Preparation method and application of cell membrane modified ultrasonic response type copper / ferroptosis activated nano-carrier

Abstract

The invention discloses a preparation method of a cell membrane modified ultrasonic response type copper / ferroptosis activated nano-carrier. The nano-carrier is used for in-vivo and in-vitro treatment of ultrasonically activated tumors. The carrier is used as an ultrasonic activation type copper / iron death activator, and breast cancer cell membrane doped lipid is coated on the outer layer of a copper-iron bimetal organic framework to form nanoparticles with a core-shell structure. The biomimetic copper / ferroptosis activated nano-carrier disclosed by the invention is mainly characterized in that (1) the in-vivo circulation time of the nano-carrier is prolonged by modification of a breast cancer cell membrane, and accumulation of the nano-carrier in breast cancer cells and tissues is improved by virtue of homologous targeting of the nano-carrier; (2) the copper-iron double-metal-doped metal organic framework has a uniform nano structure, and has the potential of effectively catalyzing Fenton / Fenton-like reaction activity and activating a copper / iron death synergistic effect in cells; and (3) depending on the sound-sensitive agent and the oxygen carrier which are loaded by lipid, the nano-carrier has an ultrasonic signal response release behavior, and can simultaneously activate a sonodynamic effect, a copper death process and an iron death process in tumor cells, so that accurate and efficient killing of tumor tissues is realized.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and specifically relates to a preparation method and application of a cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier. Background Art

[0002] Malignant tumors are one of the killers that threaten human life and health, mainly manifested as uncontrolled cell proliferation and metastasis and damage to body functions. Thanks to the rapid development of tumor diagnosis and treatment technology, the incidence and mortality of tumors have dropped significantly in recent years. However, although the survival rate of malignant tumors in my country has improved compared with ten years ago, there is still a certain gap compared with developed countries, which also makes the exploration and development of new tumor treatment technologies full of urgency. Based on this, according to the pathological characteristics of malignant tumors and the actual clinical needs, the construction of an accurate and effective non-invasive treatment platform can achieve efficient killing of cancer, and can also avoid the sequelae such as systemic dysfunction caused by traditional treatments, thereby benefiting patients.

[0003] The development of new tumor treatment methods is one of the cutting-edge hot research topics. The development of a new tumor treatment platform with tumor targeting recognition function can improve the treatment effect, reduce toxic side effects, and improve the efficiency of precision treatment by combining non-invasive technology. Among them, ultrasound technology has become an ideal way to treat tumors because of its unique physical sound wave effect, as well as its convenience, real-time, safety and radiation-free advantages. It can be used for sonodynamic therapy (SDT) of tumors. As a type of non-invasive emerging tumor treatment method, SDT can activate sonosensitizers enriched in tumors through ultrasound signals to produce singlet oxygen, thereby effectively killing tumor cells. At the same time, SDT has stronger tissue penetration (more than 10 cm) and can reach deep areas of the tumor to achieve therapeutic effects. In addition, SDT can also achieve millimeter-level precision control at the target site, thereby minimizing damage to normal tissues while killing breast cancer. On this basis, the mechanical action, cavitation, and thermal effects of ultrasound-responsive oxygen carriers mediated by ultrasound signals can also promote the tumor tissue penetration of nanoformulations, and enhance the cell uptake efficiency of nanoformulations by increasing cell membrane fluidity; at the same time, controlling the intensity of ultrasound can also accurately regulate the drug release rate in nanoformulations. Therefore, the strategy of organically combining ultrasound with nanobiology technology to build a non-invasive treatment platform for breast cancer has a solid theoretical basis and broad application prospects.

[0004] Although ultrasound therapy shows many advantages mentioned above, it still has certain limitations in practical applications. For example, ultrasound-responsive oxygen-carrier contrast agents usually have a low boiling point and poor water solubility, so it is extremely challenging to design nanocarriers to achieve their stable and efficient delivery in vivo. At the same time, traditional small molecule sonosensitizers are limited by their low water solubility, poor targeting and stability, which limits their further application in vivo. Therefore, compared with a single treatment mode, combining ultrasound diagnosis and treatment with other treatment methods and developing a combined diagnosis and treatment strategy for tumors can significantly improve the anti-tumor efficiency. On this basis, an intelligent nano-delivery system is designed and constructed to achieve efficient co-delivery of each treatment module, which can provide advanced strategies for promoting the development of ultrasound therapy for tumors.

[0005] In addition to traditional treatments commonly used in clinical practice (such as surgery, radiotherapy, and chemotherapy), medical and scientific researchers are currently actively exploring new mechanisms that can lead to tumor death in order to develop new treatment models to improve anti-tumor effects. Ferroptosis is an iron-dependent non-apoptotic cell death process mediated by the Fenton reaction. Ferroptosis inducers cause the inactivation of ferroptosis-related proteins (including glutathione peroxidase-4, cystine / glutamate antiporter, etc.) in tumor cells, resulting in the accumulation of lipid peroxides, thereby inducing oxidative damage to tumor cells. This process is closely related to the level of glutathione (GSH) in tumor cells. The large amount of reactive oxygen species (ROS) produced during the SDT process can consume the high concentration of GSH in tumor cells, thereby sensitizing the ferroptosis process of tumor cells. Similar to ferroptosis, copper death is a process that induces non-apoptotic death of tumor cells. Its main process is that overloaded copper ions bind to the acylated components in the tricarboxylic acid cycle of tumor cell mitochondrial respiration, leading to the aggregation of acylated proteins (such as dihydrolipoyl transacetylase, etc.) and the downregulation of iron-sulfur cluster proteins, causing tumor cells to undergo protein toxic stress and eventually die. It should be noted that high concentrations of GSH in tumor cells can chelate with copper ions through sulfhydryl groups, thereby reducing the effect of copper death. Therefore, the GSH consumption strategy based on SDT can also sensitize the copper death process to a certain extent. Not only that, chemical kinetic reactions based on Fenton / Fenton-like reactions can also be generated in the process of copper death and ferroptosis, which synergizes with SDT to enhance the effect of cell apoptosis by increasing the level of ROS in tumor cells. This shows that the linkage of SDT, ferroptosis and copper death enables the three to synergize with each other, thereby enhancing the sensitivity of tumor cells to ferroptosis and copper death processes, while achieving synergistic regulation of redox homeostasis in tumor cells, which can produce a multiplied tumor treatment effect. Therefore, constructing a combined tumor treatment plan based on the "ternary linkage" strategy of SDT-ferroptosis-copper death is of great significance for designing ultrasound treatment platforms and leading new tumor treatment strategies.

[0006] In order to achieve the above goals, there is an urgent need for suitable carriers with drug co-delivery capabilities and specific tumor targeting properties. Bionic nanomaterials have attracted great attention from researchers because of their advantages in the structure and function of natural materials, and have shown great potential in tumor treatment. The main characteristics of bionic nanomaterials are that they have various functions similar to those of organisms and good biocompatibility, which is more conducive to in vivo application. This nano-bionic platform is usually an inorganic / organic composite nanosystem to achieve the organic fusion of multiple functions, among which the development of materials for biomimetic simulation of organisms is increasing. For example, extracting natural cell membranes and modifying them on the surface of nanomaterials is a common method to give the therapeutic system lesion targeting. This biomimetic strategy of using endogenous substances in organisms is widely used. The cell membranes of tumor cells or inflammatory cells are usually selected because they can recognize tumor endothelial tissue and have a strong tumor homing effect. At the same time, the modification of cell membranes can prolong the in vivo circulation time of nanosystems, so they can be more recruited in the lesion site. Among them, tumor cell membranes are considered to be promising candidates for the development of biomimetic nanocarriers because they have a high degree of homology with tumors, providing an effective method for optimizing the performance of nanomaterials / drugs. Summary of the invention

[0007] In view of the above technical background, in order to develop a highly integrated ultrasound-mediated bionic nano-tumor treatment platform, the present invention provides a preparation method and application of a cell membrane-modified ultrasound-responsive copper / iron death-activating nanocarrier.

[0008] The object of the present invention is achieved as follows: a cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier, characterized in that the carrier, as an ultrasound-activated copper / iron death activator, is a breast cancer cell membrane-doped lipid loaded with sonosensitizer and oxygen carrier coated on the outer layer of a copper-iron bimetallic organic framework to form a core-shell structured nanoparticle.

[0009] The copper / iron bimetallic doped metal organic framework is prepared by a hydrothermal synthesis method.

[0010] Preferably, the cell membrane is derived from one of the mouse breast cancer cell lines 4T1, EMT6, JC or the human breast cancer cell lines MCF-7, MDA-MB-231, T-47D, BT-474.

[0011] Preferably, the copper / iron bimetallic doped metal organic framework has a particle size distribution of 80-200 nm.

[0012] Preferably, the preparation method of the liposome includes but is not limited to one of a thin film dispersion method, a co-extrusion method and a microfluidic mixing method.

[0013] Preferably, the lipid components of the liposome include, but are not limited to, three or more of soybean lecithin, hydrogenated soybean lecithin, cholesterol, cholesterol ester, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

[0014] Preferably, the sonosensitizer encapsulated by the liposome includes, but is not limited to, one of verteporfin, dihydrochlorin e6, and hematoporphyrin.

[0015] Preferably, the oxygen carrier encapsulated by the liposome includes, but is not limited to, one of tetradecafluorohexane, hexafluoroheptane, and perfluoro(methylcyclohexane).

[0016] Another object of the present invention is to provide the use of the above-mentioned cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier in tumor treatment.

[0017] Preferably, the use of the cell membrane modified ultrasound-responsive copper / iron death-activated nanocarrier is characterized in that the ultrasound-responsive engineered exosomes can be used in the treatment of, including but not limited to, breast tumors.

[0018] Preferably, the application of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier is characterized in that the ultrasonic frequency range used is 0.1-30 MHz and the power range is 0.1-5 W / cm 2 , ultrasonic time is 0.1-60min.

[0019] Working principle of the present invention

[0020] The cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier described in the present invention has the following working principles: (1) The modification of the breast cancer cell membrane prolongs the circulation time of the nanocarrier in the body, and relies on its homologous targeting to improve the accumulation of the nanocarrier in breast cancer cells and tissues; (2) The copper-iron bimetallic doped metal organic framework has a uniform nanostructure, effective catalytic Fenton / Fenton-like reaction activity and the potential to activate the synergistic effect of copper / iron death in cells; (3) Relying on the loaded sonosensitizer and oxygen carrier, the nanocarrier has a release behavior in response to ultrasound signals, and can simultaneously activate the sonodynamic effect, copper death process and iron death process in tumor cells, thereby achieving a highly efficient synergistic killing effect on tumor cells.

[0021] Beneficial effects of the present invention:

[0022] 1. The present invention provides a cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier, which has a simple structure and composition, a simple preparation process, and good biocompatibility.

[0023] 2. The cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier provided by the present invention is based on homologous tumor cell membrane modification, has good tumor targeting ability, and can be effectively enriched at the tumor site to achieve the purpose of precise diagnosis and treatment of tumors.

[0024] 3. The design of a cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier provided by the present invention selects three treatment methods: SDT, ferroptosis and copper death. By utilizing the organic coordination among the three, SDT increases the level of ROS in tumor cells and consumes GSH, thereby enhancing the two non-apoptotic cell death processes of ferroptosis and copper death, while feedback increases the level of amplified oxidative stress in tumor cells, thereby achieving a "ternary linkage" anti-tumor effect mediated by ultrasound signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The particle size distribution and Zeta potential distribution test of the cell membrane modified ultrasound responsive copper / iron death activated nanocarrier and precursor nanoparticles prepared in Example 1;

[0026] Figure 2 This is a transmission electron microscope image of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier prepared in Example 1;

[0027] Figure 3 The storage stability test of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier prepared in Example 1;

[0028] Figure 4 The ultrasound-responsive oxygen release curve of the cell membrane-modified ultrasound-responsive copper / iron death-activated nanocarrier prepared in Example 1;

[0029] Figure 5 The in vitro sonodynamic effect test of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier prepared in Example 1;

[0030] Figure 6 In vitro breast cancer cell uptake detection of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier prepared in Example 1;

[0031] Figure 7 This is the in vitro breast cancer cell killing effect test of the cell membrane modified ultrasound-responsive copper / iron death-activated nanocarrier prepared in Example 1. DETAILED DESCRIPTION

[0032] The present invention is further described below by way of examples, but the following examples do not limit the scope of rights of this patent.

[0033] Example 1

[0034] The preparation method of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier is as follows:

[0035] (1) Extraction of tumor cell membranes: 4T1 cells of mouse breast cancer cell line cultured in vitro were collected and pre-cooled Tris-MgCl2(TM) buffer (containing 10 mM Tris and 1 mM MgCl2, pH 7.4) was added to make the final cell concentration 5×10 7 / mL. The cells were then repeatedly squeezed using a liposome extruder (without a polycarbonate membrane). Pre-cooled sucrose solution was added to the collected cell lysate to make the final concentration of sucrose 0.25M, followed by a series of gradient centrifugation operations at 4°C, including: (1) centrifugation at 3000rpm for 30min to obtain a precipitate; (2) resuspending in pre-cooled TM buffer-sucrose solution (sucrose concentration of 0.25M), centrifuging at 3000rpm for 30min, and obtaining a precipitate to obtain 4T1 breast cancer cell membranes.

[0036] (2) Preparation of copper-iron metal organic framework: FeCl3·6H2O (1.62 g), CuCl2·2H2O (0.68 g) and 1,4-benzodicarboxylic acid (1.50 g) were fully dissolved in 70 mL of N,N-dimethylformamide and reacted at 100°C for 12 h. The system was washed and centrifuged, and the precipitate was collected to prepare a copper-iron metal organic framework (named Fe-Cu-MOFs).

[0037] (3) Preparation of cell membrane modified ultrasound-responsive copper / iron death-activating nanocarriers: 50 mg soybean lecithin, 12.5 mg cholesterol, 5 mg distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and 20 μg verteporfin were dissolved in a chloroform-methanol mixture (1:4, v / v), and evaporated under reduced pressure in a 45°C water bath to form a dry lipid film. Subsequently, 10 mL of normal saline was added, and the mixture was hydrated in a 42°C water bath for 30 min. The suspension was subjected to probe ultrasonic treatment for 90 cycles in an ultrasonic cell disruptor in an ice bath (ultrasonic power: 2000 W, each ultrasonic cycle contained 1 s ultrasonic treatment and 2 s interval). The suspension was then filtered using a 0.22 μm mixed cellulose membrane to remove large particles and unencapsulated verteporfin to obtain nanoparticles LV. The prepared LV was mixed with Fe-Cu-MOFs (30 mg) and tetradecafluorohexane (150 μL), and then extruded in sequence in the order of 800, 400, and 200 nm using a liposome extruder to obtain L-VMP. The L-VMP was then mixed with the extracted 4T1 breast cancer cell membrane and extruded in sequence in the order of 800, 400, and 200 nm. The oxygen chamber was filled with oxygen at 1 min (oxygen flow rate of 5 L / min) to obtain the cell membrane modified ultrasound-responsive copper / iron death-activated nanocarrier Sono-Cell.

[0038] Example 2

[0039] Particle size distribution and Zeta potential distribution test of cell membrane modified ultrasound-responsive copper / iron death-activating nanocarriers and their precursor preparations:

[0040] The nanoformulations LV, L-VMP and ultrasound-responsive copper / iron death-activated nanocarrier Sono-Cell prepared in Example 1 were measured for particle size and potential by dynamic light scattering. The Fe-Cu-MOFs prepared in Example 1 were characterized for their microstructure by transmission electron microscopy. The results are as follows: Figure 1 As shown in Figure 2, the particle sizes of LV, L-VMP and Sono-Cell are 127.7±4.5nm, 139.1±6.2nm and 165.0±5.3nm, respectively, and the Zeta potentials are -18.8±2.5mV, -15.6±3.7mV and -29.5±4.1mV, respectively. The microstructure of Sono-Cell was then observed using a transmission electron microscope. The results are shown in Figure 2. Figure 2 As shown, it is a nano-scale particle with a core-shell structure of about 150 nm in size and a uniform size distribution.

[0041] Example 3

[0042] Storage stability test of cell membrane modified ultrasound-responsive copper / iron death-activating nanocarriers:

[0043] The cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier Sono-Cell prepared in Example 1 was placed at 4°C, and its particle size was measured by dynamic light scattering method on days 0, 3, 6, 9, 12, and 15, and a particle size change curve was plotted. The results are shown in Figure 3 As shown, the particle size of Sono-Cell did not change significantly within 15 days and was around 170 nm, showing good storage stability.

[0044] Example 4

[0045] Ultrasound-responsive oxygen release curve of cell membrane-modified ultrasound-responsive copper / iron death-activating nanocarriers:

[0046] Take the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier Sono-Cell prepared in Example 1, fill it with nitrogen to remove oxygen, and use an ultrasound probe to stimulate the system with an ultrasound signal (0.35 W / cm 2 ,1MHz,3min), the oxygen content in the solution is measured by the dissolved oxygen electrode, and the oxygen content-time curve is shown in Figure 4The results show that under ultrasound stimulation, Sono-Cell can quickly release a large amount of oxygen. Ultrasound can make the oxygen content in the system reach 10 mg / L within 90 seconds, significantly increasing the oxygen content in the medium.

[0047] Example 5

[0048] In vitro sonodynamic effect test of ultrasound-responsive oxygen release curve of cell membrane-modified ultrasound-responsive copper / iron death-activating nanocarriers:

[0049] The ultrasound-responsive copper / iron death-activating nanocarrier Sono-Cell and free verteporfin solution prepared in Example 1 were dispersed in air-saturated water and nitrogen-saturated water, respectively, and singlet oxygen indicator SOSG was added for co-incubation. The system was stimulated with an ultrasonic signal (0.35 W / cm 2 ,1MHz,3min), and then detect the change of SOSG fluorescence intensity (Ex=504nm,Em=525nm). Figure 5 As shown, it can be seen that in the anaerobic state, free verteporfin hardly produces sonodynamic effect, while Sono-Cell can release a large amount of oxygen under ultrasound to provide raw materials for the sonodynamic effect, thus significantly increasing the fluorescence intensity of SOSG and having a significant sonodynamic effect.

[0050] Example 6

[0051] Tumor cell uptake behavior of cell membrane modified ultrasound-responsive copper / iron death-activating nanocarriers:

[0052] In the process of preparing the ultrasound-responsive copper / iron death-activating nanocarrier Sono-Cell in Example 1, 5 mg of coumarin 6 fluorescent dye was added during the process of adding the lipid component to dissolve in step (3) to prepare coumarin 6 green-labeled L-VMP and Sono-Cell. 4T1 cells were cultured at 1×10 5 The cells were seeded at a density of 100 / well in a 24-well plate, and the cells were incubated with coumarin 6 green-labeled L-VMP and Sono-Cell (containing 100 ng / mL coumarin 6) for 0, 2, 4, 6, and 8 hours, respectively. After the incubation, the cells were washed three times with pre-cooled PBS, and the single cell suspension was prepared by adding trypsin-EDTA digestion solution. The fluorescence content of the cells was detected in a flow cytometer. The excitation wavelength was set to 465 nm and the emission wavelength was set to 502 nm. The results are shown in Figure 2. Figure 6 As shown, it can be seen that the uptake of fluorescently labeled Sono-Cell is significantly increased compared with L-VMP without cell membrane wrapping, and the fluorescence intensity is enhanced by about 0.6 times within 4 hours, indicating that the modification of tumor cell membrane significantly improves the uptake behavior of Sono-Cell, laying the foundation for achieving good anti-tumor effects.

[0053] Example 7

[0054] Tumor cell killing effect of cell membrane modified ultrasound-responsive copper / iron death-activating nanocarriers:

[0055] 4T1 cells were cultured at 5 × 10 4 The cells were inoculated at a density of 100 cells / well in a 96-well plate. The ultrasound-responsive copper / iron death-activating nanocarrier Sono-Cell and L-VMP (containing 2.5 μg / mL verteporfin) prepared in Example 1 were incubated with the cells under normoxic / hypoxic conditions for 4 h, and complete culture medium was used as a negative control. After the incubation, an ultrasound probe was used to stimulate the cells with ultrasound signals (0.35 W / cm 2 ,1MHz,3min), and then continue to culture for 24h, with the group without ultrasound as the control. Then, 20μL of 5mg / mL MTT PBS solution was added to each well. After incubation for 4h, the supernatant was discarded and 150μL DMSO was added to dissolve the formazan crystals. The absorbance of each well was measured at UV-Vis 570nm using an enzyme marker, and the cell survival rate was calculated. The results are shown in Figure 7 As shown, it can be seen that Sono-Cell exhibits obvious anti-tumor effects in a hypoxic environment under the action of laser irradiation, and can kill 74.33% of tumor cells; while the nanoformulation L-VMP without tumor cell membrane modification has a weakened anti-tumor effect due to its less intake than Sono-Cell, killing only 41.6% of tumor cells; at the same time, the Sono-Cell group without ultrasound stimulation has almost no cytotoxicity, indicating that the SDT / copper death / iron death effects induced by Sono-Cell are completed under the premise of release triggered by ultrasound stimulation, indicating the stability of Sono-Cell in the systemic circulation process, and will not cause obvious toxicity to tissues, and has good biosafety.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cell membrane modified ultrasound responsive copper / iron death-activating nanocarrier, characterized in that: The ultrasound-activated nanocarrier tumor therapeutic agent comprises a core-shell structure, which comprises: (1) a tumor cell membrane doped with lipids loaded with sonosensitizer and oxygen carrier as an outer shell layer; and (2) a copper-iron bimetallic organic framework as an inner core layer.

2. The cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier according to claim 1, characterized in that: The cell membrane is one of mouse breast cancer cell lines 4T1, EMT6, JC or human breast cancer cell lines MCF-7, MDA-MB-231, T-47D, BT-474.

3. The cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier according to claim 1, characterized in that: The copper / iron bimetallic doped metal organic framework is prepared by a hydrothermal synthesis method from one of ferric chloride hexahydrate, ferric nitrate nonahydrate, and ferric sulfate and one of cupric chloride dihydrate, cupric nitrate, and cupric sulfate pentahydrate.

4. The copper / iron bimetallic doped metal organic framework according to claim 3, characterized in that: The copper / iron bimetallic doped metal organic framework has a particle size distribution of 80-200 nm.

5. The copper / iron bimetallic doped metal organic framework according to claim 3, characterized in that: The copper / iron bimetallic doped metal organic framework is modified by liposomes loaded with sonosensitizers and oxygen carriers to enhance water dispersibility. The preparation method of the liposomes includes but is not limited to one of a thin film dispersion method, a co-extrusion method and a microfluidic mixing method.

6. The liposome according to claim 5, characterized in that: The lipid components of the liposome include, but are not limited to, three or more of soybean lecithin, hydrogenated soybean lecithin, cholesterol, cholesterol ester, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

7. The liposome according to claim 5, characterized in that: The sonosensitizer encapsulated by the liposome includes, but is not limited to, one of verteporfin, dihydrochlorin e6, and hematoporphyrin.

8. The liposome according to claim 5, characterized in that: The oxygen carrier encapsulated by the liposome includes, but is not limited to, one of tetradecafluorohexane, hexadecafluoroheptane, and perfluoro(methylcyclohexane).

9. The use of a cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier according to claim 1, characterized in that: The ultrasonic frequency range of the cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier is 0.1-30 MHz, and the power range is 0.1-5 W / cm 2 , ultrasonic time is 0.1-60min.

10. The use of a cell membrane modified ultrasound-responsive copper / iron death-activating nanocarrier according to claim 1, characterized in that: The ultrasound-responsive engineered exosomes can be applied to treatments including but not limited to breast tumors.

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