PVP, Mw = 40,000) was provided by Shanghai Yuanye Biotechnology Co., Ltd. Hydrochloric acid (HCl) was purchased from Yantai Far East Fine Chemical Co., Ltd. Perfluorohexyl bromide (PFHB) was purchased from Saien Chemical Technology Co., Ltd. Anhydrous ethanol (C2H5OH) was supplied by Sinopharm Chemical Reagent Co., Ltd. Sulfur hexafluoride microbubbles for injection (SonoVue) were from Bracco Suisse SA, Switzerland. Methylene blue (MB) and 1,3-diphenylisobenzofuran (DPBF) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Hypoxia-inducible factor-1α (HIF-1α) antibody was purchased from Beijing Bioson 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 @R 647 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 purchased from Shenzhen Dakoway Biotechnology Co., Ltd. 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 provided by Wuhan Saiwei Biotechnology Co., Ltd. Cell Counting Kit-8 (CCK-8) was provided by Shanghai Titan Technology Co., Ltd. Deionized (DI) water (Millipore Milli-Q grade, 18.2 MΩ) was used in all experiments.

[0074] 2. Preparation of nanobubble materials:

[0075] (1) Preparation of mesoporous titanium dioxide nanomaterials (MTO):

[0076] First, 55.2 mL of anhydrous ethanol, 8 mL of a 9.75 mg / mL PVP aqueous solution, 500 μL of 0.1 M hydrochloric acid, and 5 mL of a 40 mM TiF₄ solution were added sequentially to a reaction vessel and stirred at room temperature for 1 hour. The mixture was then added to an autoclave and incubated at 180°C for 3 hours. The reaction solution was cooled to room temperature, centrifuged, and washed three times with water or ethanol. Finally, the purified solution was dried in a freeze dryer and both the solution and freeze-dried powder were stored at 4°C to produce mesoporous titanium dioxide nanomaterials, labeled MTO.

[0077] (2) Preparation of PFHB-loaded MTO nanomaterials (TP):

[0078] 3 mg of MTO was placed in a 50 mL centrifuge tube fitted with a rubber stopper. The tube was evacuated with a vacuum pump, and 30 μL of PFHB was quickly injected into the tube. The tube was ultrasonicated in ice water for 10 minutes to prepare PFHB-loaded MTO nanomaterials, labeled TP. The nanomaterials were then dispersed in 1 mL of PBS for later use.

[0079] (3) Extraction of mouse breast cancer cell membrane (CCM):

[0080] Mouse breast cancer (4T1) cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% CO₂. After 36 hours of growth, when the 4T1 cancer cells reached 80–90% of the dish confluence, they were trypsinized and centrifuged at 800 rpm for 5 minutes to collect the cell pellet. The cell pellet was then washed one to two times with PBS and resuspended in hypotonic lysis buffer containing membrane protein extraction reagent and PMSF and incubated on ice for 15 minutes. Following the ice bath, the cell suspension was frozen in liquid nitrogen and thawed at room temperature twice using a freeze-thaw cycle. The freeze-thawed suspension was centrifuged at 700 g for 12 minutes at 4°C. The supernatant was collected and centrifuged again at 14,000 g for 40 minutes to obtain the mouse breast cancer cell membrane (CCM) pellet, which was resuspended in PBS for later use.

[0081] (4) Preparation of CCM-coated TP nanomaterials (TP@CCM):

[0082] First, the obtained CCM fragment suspension (resuspended in PBS) was extruded through 800 nm and 400 nm polycarbonate membranes at least 10 times to obtain CCM vesicles. The obtained CCM vesicles were then mixed with TP (at a mass ratio of 2:1) and sonicated for 5 minutes. The mixed solution was then co-extruded through a 400 nm polycarbonate membrane at least 10 times to obtain CCM-coated TP nanomaterials, labeled TP@CCM.

[0083] (5) Preparation of oxygen-saturated TP nanomaterials (TPO) and oxygen-saturated TP@CCM nanobubble materials (TPO@CCM):

[0084] O2 was introduced into the obtained TP and TP@CCM suspensions until they were saturated with oxygen (until no further oxygen could be added), thereby obtaining oxygen-saturated TP nanomaterials and oxygen-saturated TP@CCM nanobubbles, which were labeled as TPO and TPO@CCM nanobubbles, respectively.

[0085] Performance Characterization

[0086] 1. Characterization of Nanobubble Materials

[0087] 1. Morphology characterization:

[0088] MTO, TP, and TP@CCM powders were dissolved in deionized water (DW) to prepare a 1 mg / mL stock solution. Each stock solution was then diluted 50-fold and transferred to a 1.5 mL centrifuge tube. The tube was then ultrasonically cleaned for 20 minutes. 10 μL of the evenly dispersed dilution was then dropwise added to a copper grid and dried overnight in an electronic dehumidifier. Transmission electron microscopy (TEM) was used to observe the morphology of the nanoparticles. Figure 2 Transmission electron microscopy images of MTO, TP, and TP@CCM, including (a) MTO, (b) TP, and (c) TP@CCM.

[0089] like Figure 2 As shown in (a), the prepared MTO exhibits a typical spherical morphology with an average size of about 220 nm and a typical mesoporous structure, proving the successful synthesis of MTO. The morphology and size of TP are shown in Figure 2 As shown in (b), 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 (c) shows the morphology of TP@CCM. A layer of cell membrane-like coating can be clearly observed on the surface of TP, proving that CCM is successfully wrapped on the surface of TP to obtain TP@CCM.

[0090] 2. Characterization of hydrated particle size and zeta potential:

[0091] MTO, TP, and TP@CCM powders were dissolved in deionized water to prepare a 1 mg / mL stock solution. Each stock solution was then diluted 10-fold, and 3 mL of the diluted solution was transferred to a 5 mL centrifuge tube. Ultrasonication was performed for 20 minutes. The hydrated particle size and zeta potential of the nanoparticles were measured using a nanoparticle size potentiometer. This was repeated three times. Figure 3 The hydrated particle size distribution diagram and Zeta potential diagram, including (a) hydrated particle size distribution diagram, (b) Zeta potential diagram.

[0092] like Figure 3 As shown in (a), the hydrated particle sizes of MTO, TP, and TP@CCM gradually increase, proving the successful preparation of TP@CCM. Figure 3 Figure (b) shows the changes in the zeta potential of MTO, TP, and TP@CCM. Compared with MTO, the potential of TP is significantly larger, and the potential of TP@CCM is reversed, showing the same potential characteristics as pure CCM, indicating the successful synthesis of the nanoparticles.

[0093] 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment:

[0094] SDS-PAGE was performed on TP, CCM, and TP@CCM to analyze the protein composition of their surfaces and verify whether TP@CCM retained the protein profile characteristics of CCM. If TP@CCM and CCM showed the same protein banding pattern, it indicated that CCM was successfully coated on the TP surface. Figure 4 SDS-PAGE protein spectra of TP, CCM and TP@CCM.

[0095] from Figure 4 It can be seen that there is no protein on the pure TP surface, but after coating CCM on the TP surface, the protein spectrum of TP@CCM obtained is almost the same as that of pure CCM, which once again proves the successful loading of CCM.

[0096] 4. TPO oxygen release experiment:

[0097] The experiment was divided into six groups: deoxygenated water (DOW), DOW + US, oxygen-saturated MTO (MTO@O2), MTO@O2 + US, TPO, and TPO + US. Deoxygenated water was first prepared by bubbling N2 into deionized water. Each sample was then dispersed into the deoxygenated water to a final working concentration of 250 μg / mL. A dissolved oxygen meter was then used to record the oxygen concentration in each aqueous solution. After 180 seconds of recording, each experimental group requiring ultrasonic treatment was subjected to 420 seconds of ultrasound (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle). The oxygen concentration was continuously recorded during the ultrasound period. After the ultrasound period, the oxygen concentration in each aqueous solution was continuously recorded until the end of the experiment. Figure 5 is the oxygen release performance of TPO under US stimulation.

[0098] 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 strongest explosive increase, which indicates that TPO is very sensitive to external US stimulation, has excellent US responsiveness, and can release a large amount of O2 in a short time.

[0099] 5. Oxygen release experiment of TPO@CCM:

[0100] The experimental procedures were the same as those in Part 4, but this time only the differences among the DOW, TPO + US, and TPO@CCM + US groups were compared to investigate whether the presence of CCM would affect the release of oxygen. Figure 6 Oxygen release performance of TPO@CCM under US stimulation.

[0101] from Figure 6 It can be seen that during US irradiation, the dissolved oxygen concentration released by TPO@CCM is only slightly lower than that of TPO, and the difference is negligible. This result shows that CCM can be effectively coated on the TPO surface without significantly affecting the O2 loading and release behavior, confirming the potential application value of TPO@CCM in in vivo tumor treatment.

[0102] 2. In vitro experiments of TPO:

[0103] 1. In vitro US imaging experiment:

[0104] The in vitro US imaging performance of TPO was tested using a homemade agar phantom. Agar, glycerol, and distilled water (mass ratio 3:11:86) were uniformly mixed and heated until the agar was completely dissolved. The resulting hot mixture was then poured into a shaped container and allowed to set at room temperature. A silicone tube was placed on the shaped agar surface, and the hot mixture was poured over the tube to cover it. The tube was then allowed to set again at room temperature, so that the tube was embedded in the agar phantom. The silicone tube was then injected with different samples (PBS, MTO, TP, TPO, and SonoVue) at the same concentration (250 μg / mL), with PBS serving as a negative control and the clinical contrast agent SonoVue serving as a positive control; or with different concentrations of TPO (0, 0.125, 0.25, 0.5, and 1 mg / mL). The prepared agar phantoms were imaged using an ultrasound imaging system (system parameters remained constant throughout the acquisition process) to evaluate the in vitro US imaging performance of the different samples and TPO concentrations. Each experiment was repeated three times. Figure 7 In vitro US imaging images and grayscale value quantification of different materials and in vitro US imaging images and grayscale value quantification of different concentrations of TPO, including (a) in vitro US imaging images and grayscale value quantification of different materials, (b) in vitro US imaging images and grayscale value quantification of different concentrations of TPO.

[0105] Given the good oxygen loading and release capabilities of TPO, the in vitro US imaging performance of TPO was evaluated using a homemade agar model embedded with silicone tubes. Figure 7 As shown in (a), the grayscale 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 grayscale value of the TPO group increased significantly, and its value was close to that of the clinical contrast agent SonoVue. However, the grayscale values ​​of the MTO and TP groups were not much different from those of the PBS group. This proves that the application effects of MTO and TP in US imaging are limited, and TPO can significantly improve the contrast and resolution of US imaging with its excellent oxygen loading capacity and controlled release characteristics. We also explored the relationship between the grayscale value of US imaging and the concentration of TPO. As shown in Figure 7As shown in (b), the grayscale value of the group with a concentration of 0 is set to 1, and the ratio of each group to the group with a concentration of 0 is examined. As the TPO concentration increases, the grayscale value of the US imaging also increases. This indicates that high TPO concentrations can generate a large number of oxygen microbubbles, significantly enhancing the material's scattering effect on ultrasound, thereby significantly improving its US imaging capabilities.

[0106] 2. Cavitation bubble generation experiment:

[0107] Prepare suspensions of different samples (MTO, TP, and TPO) at the same concentration (250 μg / mL) and deionized water as a control. Then, drop 20 μL of each solution onto a glass slide, cover with a coverslip, and apply ultrasound (1.0 MHz, 1.5 W / cm) through the slide. 2 , 50% duty cycle, 30 s), and the cells were observed and photographed using a confocal microscope immediately after sonication.

[0108] The effect of SDT depends on the ability of the sonosensitizer and the intensity of the cavitation effect. When the sonosensitizer is determined, enhancing the cavitation intensity is the most direct and effective way to improve the SDT effect. There are two ways to enhance the cavitation intensity, namely lowering the cavitation threshold and increasing the number of cavitation nuclei. MTO, as a sonosensitizer, can produce a variety of ROS under US stimulation. Its mesoporous structure means a large specific surface area, which provides more nucleation sites for lowering the cavitation threshold. The introduction of PFHB and O2 increases the number of cavitation nuclei and increases the possibility of cavitation bubble generation. In order to further determine whether the cavitation intensity of the system is improved, we evaluated the number of cavitation bubbles generated in solutions of different materials under the same US conditions. Figure 8 These are bright field photos of cavitation clusters generated by different materials under US excitation. Figure 8 As shown in the figure, the number of cavitation bubbles in the TPO group is the largest, while the number of cavitation bubbles in the TP, MTO, and DW groups decreases in that order. These differences indicate that the mesoporous structure of MTO, the vaporization of PFHB under US, and the introduction of O2 can all enhance the intensity of cavitation, which is beneficial to improving the efficiency of SDT.

[0109] 3. ROS generation experiment:

[0110] (1) The ability of TPO NPs to generate hydroxyl radicals (·OH) was evaluated by detecting the degree of MB degradation. First, 50 μL of MB solution (1 mM) was evenly mixed with 1.55 mL of deionized water. Then, 50 μL of deionized water (control group) or 250 μg / mL samples (MTO, TP, TPO) were added to the mixture and mixed evenly. The mixture was irradiated with US, and the absorbance was measured every 0.5 min. The degree of MB degradation was quantified based on the change in absorbance at 664 nm.

[0111] (2) Evaluate the generation of singlet oxygen by TPO by detecting the degradation degree of DPBF ( 1 O2) capacity. First, 50 μL of a 5 mM DPBF solution was mixed evenly with 1.55 mL of deionized water. Then, 50 μL of deionized water (control) or a sample (MTO, TP, TPO) at a concentration of 250 μg / mL was added to the mixture and mixed thoroughly. The mixture was irradiated with US, and the absorbance was measured every 1 minute. The degree of DPBF degradation was quantified based on the change in absorbance at 398 nm. Each experiment was repeated three times. Figure 9 Degradation of MB and DPBF by different materials under US excitation, including (a) MB and (b) DPBF.

[0112] The SDT performance of TPO was evaluated by detecting the generation of ROS. First, we used the MB degradation experiment to detect the generation of ·OH, setting its concentration at minute 0 as 1, and examined the ratio of the concentration at each minute of each group to the concentration at minute 0. Figure 9 As shown in (a) of Figure 3, under the same US conditions, MB degradation in the TPO group was the highest, while MB degradation in the TP and MTO groups decreased in turn. This indicates that TPO has the strongest ·OH generation capacity. 1 The generation of O2. Figure 9 As shown in (b), the degradation of DPBF in each experimental group is similar to that of MB, and TPO shows the strongest 1 O2 generation capacity.

[0113] 4. In vitro anticancer experiment:

[0114] 4T1 cells were seeded into 96-well plates (approximately 5,000 cells per well) and cultured overnight in anoxic conditions. The old culture medium was then removed and fresh culture medium containing PBS, MTO, TP, or TPO (all at 100 μg / mL) was added, followed by anoxic culture for another 8 h. The cells were then sonicated for 1 min (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle), and continue hypoxic culture for 12 hours. Remove the old culture medium, wash twice with PBS, and incubate with CCK-8 reagent for 1-4 hours. Finally, measure the absorbance at 450 nm using a microplate reader. Calculate the viability of 4T1 cells based on the absorbance value. Repeat this experiment three times.

[0115] Based on the high SDT performance of TPO, we used 4T1 breast cancer cells cultured in hypoxia as a representative and used CCK-8 reagent to evaluate the cell activity of each group after different treatments. Figure 10 is the survival rate of 4T1 cells after different treatments. Figure 10 As shown in the figure, the survival rate of 4T1 cells in the TPO + US group was the lowest compared with other experimental groups. This indicates that TPO has the strongest killing ability on 4T1 cells under US stimulation.

[0116] 5. In vitro anti-cancer mechanism exploration experiment:

[0117] (1) Cell hypoxia relief detection:

[0118] (i) 4T1 cells were plated into confocal microplates (approximately 10 cells per dish). 5 (ii) anoxic culture until the cells grew to about 80%; (iii) the old culture medium was removed and fresh culture medium containing PBS, MTO, TP, or TPO (all at 100 μg / mL) was added; (iv) anoxic culture was continued for 8 h; (v) the cells were sonicated for 1 min (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle); (vi) continue hypoxic culture for 12 h; (vii) remove old culture medium and wash with PBS 2-3 times, add 4% paraformaldehyde to fix the cells for 30 min; (viii) remove paraformaldehyde and wash with PBS 3 times, each time for no less than 3 min, then add PBS containing 0.5% Triton X-100 to permeabilize the cells for 15 min; (ix) remove permeabilization solution and wash with PBS, add PBS containing 5% bovine serum albumin to block the cells for 1 h; (x) remove blocking solution, add diluted anti-HIF-1α antibody (diluted at a ratio of 1:300), and incubate at 4°C overnight; (xi) remove primary antibody and wash with PBS 2-3 times, add diluted AlexaFluor 488-conjugated goat anti-mouse IgG secondary antibody, and incubate at room temperature in the dark for 1 h; (xii) remove secondary antibody and wash with PBS, add DAPI staining solution, and continue incubation at room temperature in the dark for 5 min; (xiii) aspirate the DAPI staining solution and wash with PBS, then observe and photograph using a confocal microscope. The above experiment was repeated three times.

[0119] To elucidate the anti-cancer mechanism of TPO in vitro, we first evaluated the hypoxia status of cells in each group using hypoxia-inducible factor HIF-1α immunofluorescence staining. Figure 11 These are images of HIF-1α immunofluorescence staining of 4T1 cells after different treatments.

[0120] like Figure 11As shown, the PBS, TPO, US, MTO + US, and TP + US groups all showed strong green immunofluorescence, indicating significant HIF-1α accumulation in these groups, suggesting that the 4T1 cells were in a state of hypoxia. In contrast, the immunofluorescence signal in the TPO + US group was very weak, indicating that the expression level of HIF-1α in this group of cells was significantly reduced, and the hypoxic state was effectively alleviated.

[0121] (2) Intracellular ROS content test:

[0122] (i)-(vi) Same as in the "Cell Hypoxia Relief Assay" section. (vii) Remove old culture medium and wash cells with PBS. Stain cells according to the instructions of the reactive oxygen species detection kit. (viii) Wash off excess staining solution with PBS, observe and photograph cells under a confocal microscope. Repeat the above experiment three times.

[0123] The DCFH-DA probe was used to detect ROS levels in 4T1 cells. The DCFH-DA probe can be oxidized by intracellular ROS to produce green fluorescent 2',7'-dichlorofluorescein, whose fluorescence intensity is positively correlated with ROS levels. Figure 12 The following images show ROS fluorescence staining and quantitative results for 4T1 cells after different treatments. Because absolute fluorescence values ​​are affected by multiple factors, such as instrumentation and laser intensity, relative values ​​are used to facilitate data consistency and comparison. Relative values ​​are typically expressed in arbitrary units (au), which have no practical meaning in fluorescence intensity.

[0124] like Figure 12 As shown, under US stimulation, TPO-treated cells exhibited stronger green fluorescence than cells in other groups, indicating that the level of ROS in these cells was extremely high. This high level of ROS triggered strong oxidative damage in 4T1 cells, leading to cell death, further revealing the potential mechanism by which TPO exerts its potent anti-cancer effects by inducing ROS generation under US stimulation.

[0125] 6. Detection of induced dendritic cell (DC) maturity:

[0126] First, 4T1 cells were seeded into six-well plates (approximately 2 × 10 5 The old culture medium was removed and 4T1 cells were placed in a culture medium containing PBS or TPO and continued to be cultured in anoxic conditions for 8 h. The cells were then sonicated for 1 min (1.0 MHz, 1.5 W / cm 2, 50% duty cycle) and continue culturing for 12 hours. The supernatant from each culture dish was aspirated and transferred to a culture dish containing DC 2.0 cells. The DC cells were cultured for another 24 hours. The DC cell supernatant was then removed, and the cells were washed with PBS. The cells were centrifuged at 4°C, 500 rpm for 5 minutes to obtain a cell pellet. Anti-CD11c-FITC, anti-CD86-PE-Cy7, and anti-CD80-Alexa Fluor diluted in 5% FBS were added to the DC cell pellet. @R The cells were incubated with the 647 antibody at room temperature in the dark for 45 minutes. Finally, the cells were centrifuged and washed with PBS. After resuspending, they were immediately analyzed by flow cytometry. This experiment was repeated three times.

[0127] Given that cancer cell fragments can act as tumor-associated antigens to induce DC cell maturation, we used flow cytometry to detect the effects of 4T1 cell culture supernatant under different treatment conditions on DC cell maturation. Figure 13 This flow cytometric analysis quantitatively analyzes DC maturation after different treatments. CD80 and CD86 are the names of proteins expressed on cells. The value in Q2 (double positive) represents the expression of CD86 and CD80 molecules. A higher value indicates greater expression of CD86 and CD80 molecules and higher DC maturation. The opposite is true for Q4 (double negative).

[0128] like Figure 13 As shown in the results, in the absence of US stimulation, culture supernatants from 4T1 cells treated with PBS and TPO failed to induce DC maturation. However, under US stimulation, culture supernatants from the PBS group only induced a small number of DC maturation, while culture supernatants from the TPO group significantly promoted the maturation of a large number of DCs. This result reveals that TPO can promote DC maturation by inducing immunogenic cell death (ICD) under US stimulation, providing important experimental evidence for the potential application of TPO in anti-tumor immunotherapy.

[0129] 3. In vivo experiments of TPO@CCM:

[0130] 1. Establishment of mouse breast cancer model:

[0131] Mice (BALB / c, 6 weeks, female) were purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd. and acclimated to the laboratory for 1 week. All animal experiments were performed in accordance with protocols approved by the Experimental Animal Center of Shandong University. Cultured 4T1 cells were first collected by digestion and centrifugation, resuspended in PBS, and placed on ice until ready for use. The hair on the right back of the mouse was then removed, and the 4T1 cell suspension (approximately 10 7 cells). When the tumor grows to 80-100 mm 3After about one week of growth, subsequent experiments can be carried out.

[0132] 2. Tumor US imaging experiment:

[0133] (1) In situ tumor US imaging: Six days after subcutaneous injection of 4T1 cells (this day was defined as day 1), tumor-bearing mice were randomly divided into four groups. PBS, MTO, TPO, and TPO@CCM (all at a concentration of 15 mg / kg) were then injected into the mice via the tail vein. 12 hours later, the primary tumor sites of the mice were imaged using US imaging equipment. The above experiment was repeated three times.

[0134] (2) US imaging of metastatic tumors: The experimental method was the same as that in “US imaging of in situ tumors”, except that the experiment was performed 44 days after subcutaneous injection of 4T1 cells, and the US imaging site was changed to the mouse liver. The above experiment was repeated three times.

[0135] Based on TPO's excellent performance in in vitro US imaging, we evaluated the US imaging performance of tumors at different locations in vivo using the tumor-targeting TPO@CCM at different time points to monitor tumor growth and metastasis in real time. We first systematically evaluated its US imaging performance in primary tumors. Figure 14 US imaging and gray value quantification of primary tumors in different treatment groups, including (a) US imaging and (b) gray value quantification.

[0136] like Figure 14 As shown in (a), the grayscale value of the TPO group increased significantly, while the MTO group showed little change 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 on homologous cancer cell lines, which promoted 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 grayscale value of the tumor area marked by the red circle in the figure. Figure 14 As can be seen in (b), consistent with the results discussed above, the grayscale value of the TPO@CCM group is the highest, indicating that its excellent oxygen carrying capacity and tumor targeting performance significantly improve the US imaging effect.

[0137] To further explore the potential of TPO@CCM for microtumor detection, we evaluated its US imaging performance in metastatic tumors. Given that the lung is a primary target organ for breast cancer metastasis, it would have been the preferred target. However, the air-filled nature of the lung significantly attenuates ultrasound energy, making it difficult to achieve ideal imaging results. Therefore, we selected the liver as a representative target for metastatic tumors. Figure 15 Typical tumor-like tissue in mouse liver and sagittal and transverse US imaging images of the liver in different treatment groups, including (a) tumor-like tissue, (b) liver US imaging images.

[0138] like Figure 15 As shown in (a), typical tumor-like tissue was observed in the liver of mice 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 in (b) show that the grayscale difference between liver tumors and normal liver tissue in the TPO and TPO@CCM groups was significantly higher than that in the PBS and MTO groups. This finding confirms the applicability of TPO@CCM in detecting micrometastatic tumors and provides an important basis for early diagnosis of tumors and the development of clinical treatment strategies.

[0139] 3. Treatment of breast cancer model mice:

[0140] Five days after subcutaneous injection of 4T1 cells (defined as day 0), tumor-bearing mice were randomly divided into five groups: PBS, TPO@CCM, αPD-L1, TPO@CCM + US, and TPO@CCM + αPD-L1 + US. TPO@CCM (15 mg / kg) was delivered via the tail vein on days 0, 2, and 4, and 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) were 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 The data were recorded for 16 days (a / 2, where a and b are the length and width of the tumor, respectively). After 16 days, the mice were euthanized, and their blood, solid tumors, and lungs were collected for analysis. Each group consisted of ≥5 mice, and the experiments were repeated three or more times.

[0141] Figure 16 is the change of tumor volume in each group of mice. Figure 16As shown, the tumor progression rate in the TPO@CCM group was comparable to that in the PBS group, indicating that TPO@CCM alone was ineffective in inhibiting tumor growth. Furthermore, injection of αPD-L1 alone also failed to significantly inhibit tumor progression, suggesting that immune checkpoint blockade (ICB) has limited efficacy against refractory, immune-cold 4T1 tumors. In contrast, the TPO@CCM + US group significantly inhibited tumor growth by enhancing the ultrasound cavitation effect and alleviating the tumor's hypoxic microenvironment, validating the superior efficacy of SDT. However, complete tumor regression was not achieved, suggesting that tumor cells may have intrinsic immune evasion mechanisms. Notably, the TPO@CCM + US + αPD-L1 group demonstrated the most significant tumor growth inhibition of all experimental groups. This is due to the significant advantages of synergistic SDT and immunotherapy, providing a new approach to overcoming tumor immune escape and achieving complete tumor elimination.

[0142] After 16 days of treatment, we euthanized the mice and extracted tumor tissues for photography and weight measurement. Figure 17 The tumor weights and corresponding visual photos of mice in each group after 16 days of treatment are shown in Figure 1, where (a) is the tumor weight and (b) is the visual photo. Figure 17 As shown in (a), the average tumor mass in the PBS group and the TPO@CCM group was the largest, and the tumor mass in the αPD-L1 group, the TPO@CCM + US group, and the TPO@CCM + αPD-L1 + US group decreased in sequence. This result is highly consistent with the tumor volume growth trend. Figure 17 (b) shows photos of in vitro tumors in different treatment groups. 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 therapeutic effect of TPO@CCM combined with αPD-L1 under ultrasound irradiation.

[0143] To further explore the key mechanisms of action of sonoimmunotherapy, we systematically evaluated key immune indicators such as DC cell maturation and T cell infiltration in mice. Figure 18 Flow cytometry analysis of DC and T cell counts in tumor tissues of mice in each group. CD4, CD8, CD80, and CD86 are all protein molecules expressed on cells. In the first row, a larger value in Q2 indicates higher expression of CD80 and CD86, and thus greater DC maturity; the opposite is true for Q4. In the second row, Q1 represents CD8 expression, and Q3 represents CD4 expression. Higher values ​​for these two components indicate greater T cell maturity.

[0144] like Figure 18As shown in the figure, the composition of immune cells in the tumor microenvironment of each group of mice was analyzed by flow cytometry. 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. It is worth noting that the TPO@CCM + US + αPD-L1 treatment group showed the highest DC maturation ratio, which confirmed that the synergistic effect of SDT and αPD-L1 can 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 CD4 + and CD8 + The T cell infiltration ratios all reached the highest levels, indicating that SDT stimulated 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.

[0145] In addition, we collected the lungs of surviving mice on day 30 and systematically analyzed the lung metastasis of mice in each group by H&E staining. Figure 19 The following are the visualization photos and corresponding H&E staining results of the lungs of mice in each group after 30 days of treatment, including (a) visualization photos and (b) corresponding H&E staining results. Figure 19 (a) and Figure 19 As shown in (b), distinct tiny transparent bubbles were observed in the lungs of mice in the PBS, TPO@CCM, and αPD-L1 groups, indicating the occurrence of tumor metastasis. H&E staining further confirmed these observations, with all groups displaying significant lung metastatic nodules (nodules marked with red circles). It is important to emphasize that mice in the TPO@CCM + US + αPD-L1 group showed almost no metastatic nodules in their lungs, in stark contrast to the other groups. These results demonstrate that cavitation-enhanced SDT combined with αPD-L1-mediated ICB therapy achieves the dual therapeutic goals of tumor ablation and metastasis inhibition by effectively alleviating the immunosuppressive microenvironment and promoting the massive production of ROS, opening up a new therapeutic path for clinical tumor treatment.

[0146] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An ultrasonic confined cavitation nanobubble, characterized in that: It has a core-shell structure, including a core of mesoporous titanium dioxide nanoparticles loaded with perfluorohexyl bromide and a tumor cell membrane shell coated on the surface of the core; The core in the shell is in an oxygen saturated state; The ratio of perfluorohexyl bromide to mesoporous titanium dioxide nanoparticles is (10-15) μL:1 mg; The mass ratio of the perfluorohexyl bromide-loaded mesoporous titanium dioxide nanoparticles to the tumor cell membrane is 1:(1-3); The mesoporous titanium dioxide nanoparticles are spherical, with a particle size of 200-250 nm and a pore size of 2-20 nm; The mesoporous titanium dioxide nanoparticles lower the cavitation threshold and provide nucleation sites, and the introduction of perfluorohexyl bromide and oxygen increases the number of cavitation nuclei.

2. The ultrasonic confined cavitation nanobubble according to claim 1, characterized in that: The tumor cell membrane is a breast cancer cell membrane.

3. A method for preparing ultrasonic confined cavitation nanobubbles according to claim 1 or 2, characterized in that: The following steps are involved: (1) ultrasonically mixing mesoporous titanium dioxide nanoparticles with 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 was performed, and the mixture was 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 ultrasonically confined cavitation nanobubbles.

4. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 3, wherein: 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 were stirred and mixed at room temperature, and then reacted. After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed and freeze-dried to obtain mesoporous titanium dioxide nanoparticles.

5. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 4, 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.

6. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 3, 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; The breast cancer cell membrane fragment suspension solvent is PBS, 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 breast cancer cell pellets were obtained after one centrifugation. Breast cancer cell membrane fragments were obtained after washing, resuspending, ice bathing, freezing and thawing, and secondary centrifugation.

7. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 3, 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.

8. The method for preparing ultrasonic confined cavitation nanobubbles according to claim 6, characterized in that: The breast cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum under the following conditions: a temperature of 37°C and 5% CO2. The centrifugation speed is 770-850 rpm and the time is 3-6 minutes; The washing step is to wash 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 was performed at 650-750 g for 10-15 minutes at 4 °C, and the supernatant was collected and centrifuged again at 13000-15000 g for 30-40 minutes.

9. Use of the ultrasonic confined cavitation nanobubbles according to claim 1 or 2 in the preparation of drugs for treating tumors and ultrasonic imaging drugs.

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