Tumor cell membrane ultrasound microbubble, preparation method therefor, and use thereof in enhancing tumor immunotherapy

Abstract

A tumor cell membrane ultrasound microbubble, a preparation method therefor, and use thereof in enhancing tumor immunotherapy. The tumor cell membrane ultrasound microbubble comprises a phospholipid bilayer shell composed of a tumor cell membrane, and an inert gas is encapsulated within the shell. The tumor cell membrane ultrasound microbubble is prepared by the following method: mixing the tumor cell membrane with a glycerol-containing phosphate buffer solution, carrying out ultrasonic treatment to obtain a tumor cell membrane suspension, and transferring the tumor cell membrane suspension into a container; adding perfluorotributylamine, and sealing the container; and extracting air in the container, injecting the inert gas, and carrying out oscillation treatment to obtain the tumor cell membrane ultrasound microbubble. The microbubble can be used as a tumor vaccine to stimulate organism immunity, thereby solving the problems of a single antigen and the low immunogenicity of traditional tumor vaccines. The microbubble is of a typical hollow structure, can be used for enhancing ultrasonic contrast imaging and promoting the maturation of dendritic cells in vivo and in vitro, and has application prospects in tumor immunotherapy.

Description

Tumor cell membrane ultrasound microbubbles, preparation method thereof, and application thereof in enhancing tumor immunotherapy Technical Field

[0001] The present invention relates to the technical field of ultrasound microbubbles and tumor immunotherapy drugs, and in particular to a tumor cell membrane ultrasound microbubble and a preparation method thereof and application in enhancing tumor immunotherapy. Background Art

[0002] Tumor immunotherapy is a new type of tumor treatment that has demonstrated strong anti-tumor activity in a variety of tumor types. As one of these immunotherapy strategies, tumor vaccines are a highly promising treatment approach that infuses tumor-associated antigens to increase the exposure of antigens to antigen-presenting cells, thereby enhancing tumor-specific immune responses. Currently, in order to more effectively activate the body's immunity and ensure the occurrence of anti-tumor immune responses in vivo, researchers have developed a variety of tumor vaccines, mainly including: ① tumor-associated antigens derived from peptides and proteins; ② nucleic acid-based tumor vaccines, such as tumor DNA vaccines and RNA vaccines; ③ in situ tumor vaccines, which use different therapeutic methods to convert autologous tumor cells in situ into tumor-associated antigens. Although the above vaccines have achieved certain results in tumor immunity, most traditional tumor vaccines have a series of shortcomings that affect their preclinical and clinical therapeutic efficacy, such as their single antigen composition, poor in vivo stability, and high systemic toxicity.

[0003] Studies have confirmed that tumor cells containing multiple antigens can be used as tumor vaccines to induce tumor-specific immune responses. Tumor cells have a variety of specific antigens and are an ideal source of tumor vaccines. Autologous or allogeneic tumor cells are treated by physical, chemical, and other means to render them incapable of proliferation and invasiveness. Tumor vaccines using tumor cell lysates as tumor-associated antigens are an important type of tumor vaccine. However, most tumor cell lysates used as tumor vaccine antigens are unable to induce a strong anti-tumor immune response because most of the tumor cell lysates are non-tumor-associated antigen components. In contrast, tumor cell membranes have a high proportion of tumor antigens and can be used in the preparation of tumor vaccines. There is an urgent need to develop a technical solution for preparing tumor cell membranes into tumor vaccines to stimulate the body's immunity, in order to solve the problem of traditional tumor vaccines with a single antigen and low immunogenicity. Summary of the Invention

[0004] The present invention aims to provide a tumor cell membrane ultrasound microbubble to solve the technical problems of traditional tumor vaccine with single antigen and low immunogenicity.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention discloses a tumor cell membrane ultrasound microbubble, which comprises a phospholipid bilayer shell composed of the tumor cell membrane, wherein the phospholipid bilayer shell is enclosed by an inert gas.

[0007] This scheme also provides a method for preparing tumor cell membrane ultrasonic microbubbles, comprising the following steps in sequence: mixing the tumor cell membrane with a phosphate buffer containing glycerol, obtaining a tumor cell membrane suspension after ultrasonic treatment, and transferring it into a container; then adding perfluorotributylamine and sealing the container; extracting the air in the container and injecting an inert gas, and obtaining tumor cell membrane ultrasonic microbubbles after shaking.

[0008] This solution also provides an application of tumor cell membrane ultrasound microbubbles in the preparation of tumor immunotherapy preparations.

[0009] Furthermore, the phospholipid bilayer shell is processed in the following manner: the tumor cell membrane is mixed with a phosphate buffer solution containing glycerol, and the tumor cell membrane suspension is obtained after ultrasonic treatment; and the tumor cell membrane suspension is mixed with perfluorotributylamine.

[0010] Furthermore, the volume ratio of the tumor cell membrane suspension to perfluorotributylamine is 0.2 mL:20-50 μL.

[0011] Furthermore, the tumor cell membrane is extracted from CT26 cells.

[0012] Furthermore, the inert gas is C3F8.

[0013] Furthermore, in the phosphate buffer containing glycerol, the mass percentage of glycerol is 10%, the specification of the phosphate buffer is 0.01 M, and the pH value is 7.4.

[0014] Furthermore, the power of the ultrasonic treatment was 50 W, the time was 1 min, and the ultrasonic treatment was performed according to a procedure of working for 5 s and pausing for 5 s.

[0015] Furthermore, the container is a 2 mL vial; 0.2 mL of the tumor cell membrane suspension is added to the vial, and then 20-50 μL of perfluorotributylamine is added.

[0016] In summary, the principles and beneficial effects of this technical solution are:

[0017] The inventors have developed a novel tumor cell membrane ultrasound microbubble system using a mechanical oscillation method for preparing lipid ultrasound microbubbles. This type of ultrasound microbubble system using tumor cell membrane as the shell material has not been reported before. These novel tumor cell membrane ultrasound microbubbles can be used as tumor vaccines to stimulate immunity, addressing the problem of traditional tumor vaccines with a single antigen and low immunogenicity. In vitro experiments have demonstrated that the prepared tumor cell membrane microbubbles exhibit a typical hollow structure, which can not only be used to enhance ultrasound contrast imaging but also promote dendritic cell maturation in vitro and in vivo in tumor tissue, spleen, and lymph nodes.

[0018] Ultrasonic microbubbles are most effective in promoting immune cell maturation when their lipid shells are composed entirely of tumor cell membranes. However, conventional methods have proven difficult to successfully prepare when the lipid shells of these microbubbles are composed entirely of tumor cell membranes. Through extensive experimentation, the inventors discovered that adding a specific liquid fluorocarbon, PFTBA, to the preparation process can address this issue.

[0019] In addition, in terms of promoting the maturation of dendritic cells, the addition of liquid fluorocarbon PFTBA also effectively promoted the increase in the maturation ratio of dendritic cells in the tumor site. When ultrasonic treatment was applied, ultrasonic blasting promoted the concentration of microbubbles in the tumor site, which could further significantly promote the maturation of dendritic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is an electron micrograph of different microvesicles prepared from tumor cell membranes with or without the addition of PFTBA in Example 1 (left: PFTBA tumor cell membrane; right: microvesicles without the addition of PFTBA).

[0021] FIG2 shows flow cytometry analysis images (a) and quantitative analysis results (b) of in vitro stimulation of dendritic cell maturation by mixed microbubbles of tumor cell membranes and phospholipids at different ratios in Example 2 (in the statistical graph on the right, data are expressed as mean±SD, n=4).

[0022] Figure 3 shows flow cytometry analysis images and quantitative analysis results of (a, b) lymph node maturation and (c, d) tumor tissue dendritic cell maturation in CT-26 transplanted tumor mice stimulated by different ratios of tumor cell membrane-phospholipid mixed microbubbles in Example 2 (in the statistical graph on the right, the data are expressed as mean ± SD, n = 3).

[0023] FIG4 is an in vitro enhanced ultrasound imaging diagram and echo intensity quantitative analysis of Example 3.

[0024] FIG5 is a light microscopic image and a fluorescence imaging image of the tumor cell membrane microvesicles in Example 3.

[0025] FIG6 shows flow cytometry analysis images (a) of the tumor cell membrane microvesicles promoting dendritic cell maturation in vitro and quantitative analysis (b) of the dendritic cell maturation ratio of Example 4 (in the statistical graph on the right, data are expressed as mean±SD, n=5).

[0026] Figure 7 shows flow cytometry images (a) and quantitative analysis of the maturation ratio of dendritic cells in tumor tissues, lymph nodes and spleen promoted by tumor cell membrane microvesicles in Example 5 (bd) (in the statistical graph on the right, data are expressed as mean ± SD, n = 5). DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0028] Example 1: Ultrasonic microbubble production method

[0029] (1) Tumor cell membrane extraction

[0030] In order to obtain tumor cell membranes, CT26 (mouse colon cancer cell) tumor cell membranes were extracted according to the method provided by the membrane protein extraction kit. First, the collected cells (1×10 8 ) were resuspended in 3 mL of membrane protein extraction solution (Shanghai Beyotime Biotechnology Co., Ltd., Product No. P0033) and supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF; protease inhibitor, Shanghai Beyotime Biotechnology Co., Ltd., Product No. ST505). The cells were incubated on ice for 15 minutes. The cell suspension was then freeze-thawed three times in liquid nitrogen and centrifuged (2000 rpm, 10 min) to collect the supernatant. The cell supernatant was then centrifuged again (14,000 rpm, 30 min) to collect the precipitated cell membrane fragments. Finally, the pellet was collected and lyophilized (-80°C, 8-12 h, 10 Pa) for further use.

[0031] (2) Ultrasonic microbubble preparation

[0032] 10 mg of the tumor cell membranes obtained above (using only the tumor cell membranes as the raw material for the phospholipid bilayer shell) was added to 1 mL of phosphate buffer (0.01 M, pH 7.4) containing 10% glycerol. The suspension was then ultrasonically treated (50W, 5s on, 5s off) for 1 min to obtain a tumor cell membrane suspension. Using a pipette, 0.2 mL of the mixture was transferred to a 2 mL vial. 20 μL of perfluorotributylamine (PFTBA) was added to the vial. After sealing with parafilm, the air in the vial was aspirated using a syringe. Immediately, 2-3 mL of C3F8 inert gas was introduced using another syringe (for subsequent experiments, the amount of C3F8 introduced was approximately 3 mL). The suspension was then mechanically shaken for 50 seconds using a silver-mercury capsule blender (frequency: 4300 cpm) to obtain MBs@PFTBA tumor cell membrane microvesicles. As a control, tumor cell membrane microbubbles were prepared without adding 20 μL of PFTBA. The sonicated mixture was added to a vial, and then the subsequent inert gas injection and mechanical shaking were directly performed.

[0033] The morphology and structure of tumor cell membrane microbubbles were observed under a light microscope, and the differences in microbubbles between the two groups were compared with those of tumor cell membranes without PFTBA added. The results showed that, under the same concentration conditions, tumor cell membranes with PFTBA added formed a large number of round microbubbles of varying sizes after mechanical vibration, while tumor cell membranes without PFTBA only formed a very small number of microbubbles, with more tumor cell membrane fragments observed under the microscope (see Figure 1). These experimental results indicate that when using single tumor cell membranes for ultrasonic microbubble generation, the addition of a certain amount of liquid fluorocarbon (e.g., PFTBA) is necessary to ensure the formation of ultrasonic microbubbles. Traditional ultrasonic microbubble generation methods do not use the addition of a small amount of liquid fluorocarbon, but instead directly inject gas. For example, Chinese patent CN111420073A (Multimodal targeted nanobubbles carrying AMD070 and ICG and their preparation method) uses a lipid suspension (dipalmitoylphosphatidic acid, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine, etc.) as the outer shell (shell membrane material) of ultrasonic microbubbles. This allows for direct gas injection without the addition of liquid fluorocarbon, ultimately forming ultrasonic microbubbles. However, this technical solution directly uses tumor cell membranes as the shell membrane material for the microbubbles. If prepared using traditional methods, ultrasonic microbubbles cannot be effectively formed due to problems such as the poor hardness of the natural cell membrane. After extensive research, the inventors discovered that adding a small amount of liquid fluorocarbon can resolve this issue. The inventors speculate that the addition of liquid fluorocarbon ensures microbubble formation primarily because the polarity of the liquid fluorocarbon itself affects the overall microbubble formation effect.

[0034] In addition to PFTBA, the inventors also experimented with other types of liquid fluorocarbons, such as perfluoropentane (PFP) and perfluorohexane (PFH). These other types of liquid fluorocarbons were substituted in equal amounts for the PFTBA used in this protocol, and ultrasonic microbubbles were prepared according to the above method. These are referred to as PFP microbubbles, PFH microbubbles, and PFTBA microbubbles. Microscopic observation of the prepared ultrasonic microbubbles revealed that the microbubbles' stability was suboptimal, with phase transitions occurring due to environmental influences. In particular, microbubbles undergoing phase transitions were highly likely to cause mouse mortality after injection into mice. At the same injection dose, intravenous injection of PFP and PFH microbubbles resulted in a mortality rate exceeding 50%, while bolus injection of PFTBA microbubbles resulted in a survival rate of 95%. The microbubbles obtained according to this technical solution exhibited excellent stability, with microscopic observation demonstrating virtually no phase transitions due to environmental influences. Moreover, after being injected into mice, the microbubbles will basically not undergo phase transition unless specific stimulation to guide phase transition is used, and the probability of mice dying from injection of the microbubbles of this protocol is low (excluding improper operation).

[0035] In addition, when preparing ultrasonic microbubbles containing PFTBA, the inventors also investigated the effects of varying PFTBA dosages on microbubble formation. These studies revealed that a PFTBA dosage greater than 20 μL was most effective in promoting microbubble formation. The number of microbubbles in each type of ultrasonic microbubble prepared using different PFTBA dosages was counted (each microbubble was counted in three microscopic fields and the average was taken). No significant differences were found between the number of microbubbles (20 μL PFTBA dosage), the number of microbubbles (30 μL PFTBA dosage), and the number of microbubbles (50 μL PFTBA dosage) (t-test, p>0.05). The number of ultrasonic microbubbles (5 μL PFTBA added) and the number of ultrasonic microbubbles (10 μL PFTBA added) were significantly different from the number of ultrasonic microbubbles (20 μL PFTBA added) (t-test, p < 0.05). The number of ultrasonic microbubbles (5 μL PFTBA added) and the number of ultrasonic microbubbles (10 μL PFTBA added) were approximately 46% and 69% of the number of ultrasonic microbubbles (20 μL PFTBA added), respectively. These experiments demonstrate that the type of liquid fluorocarbon is crucial for the formation and efficacy of ultrasonic microbubbles, and that an inappropriate amount of PFTBA can significantly reduce the amount of ultrasonic microbubbles formed. Therefore, using pure tumor cell membranes as the lipid shell of ultrasonic microbubbles, the key technical considerations for ensuring the success rate of ultrasonic microbubble preparation lie in whether to add a certain amount of liquid fluorocarbon and the specific type of liquid fluorocarbon. These key technical considerations are not reported in the prior art and are the result of extensive independent research by the inventors. Example

[0036] The tumor cell membrane in Example 1 was replaced with a mixture of phospholipids (distearoylphosphatidylethanolamine-polyethylene glycol and dipalmitoylphosphatidylcholine, with a mass ratio of 5:3) and tumor cell membranes, and microbubbles were prepared according to the method of Example 1.

[0037] Microbubbles containing different ratios of tumor cell membrane-lipid mixtures were prepared. Phospholipids used to prepare lipid microbubbles were mixed with tumor cell membranes to form mixed microbubbles. The ability of these mixed microbubbles to stimulate dendritic cell maturation was assessed by flow cytometry. The experiment consisted of two parts: testing the ability of the mixed microbubbles to stimulate the maturation of bone marrow dendritic cells (immature dendritic cells) in vitro; and testing their ability to stimulate the maturation of dendritic cells in the lymph nodes and tumor tissues of mice bearing CT26 colorectal cancer xenografts in vivo. The lipid shells of the ultrasound-activated microbubbles used in these tests consisted of a ratio of tumor cell membrane to phospholipid of 0:3.2 by volume, a ratio of tumor cell membrane to phospholipid of 0.5:2.4 by volume, a ratio of tumor cell membrane to phospholipid of 1:1.6 by volume, and a ratio of tumor cell membrane to phospholipid of 2:0 by volume. The remaining preparation steps were similar to those in Example 1.

[0038] The specific process of in vitro testing is as follows: conventional culture of bone marrow-derived dendritic cells, i.e. immature dendritic cells, at a rate of 1×10 6 Bone marrow-derived dendritic cells were seeded into a 12-well plate at a ratio of 1:1 / well and cultured in an incubator for 24 hours. Then, 200 μL of microbubbles prepared by the method of Example 1 with different tumor cell membrane and phospholipid mass ratios were added. After incubation for 24 hours, the cells were collected and APC-labeled anti-CD80 antibody (0.06 μg / test) and PE-labeled anti-CD86 antibody (0.125 μg / test) were added for co-staining, and the ability of mixed microbubbles with different mass ratios to stimulate dendritic cell maturation was detected by flow cytometry.

[0039] The specific process of in vivo testing is as follows: a mouse colorectal cancer tumor model was established. After 200 μL of mixed microbubbles of different mass ratios were injected into the tail vein, the tumor tissue was immediately irradiated with low-intensity ultrasound for 5 minutes at a frequency of 1 MHz. The above treatment was repeated every 2 days for a total of 3 times. On the 7th day after treatment, the mouse tumor tissue and lymph nodes were collected and prepared into single-cell suspensions. The cells were stained with APC-labeled anti-CD80 antibody (0.06 μg / test) and PE-labeled anti-CD86 antibody (0.125 μg / test), respectively. The ability of mixed microbubbles of different mass ratios to stimulate dendritic cell maturation in vivo was detected by cytometry.

[0040] The results showed that when no tumor cell membranes were added (i.e., when the ratio of tumor cell membranes to phospholipids was 0:3.2, meaning the lipid shell of the ultrasound microbubbles was composed entirely of phospholipids), only a small number of bone marrow dendritic cells were stimulated to mature into mature dendritic cells. However, when tumor cell membranes were added, a significant number of dendritic cells were stimulated to mature, and the maturation rate was concentration-dependent (Figure 2). The ultrasound microbubbles were most effective in promoting immune cell maturation when the lipid shell was composed entirely of tumor cell membranes. However, conventional methods proved difficult to successfully prepare ultrasound microbubbles when the lipid shell was composed entirely of tumor cell membranes. After extensive experimentation, the inventors discovered that adding a specific liquid fluorocarbon, PFTBA, to the preparation process could resolve this problem. Consistent with the in vitro results, in vivo experiments also confirmed that increasing the proportion of tumor cell membranes significantly increased the proportion of mature dendritic cells in lymph nodes and tumor tissues (Figure 3). Example

[0041] The light microscopy and fluorescence imaging images of the ultrasound microbubbles prepared in Example 1 are shown in Figure 5. In vitro experiments have shown that the prepared tumor cell membrane microbubbles have a typical hollow structure. Ultrasound contrast imaging studies were performed on the ultrasound microbubbles, and the experimental results are shown in Figure 4. Example

[0042] Flow cytometry was used to analyze the in vitro promotion of dendritic cell maturation at different microvesicle dosages. The experimental method was similar to that in Example 2, and the experimental results are shown in Figure 6. The experimental results of this example show that tumor cell membrane microvesicles can promote dendritic cell maturation. Example

[0043] This example investigated the effect of tumor cell membrane microvesicles on the maturation of dendritic cells in tumor tissue, lymph nodes, and spleen. A mouse colorectal cancer model was established and randomly divided into five groups: a blank control group (control), an ultrasound-irradiated group (US), a group containing tumor cell membrane microvesicles without PFTBA (MBs), a group containing PFTBA (PFTBA@aMBs), and a group containing PFTBA combined with ultrasound (PFTBA@aMBs + US). Treatments were administered according to the groupings. The microbubble injection dose was 200 μL at 2 mg / mL, and the ultrasound irradiation time was set at 5 minutes at a frequency of 1 MHz. These treatments were repeated every two days for a total of three times. On the 7th day after treatment, mouse tumor tissues, spleens, and lymph nodes were collected and prepared into single-cell suspensions. The cells were stained with APC-labeled anti-CD80 antibody (0.06 μg / test) and PE-labeled anti-CD86 antibody (0.125 μg / test), respectively. The mature proportion of dendritic cells in tumor tissues, spleens, and lymph nodes was detected by cytometry.

[0044] The experimental results are shown in Figure 7. The experimental results show that the ultrasound microbubbles of this scheme can stimulate the maturation of dendritic cells in tumor tissue, spleen and lymph nodes in vivo. For Figure 7B, the specific data of each group are as follows:

[0045] Control group: 15.36±3.09%; US group: 15.20±3.32%; MBs group: 23.32±2.66%; PFTBA@MBs group: 27.66±2.40%; PFTBA@MBs+US group: 43.52±8.00%. Ultrasonication alone did not increase the proportion of DC maturation (US group). However, the use of ultrasound-treated microbubbles without PFTBA, due to the introduction of cancer cell membranes, promoted DC maturation to a certain extent. The addition of PFTBA to the preparation of ultrasound-treated microbubbles resulted in improved quality and integrity of the microbubbles, further increasing the proportion of DC maturation. Furthermore, statistically significant differences were observed between the MBs and PFTBA@MBs groups, indicating that PFTBA plays a significant role in increasing the proportion of DC activation in tumors and other sites. Therefore, PFTBA plays a dual role in promoting immune processes and ensuring microbubble formation in this protocol. Furthermore, ultrasound treatment of PFTBA@MBs significantly increases the proportion of mature DCs, boosting the immune response and creating conditions for further enhancing the effectiveness of tumor immunotherapy. The inventors attribute this to the fact that ultrasound blasting promotes the accumulation of microbubbles at the tumor site, effectively promoting DC maturation.

[0046] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A tumor cell membrane ultrasound microbubble, characterized in that: It includes a phospholipid bilayer shell composed of tumor cell membranes, and an inert gas is enclosed in the phospholipid bilayer shell.

2. The tumor cell membrane ultrasound microbubble according to claim 1, characterized in that: The phospholipid bilayer shell is processed in the following manner: the tumor cell membrane is mixed with a phosphate buffer solution containing glycerol, and a tumor cell membrane suspension is obtained after ultrasonic treatment; and the tumor cell membrane suspension is mixed with perfluorotributylamine.

3. The tumor cell membrane ultrasound microbubble according to claim 2, characterized in that: The volume ratio of tumor cell membrane suspension to perfluorotributylamine is 0.2mL:20-50μL.

4. The tumor cell membrane ultrasound microbubble according to claim 1, characterized in that: The tumor cell membrane is extracted from CT26 cells.

5. The tumor cell membrane ultrasound microbubble according to claim 1, characterized in that: The inert gas is C3F8.

6. The method for preparing tumor cell membrane ultrasound microbubbles according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: mixing tumor cell membranes with phosphate buffer solution containing glycerol, obtaining a tumor cell membrane suspension after ultrasonic treatment, and transferring the suspension into a container; then adding perfluorotributylamine and sealing the container; extracting the air in the container and injecting an inert gas, and obtaining tumor cell membrane ultrasonic microbubbles after oscillation treatment.

7. The method for preparing tumor cell membrane ultrasound microbubbles according to claim 6, characterized in that: In the phosphate buffer containing glycerol, the mass percentage of glycerol is 10%, the specification of the phosphate buffer is 0.01M, and the pH is 7.

4.

8. The method for preparing tumor cell membrane ultrasound microbubbles according to claim 6, characterized in that: The power of ultrasonic treatment was 50 W, the time was 1 min, and the ultrasonic treatment was carried out according to the procedure of working for 5 s and pausing for 5 s.

9. The method for preparing tumor cell membrane ultrasound microbubbles according to claim 6, characterized in that: The container is a 2 mL vial; 0.2 mL of tumor cell membrane suspension is added into the vial, and then 20-50 μL of perfluorotributylamine is added.

10. Use of the tumor cell membrane ultrasound microbubble according to any one of claims 1 to 5 in preparing a preparation for tumor immunotherapy.

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