Dendritic cell membrane microvesicle, preparation method thereof and application of dendritic cell membrane microvesicle in tumor immunotherapy

Abstract

The invention discloses dendritic cell membrane microvesicles DCM (at) MBs as well as a preparation method and application thereof in tumor immunotherapy, and the DCM (at) MBs is prepared by the following steps: stimulating immature dendritic cells to be mature by adopting tumor cell membrane protein, then extracting cell membranes DCM of the mature dendritic cells, mixing the cell membranes DCM with dipalmitoyl phosphatidylcholine DPPC and distearoyl phosphatidylethanolamine-polyethylene glycol DSPE-MPEG, and carrying out ultrasonic treatment to obtain the dendritic cell membrane microvesicles DCM (at) MBs. The material is prepared by adopting a mechanical oscillation method. The DCM (at) MBs can effectively activate T cells and promote the T cells to specifically kill tumor cells, a new strategy is provided for tumor immunotherapy, the immune function of dendritic cells can be recovered through the strategy, and immunosuppression in a tumor microenvironment is expected to be overcome. The DCM (at) MBs also has the capability of delivering drugs, and can be used in combination with other treatment means in the future to realize more accurate and efficient tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a dendritic cell membrane microvesicle and a preparation method thereof and an application thereof in tumor immunotherapy. Background Art

[0002] In recent years, tumor immunotherapy has become a revolutionary treatment method following traditional surgery, chemotherapy and radiotherapy. Among them, the development of tumor vaccines based on dendritic cells (DCs) has become a hot spot in tumor immunotherapy. As a professional type of antigen presenting cell (APC), DCs can recognize and process tumor antigens and present them as antigen peptide major histocompatibility complex (pMHC) molecules on the DC cell membrane. Immature DCs differentiate into mature DCs after taking up antigens or being stimulated by certain factors. Subsequently, these mature DCs activate several subsets of antigen-specific T lymphocytes to eliminate homologous tumor cells. DC vaccine is an immunotherapy that uses DC to present antigens and stimulate the immune system to attack tumor cells.

[0003] The current method for preparing DC vaccines is to first sort DCs from the mononuclear cells of the patient's blood and process them in vitro. After quality testing, the active DCs are re-infused into a site in the same patient to promote their priming of T cells in the nearest lymphoid tissue. However, intact dendritic cells (DCs) may be regulated by a variety of inhibitory signals in vivo, which may come from the tumor microenvironment or other immunosuppressive mechanisms, resulting in a weakened function of DCs in antigen presentation and T cell activation. For example, exosomes released by tumor cells can inhibit the maturation and migration of DCs, thereby promoting immunosuppression. In addition, certain pharmacological molecules can also weaken their immunostimulatory ability by changing the activation state of DCs and inducing their tolerance. Therefore, there is a need to develop safer and more effective dendritic cell-based vaccines as an alternative strategy for tumor immunotherapy.

[0004] In general, most current tumor vaccines target a single antigen target. Although current tumor vaccines have shown encouraging therapeutic effects in some melanomas and prostate cancers, they often fail to provide sufficient immunogenicity in the face of high heterogeneity and mutation rates of cancer cells. In addition, these vaccines may also promote immune escape and antigen loss. Therefore, how to identify one or a few optimal antigens with high tumor specificity among tumor antigens is a serious challenge. One strategy to address these problems is to use whole cancer cells, whole cancer cell lysates (WCLs), or tumor cell membranes as a multi-antigen source for dendritic cells (DCs) priming. This can prime the immune system against the complete antigen spectrum of tumor cells. However, the presence of a large amount of non-tumor-associated antigenic material may significantly reduce the efficacy of the immune response. Notably, tumor cell membranes are able to replicate the diversity of tumor cell surface antigens, including known and unknown tumor-specific antigens (TSAs) and a full array of tumor-associated antigens (TAAs). Applying tumor cell membrane stimulation to dendritic cells can maximize the advantages of multi-antigen vaccines in target recognition while reducing the susceptibility to immune escape. Summary of the invention

[0005] The purpose of the present invention is to provide a dendritic cell membrane microvesicle and a preparation method thereof and application thereof in tumor immunotherapy in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a dendritic cell membrane microvesicle DCM@MBs, which is prepared by using tumor cell membrane proteins to stimulate the maturation of immature dendritic cells, then extracting the cell membrane DCM of mature dendritic cells and mixing it with dipalmitoylphosphatidylcholine DPPC and distearoylphosphatidylethanolamine-polyethylene glycol DSPE-MPEG, and using a mechanical oscillation method. The mass ratio of the mixture of DCM, DPPC and DSPE-MPEG is 1:1.5-2.5, and the mass ratio of DPPC to DSPE-MPEG in the mixture of DPPC and DSPE-MPEG is 4-6:3.

[0008] The DSPE-MPEG is DSPE-MPEG2000.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned dendritic cell membrane microvesicles DCM@MBs, comprising the following steps:

[0010] S1. Activating dendritic cells: taking immature dendritic cells and culturing them for 12 to 24 hours until the immature dendritic cells adhere to the wall, adding tumor cell membrane proteins and co-incubating for 20 to 24 hours, the ratio of immature dendritic cells to tumor cell membrane proteins is based on the initial ratio of adding 35 to 45 μg of tumor cell membrane proteins for every 30,000 immature dendritic cells; after the co-incubation, mature dendritic cells are obtained;

[0011] S2. Extracting mature dendritic cell membranes: extracting cell membranes of the obtained mature dendritic cells;

[0012] S3. Preparation of DCM@MBs: Mature dendritic cell membranes were taken, mixed with DPPC and DSPE-MPEG, and DCM@MBs were prepared by mechanical oscillation.

[0013] In step S1, the culture and co-incubation conditions are: culture in an incubator at 35-37° C. and 4-5% CO 2 .

[0014] Step S3, preparing DCM@MBs: taking DCM, DPPC and DSPE-MPEG, mixing and dispersing them in a buffer solution, introducing an inert gas, and mechanically oscillating to obtain microbubbles with DCM loaded on the surface, namely DCM@MBs.

[0015] The inert gas is C3F8, and a silver mercury capsule blender is used for mechanical oscillation.

[0016] The buffer is a phosphate buffer containing 0.8-1.2 wt % glycerol.

[0017] The tumor cell membrane protein is a breast cancer cell membrane protein or a colorectal cancer cell membrane protein.

[0018] The third aspect of the present invention provides the use of the above-mentioned dendritic cell membrane microvesicles DCM@MBs in the preparation of tumor vaccines.

[0019] The fourth aspect of the present invention provides the use of the above-mentioned dendritic cell membrane microvesicles DCM@MBs in the preparation of a preparation for tumor immunotherapy.

[0020] The beneficial effects of the present invention are as follows: the dendritic cell membrane-based vaccine (DCM@MBs) provided by the present invention has shown significant potential in tumor immunotherapy. First, the preparation of DCM@MBs is simple and stable, and has good biocompatibility and immune activation ability, providing a new strategy for tumor immunotherapy. Secondly, DCM@MBs can effectively activate T cells and promote their specific killing of tumor cells, proving that this strategy can restore the immune function of dendritic cells and is expected to overcome immunosuppression in the tumor microenvironment. In addition, DCM@MBs not only has the potential to be used as a tumor vaccine, but also has the ability to deliver drugs. In the future, it can be used in combination with other treatment methods (such as chemotherapeutic drugs, immunomodulatory factors, etc.), so as to achieve more accurate and efficient tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Tumor cell membrane proteins were shown to stimulate maturation of dendritic cells (DCs).

[0022] Figure 2 It is the characterization of dendritic cell membrane microvesicles (DCM@MBs).

[0023] Figure 3 It shows the activation and specific killing ability of T cells. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0026] Research methods of the present invention: First, through tumor cell membrane protein extraction and dendritic cell (DCs) activation experiments, it was confirmed that tumor antigens can effectively stimulate the maturation of immature DCs. Then, DCM@MBs were prepared by mixing mature dendritic cell membranes and lipids, and their morphology, size and protein composition were characterized by scanning electron microscopy (SEM) and SDS-PAGE. Finally, in vitro co-culture experiments were performed to evaluate the activation effect of DCM@MBs on T cells and its ability to specifically kill tumor cells.

[0027] The results of the present invention are as follows: In the dendritic cell activation experiment in vitro, compared with the control group, the expression of MHC class II molecules on the surface of immature DCs increased significantly after stimulation with tumor cell membrane proteins; DCM@MB was spherical, about 1-2 μm in size, and had a rough surface. In the in vitro co-culture experiment, compared with the control group, the DCM@MBs group significantly promoted the activation of CD8+T cells, and the secretion of T cell INF-γ increased significantly; and the immune-specific killing effect on CT26.WT tumor cells was significantly stronger than that on 4T1 mouse breast cancer cells.

[0028] The specific research experiments are as follows:

[0029] Example 1

[0030] 1 Materials and methods

[0031] 1.1 Materials

[0032] 1.1.1 Cells

[0033] 4T1 mouse breast cancer cell line and DC2.4 mouse dendritic cell line (immature dendritic cells) were purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). CT26.WT mouse colorectal cancer cell line was purchased from Wuhan Punosai Life Science Co., Ltd. (Wuhan, China). Cells were cultured in a 5% CO2 incubator at 37°C with recommended culture medium.

[0034] 1.1.2 Main reagents

[0035] Cell membrane protein and cytoplasm protein extraction kit, BCA protein concentration determination kit, protease inhibitor (PMSF), lactate dehydrogenase cytotoxicity detection (LDH) kit, and Coomassie brilliant blue dye were purchased from Biotech Co., Ltd. (Shanghai, China). Dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) were purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China). Glycerol was purchased from Sigma-Aldrich (St. Louis, USA). Sulfonated far-red Cyanine5.5 fluorophore amine-reactive ester (Sulfo-CY-5.5NHS ester) was purchased from MCE (New Jersey, USA). The following antibodies were used in the experiment: FITC-labeled mouse anti-CD4 antibody (clone GK1.5, cat. no. 11-0041-82, eBioscience), APC-labeled mouse anti-CD8a antibody (clone 53-6.7, cat. no. 100712, BioLegend), PE-labeled mouse anti-CD3 antibody (clone 17A2, cat. no. 12-0032-82, eBioscience), FITC-labeled mouse anti-MHC class II antibody (clone GK1.5, cat. no. 11-0041-82, eBioscience). The enzyme-linked immunosorbent assay (ELISA) kit for detecting interferon-γ (IFN-γ) was from Meike Biotechnology Co., Ltd. (Shanghai, China).

[0036] 1.2 Methods

[0037] 1.2.1 Preparation of tumor cell membrane proteins

[0038] In order to prepare tumor antigens, CT26.WT mouse colorectal cancer cell membrane proteins were extracted according to the method provided by the cell membrane protein extraction kit. First, cultured CT26 cells (1x 10 8) was resuspended in 3mL of membrane protein extraction solution, and protease inhibitors were added and incubated in an ice bath for 15 minutes. Then, the cell suspension was repeatedly frozen and thawed in liquid nitrogen three times and centrifuged (2000rpm, 10min) to collect the supernatant. The cell supernatant was then centrifuged again (14,000rmp, 30min) to collect the precipitate. The collected precipitate was resuspended in 200μL of membrane protein extraction reagent, centrifuged (14,000rpm, 5min) after ice bath for 15 minutes, and the supernatant was collected as tumor cell membrane protein. The concentration of tumor cell membrane protein was quantified according to the method provided by the BCA kit. First, the tumor cell membrane protein solution was added to a 96-well plate, 20μL per well, and 3 replicate wells were set. Subsequently, 200μL BCA working solution was added to each well and placed at 37°C for 30 minutes. Finally, the absorbance at 562nm was measured with an enzyme reader, and the sample protein concentration was calculated according to the standard curve. The remaining tumor cell membrane proteins were stored at -80°C until use.

[0039] 1.2.2 Activation effect of dendritic cells

[0040] In order to evaluate whether tumor cell membrane proteins can be effectively taken up by DC2.4 cells, DC2.4 cells were inoculated in confocal dishes, with about 30,000 cells per dish. After culturing in a 37°C, 5% CO2 incubator for 24 hours, 2 mL of 20 μg / mL tumor cell membrane protein (labeled with Cy5.5) was added to each dish at different time points (0h, 1h, 3h, 6h, 24h). Finally, the phagocytosis of tumor cell membrane proteins by DC2.4 cells was observed by confocal microscopy (Nikon). In order to evaluate the maturation rate, DC2.4 cells were inoculated in 6-well plates, with about 200,000 cells per well, and 3 replicates were set for each group. Subsequently, 1 mL of different concentrations of tumor membrane protein (0, 5, 15, 25 μg / mL) was added to each well, and the cells were returned to the 37°C, 5% CO2 incubator for stimulation for 24 hours. The expression of MHC class II antigens on the surface of each group of DC2.4 cells was detected by flow cytometry. To evaluate the static process of DC2.4 activation, images were taken before and 24 h after tumor cell membrane protein stimulation, and the morphological changes of DC2.4 were observed by confocal microscopy.

[0041] 1.2.3 Extraction of mature dendritic cell membranes

[0042] Mature dendritic cell membrane (DCM) was extracted according to the method provided by the cell membrane protein extraction kit. First, mature DC cells (1x 10 8) was resuspended in 3 mL of extract, and protease inhibitors were added and incubated in an ice bath for 15 minutes. Then, the plasma cell suspension was repeatedly frozen and thawed in liquid nitrogen three times 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 precipitate was collected and freeze-dried for further use.

[0043] 1.2.4 Preparation of DCM@MBs

[0044] DCM@MBs were synthesized by a typical mechanical shaking method. Briefly, the obtained DCM (1 mg) and a mixture of 2 mg of DPPC and DSPE-PEG2000 (mass ratio of 5:3) were dispersed in 1 wt% glycerol phosphate buffer (PBS) (0.5 mL). Subsequently, the mixture was transferred (0.5 mL) to a vial, and the air in the vial was replaced with C3F8 gas using a gas exchange system. Finally, the vial was mechanically shaken using a silver mercury capsule blender (YJT-2, Shanghai, China) at a vibration frequency of 4000 times / min for 50 s. This process utilizes the lipid properties of the cell membrane, and the resulting product is named DCM@MBs. For the control experiment (i.e., without the addition of DCM), ordinary lipid microbubbles were prepared according to the same procedure, and the raw material used was a mixture of DPPC and DSPE-PEG2000 (mass ratio of 5:3) with a total mass of 3 mg.

[0045] 1.2.5 Characterization of DCM@MBs

[0046] High-resolution images of the DCM@MBs surface were taken by scanning electron microscopy (SEM) (ZEISS GeoiniSEM 300, Germany) to assess their size and shape. Protein composition in DCM@MBs was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining.

[0047] 1.2.6 In vitro co-culture

[0048] To evaluate whether DCM@MBs can activate T cells, we isolated 1x10 T cells from BALB / c mice bearing CT26 tumors. 7Spleen lymphocytes were collected and co-cultured with PBS, MBs, iDCM@MBs, and DCM@MBs (200 μg / mL), respectively. After 48 hours, the spleen lymphocytes were washed three times with PBS and stained with anti-CD3-PE, anti-CD4-FITC, and anti-CD8a-APC antibodies at 4°C for 30 min. After washing three times with cold PBS, the cell fluorescence was detected by flow cytometry. In order to determine the cytokines secreted by immune cells after stimulation, the culture medium suspension of spleen lymphocytes co-cultured with PBS, MBs, iDCM@MBs, and DCM@MBs was collected 48 hours later, and IFN-γ was determined according to the standard method of enzyme-linked immunosorbent assay (ELISA) kit. According to the instructions of the lactate dehydrogenase cytotoxicity (LDH) kit, the killing ability of DCM@MBs activated immune cells CT26 and 4T1 was detected (the ratio of activated spleen lymphocytes to target cells was 10:1).

[0049] 1.2.7 Statistical methods

[0050] GraphPad Prism 9.0 software was used for statistical analysis and drawing, and the quantitative data were presented as mean ± standard deviation (x ± s). One-way analysis of variance was used for comparison among the groups, with the test level α = 0.05.

[0051] 2 Results

[0052] 2.1 In vitro regulation of maturation of dendritic cells (DCs)

[0053] Since whether tumor antigens can activate DC cells in vitro is crucial for the preparation of DC vaccines, the maturation of DC cells was studied. First, CT26.WT tumor cells were cultured alone, and the tumor cell membrane proteins, i.e., tumor antigens, were extracted using a protein extraction kit, and the tumor cell membrane protein concentration was measured using a BCA protein concentration detection kit for subsequent use. In order to study whether tumor cell membrane proteins can be effectively taken up by DC, tumor cell membrane proteins labeled with Cy5.5 (red fluorescence, protein dye) were co-incubated with immature DCs (DAPI, nuclear dye marker). It can be clearly observed by confocal laser scanning microscopy (CLSM) that the uptake of tumor cell membrane proteins gradually increased from 0 to 24 hours, so we chose 24 hours as the co-incubation condition for subsequent experiments ( Figure 1A). In order to detect the in vitro maturation of DCs, we co-incubated different concentrations of tumor cell membrane proteins with immature DCs for 24 h, and then analyzed the expression of MHC class II antigens on the surface of DCs in each group by flow cytometry. The expression levels of the control group, 5 μg / mL group, 15 μg / mL group, and 25 μg / mL group were 14.967±1.703%, 17.367±0.013%, 19.567±0.043%, and 25.167±1.203%, respectively. Compared with the control group, the expression of MHC class II antigens on the surface of DCs in the 25 μg / mL group was significantly increased (P=0.000)( Figure 1 B). In addition, compared with the small triangles of immature DCs, the cell surface of tumor cells stimulated by membrane proteins has branch-like protrusions with irregular shapes, lengths and thicknesses ( Figure 1 C).

[0054] 2.2 Characterization of dendritic cell membrane microvesicles (DCM@MBs)

[0055] After the immature DCs were subjected to shock sensitization treatment with tumor cell membrane proteins, the mature DC cell membrane (DCM) was separated. Subsequently, DPPC, DSPEmPEG2000 and DCM were hydrated with phosphate buffered saline (PBS) containing 1wt% glycerol in a mass ratio of 5:3:4, and then the mixture was filled with C3F8, and finally DCM@MBs were prepared under mechanical vibration. Scanning electron microscopy (SEM) can clearly observe that DCM@MBs are spherical, about 1 to 2 μm in size, and have a rough surface, indicating that a large amount of membrane fragments are wrapped on the surface of the microbubble shell ( Figure 2 A). In addition, the protein composition of mature DC cell membrane (DCM) and DCM@MBs was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and it was found that the protein expression of DCM and DP@MBs was very similar, indicating that cell membrane fragments were successfully loaded during the preparation of microbubbles ( Figure 2 B). The surface zeta potential of the microbubbles was measured using an OpptroniX instrument. Analysis showed that the negative surface charge zeta value of DP@MB was -41.467±0.093mV ( Figure 2 C), compared with MBs (-33.8±4.360 mV), it is demonstrated that mature DC cell membrane (DCM) as part of the DCM@MBs composition improves the stability and dispersibility of the material.

[0056] 2.3 T cell activation and specific anti-tumor effect in vitro

[0057] To evaluate whether DCM@MBs can present tumor antigens to T cells and activate T cells, we performed in vitro co-culture. Since CD8+ cytotoxic T lymphocytes (CTLs) are the main force in killing cancer cells in immunotherapy, we measured the expression of CD8 in CD3 + Expression on the cell membrane of T cells (from mouse spleen lymphocytes), CD3 in the Control group, MBs group, iDCM@MBs group, and DCM@MBs group + The expression levels of CD8 in T cells were 14.233±0.643%, 14.467±1.213%, 17.467±0.143%, and 22.2±0.07%, respectively. + The expression of CD8 in T cells was significantly increased (P = 0.000). In addition, we found that compared with the Control group, the iDCM@MBs group also showed T cell activation (P = 0.003) ( Figure 3 A, B), which may be related to the low level of MHC class II molecules expressed on the surface of immature DCs. In addition, the content of INF-γ in the supernatant of spleen lymphocyte culture medium of each group was detected by Elisa kit. The contents of Control group, MBs group, iDCM@MBs group, and DCM@MBs group were 11.761±1.703pg / mL, 11.573±1.671pg / mL, 70.339±1.270pg / mL, and 120.574±5.179pg / mL, respectively. Compared with Control, DCM@MBs group significantly promoted the secretion of INF-γ of T cells (P=0.000)( Figure 3 C). Given that DCM@MBs can effectively induce T cell activation, we incubated spleen lymphocytes treated with DCM@MBs with CT26.WT tumor cells and 4T1 mouse breast cancer cells at a ratio of 10:1 (T cells to target cells) for 24 hours and then performed lactate dehydrogenase detection. The cell death rate of the CT26 group was 30.737±0.216%, which was 3.11 times that of the 4T1 group (9.893±0.341%). Figure 3 D), indicating that DCM@MBs have immune specificity for CT26.

[0058] 3 Analysis

[0059] Currently, the preparation and use of DC vaccines involve separation, ex vivo loading, and reinfusion of DCs into patients. Although DC-mediated immunotherapy may be effective, one obstacle faced by traditional DC vaccines is that intact dendritic cells (DCs) may be regulated by multiple inhibitory signals in vivo. However, it is worth noting that DC membranes have been shown to carry more TAA-MHC II complexes than intact DCs, thereby more effectively initiating anti-tumor immunity. In response to the above problems, the present invention proposes a DC vaccine strategy based on dendritic cell membranes and verifies its potential in tumor immunotherapy through a series of experiments.

[0060] Immature DCs are known to be unable to induce antigen-specific responses and may induce the differentiation of regulatory T cells. In contrast, mature DCs show enhanced expression of major histocompatibility complex (MHC) molecules necessary for T cell responses. Therefore, controlling DCs to reach a fully mature and activated state before vaccine administration is crucial to avoid inducing immune tolerance. The present invention found that immature DCs can effectively phagocytose tumor cell membrane proteins, and the amount of phagocytosis gradually increases over time. Through flow cytometry analysis, we found that after stimulation with tumor cell membrane antigens, the expression of MHC class II molecules on the surface of DCs increased significantly, which demonstrated the effective presentation of tumor antigens and the maturation of DCs. In addition, this result provides a theoretical basis for the preparation of subsequent DC vaccines, indicating that tumor antigens can be effectively delivered by dendritic cells and promote the activation of T cells.

[0061] DCM@MBs were prepared by mechanical vibration after mixing mature DC membrane (DCM) with lipids and filling C3F8. SEM and SDS-WB experiments proved that mature DC membrane was successfully loaded on the surface of microbubbles, and the volume of microbubbles was smaller than that of DC. Therefore, a higher vaccine yield can be produced from the same starting amount of DC cells. In addition, the Zeta potential analysis results of microbubbles showed that DCM@MBs have a negative charge, which helps to improve its stability in vivo, prevent aggregation, and ensure good biocompatibility. It is well known that microbubbles can achieve precise in vivo delivery, namely ultrasound targeted microbubble destruction (UTMD) technology. Specifically, after intravenous injection of microbubbles, these microbubbles will oscillate and collapse where ultrasound irradiation is applied, thereby achieving local acoustic cavitation effect. At the same time, the ultrasonic perforations generated by shear stress during mechanical microfluidics and microbubble destruction can enhance local vascular permeability. Therefore, the DCM@MBs designed and prepared by us have the potential to mediate precise vaccine delivery to lymphoid organs.

[0062] In the clinical application of tumor immunotherapy, the activation of T cells and tumor-specific immune response are key links. The present study first evaluated the ability of DCM@MBs to activate T cells by flow cytometry experiments. Compared with the Control group, the number of CD8+T cells in the DCM@MBs group after co-incubation increased significantly. Next, we further verified its immune-specific killing effect on tumor cells, and its killing of CT26.WT tumor cells showed a more significant cell mortality rate than that of 4T1 mouse breast cancer cells. Experiments have shown that DCM@MBs can activate T cells and achieve their immune-specific killing effect on tumor cells, and has the potential to further study T cell-mediated tumor immunotherapy in vivo.

[0063] In summary, the dendritic cell membrane-based vaccine (DCM@MBs) strategy proposed in this study has shown significant potential in tumor immunotherapy. First, the preparation of DCM@MBs is simple and stable, with good biocompatibility and immune activation ability, providing a new strategy for tumor immunotherapy. Secondly, DCM@MBs can effectively activate T cells and promote their specific killing of tumor cells, proving that this strategy can restore the immune function of dendritic cells and is expected to overcome immunosuppression in the tumor microenvironment. In addition, DCM@MBs not only has the potential to be used as a tumor vaccine, but also has the ability to deliver drugs. In the future, it can be used in combination with other treatment methods (such as chemotherapeutic drugs, immunomodulatory factors, etc.), so as to achieve more accurate and efficient tumor treatment.

Claims

1. A dendritic cell membrane microvesicle DCM@MBs, characterized in that: The method is prepared by using tumor cell membrane proteins to stimulate the maturation of immature dendritic cells, then extracting cell membrane DCM of mature dendritic cells, mixing it with dipalmitoylphosphatidylcholine DPPC and distearoylphosphatidylethanolamine-polyethylene glycol DSPE-MPEG, and adopting a mechanical oscillation method. The mass ratio of the mixture of DCM, DPPC and DSPE-MPEG is 1:1.5-2.5, and the mass ratio of DPPC to DSPE-MPEG in the mixture of DPPC and DSPE-MPEG is 4-6:

3.

2. The dendritic cell membrane microvesicles DCM@MBs according to claim 1, characterized in that: The DSPE-MPEG is DSPE-MPEG2000.

3. The method for preparing dendritic cell membrane microvesicles DCM@MBs according to claim 1 or 2, characterized in that: The steps include: S1. Activating dendritic cells: taking immature dendritic cells and culturing them for 12 to 24 hours until the immature dendritic cells adhere to the wall, adding tumor cell membrane proteins and co-incubating for 20 to 24 hours, the ratio of immature dendritic cells to tumor cell membrane proteins is based on the initial ratio of adding 35 to 45 μg of tumor cell membrane proteins for every 30,000 immature dendritic cells; after the co-incubation, mature dendritic cells are obtained; S2. Extracting mature dendritic cell membranes: extracting cell membranes of the obtained mature dendritic cells; S3. Preparation of DCM@MBs: Mature dendritic cell membranes were taken, mixed with DPPC and DSPE-MPEG, and DCM@MBs were prepared by mechanical oscillation.

4. The preparation method according to claim 3, characterized in that: In step S1, the culture and co-incubation conditions are: culture in an incubator at 35-37° C. and 4-5% CO 2 .

5. The preparation method according to claim 3, characterized in that: Step S3, preparing DCM@MBs: taking DCM, DPPC and DSPE-MPEG, mixing and dispersing them in a buffer solution, introducing an inert gas, and mechanically oscillating to obtain microbubbles with DCM loaded on the surface, namely DCM@MBs.

6. The preparation method according to claim 5, characterized in that: The inert gas is C3F8, and a silver mercury capsule blender is used for mechanical oscillation.

7. The preparation method according to claim 5, characterized in that: The buffer is a phosphate buffer containing 0.8-1.2 wt % glycerol.

8. The preparation method according to claim 3, characterized in that: The tumor cell membrane protein is a breast cancer cell membrane protein or a colorectal cancer cell membrane protein.

9. Use of the dendritic cell membrane microvesicles DCM@MBs according to claim 1 or 2 in the preparation of tumor vaccines.

10. Use of the dendritic cell membrane microvesicles DCM@MBs according to claim 1 or 2 in the preparation of a preparation for tumor immunotherapy.

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