Method for preparing cytosolic vesicles and uses thereof

Abstract

This invention discloses a method for preparing cytoplasmic vesicles and their applications. The method utilizes a dehydration process. Cells are dehydrated, causing them to shrink, and then cultured in a culture medium. This allows for the rapid secretion of large quantities of vesicles containing ribosomes, mitochondria, and other organelles, as well as the cellular matrix. These vesicles can be collected by centrifugation for further processing. This method is convenient, rapid, and not limited by cell type. It also boasts high yield, does not involve the use of toxic substances, and allows for modification of the parent cells to alter the function of the cytoplasmic vesicles. The cytoplasmic vesicles prepared by this invention can be used for disease diagnosis and clinical medication, possessing high application value and suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing cytoplasmic vesicles and their applications. Background Technology

[0002] Cellular vesicles are a biomimetic technology primarily used to prepare micron- or nano-vesicles with cell membrane-like structures. Currently, existing cellular vesicles mainly include vesicles prepared from cell membranes and extracellular vesicles secreted by cells. Cellular vesicles have proven to have many advantages in drug development and disease diagnosis, such as good biosafety, targeting of membrane proteins, and multiple drug delivery capabilities, giving them broad application prospects. However, large-scale preparation and multifunctionalization of cellular vesicles still face some challenges. In particular, the number of extracellular vesicles secreted by cells is extremely small, far less than the actual demand. Studies have shown that, under in vitro culture conditions, 10 6 The daily secretion of exosomes by individual cells is less than micrograms, which creates a research dilemma for cell-secreted exosomes (exosomes) in fields such as disease diagnosis and treatment. To overcome this shortcoming, the need for large-scale production of cell-derived micron and nanovesicles has emerged. Induced secretion vesicles, while possessing similar functions to exosomes, have a relatively simpler preparation process and yield a larger amount of vesicles. The most common method is to use chemical induction (chemical reagents or nanomaterials) to stimulate cells to secrete more vesicles [Advanced Functional Materials, 2016, 26(32):5804-5817]. Other methods include physical methods, such as photo-induced nanovesicle production [Nature Communications, 2022, 13(1):6534]. These methods can obtain a relatively sufficient number of vesicles with membrane components similar to those of the parent cell [ACS Appl. Mater. Interfaces 2021, 13, 55767-55779]. However, existing cell-secreted vesicle induction techniques are either time-consuming and have low yield improvements, or they introduce reagents with biosafety risks. Therefore, it is urgent to develop new, efficient, and safe methods for preparing cell-like vesicles.

[0003] Furthermore, existing applications of cell-like vesicles are based on existing cell membrane structures. By loading or modifying small molecule therapeutics or diagnostics within the vesicles or on the cell membrane, these vesicle-derived small molecule therapeutics or diagnostics can exhibit excellent biocompatibility and targeting. However, such vesicle loading methods still face significant challenges when loading biomolecules (proteins, mRNA, organelles), resulting in low loading efficiency. This indirectly reflects the limitations of the expandable functions of cell-like vesicles. While there are other methods for loading and in vivo delivery of biomolecules such as protein drugs and mRNA drugs, strategies for loading or delivering organelles, which are larger in size (hundreds of nanometers to tens of micrometers) and more fragile, are rarely reported. Therefore, constructing cell-like vesicles containing biomolecules and structures (especially organelles) is significant for the development of biotherapy and diagnostics and possesses potential application value. Summary of the Invention

[0004] The purpose of this invention is to provide an efficient method for preparing cytoplasmic vesicles and its applications. The preparation method of this invention is convenient, quick, inexpensive, and does not involve toxic substances, making it suitable for widespread application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a method for preparing extracellular cytoplasmic vesicles, the method comprising the step of inducing cells to generate vesicles through dehydration treatment.

[0007] In some implementations, the dehydration treatment time is 1 second to 60 minutes, for example 5 minutes to 30 minutes, 10 minutes to 30 minutes, 5 minutes to 20 minutes, 10 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 1 minute to 2 minutes, 1 second to 10 seconds, 20 minutes to 60 minutes, 30 minutes.

[0008] In some embodiments, the preparation method includes the following steps: dehydrating the cells until (e.g., observing cell shrinkage under a microscope) the cells shrink, thereby inducing them to generate vesicles.

[0009] In some implementations, the dehydration process is selected from any of the following:

[0010] I) In the absence of culture medium or buffer, the cells are left to stand at a certain temperature for a period of time; for example, at a temperature of -80°C to 80°C for 1 second to 60 minutes.

[0011] II) Place the cells in a high concentration of sodium chloride solution and / or sucrose and let them stand for a period of time; for example, 1 minute to 60 minutes.

[0012] In some implementations, the preparation method can be carried out on a small scale in a laboratory or on a large scale for industrial production.

[0013] In some implementations, in dehydration treatment method I), the settling time can be adjusted according to the settling temperature until cell shrinkage is observed under a microscope. For example, when the settling temperature is high, the settling time is reduced; when the settling temperature is low, the settling time is increased.

[0014] In some implementation schemes, dehydration treatment method I) can specifically be selected from any of the following treatment methods:

[0015] i) Without culture medium or buffer, let the cells stand at room temperature for 1 minute to 60 minutes, for example, 5 minutes to 30 minutes, 10 minutes to 30 minutes, 5 minutes to 20 minutes, 10 minutes;

[0016] ii) Without culture medium or buffer, incubate the cells at room temperature to 80°C for 1 second to 20 minutes, for example, 1 minute to 10 minutes, 1 minute to 5 minutes, 1 minute to 2 minutes, 1 second to 10 seconds, 20 seconds, 30 seconds;

[0017] iii) Without culture medium or buffer, let the cells stand for 10 to 80 minutes, for example, 20 to 60 minutes or 30 minutes, at a temperature below room temperature.

[0018] In some implementations, i) the room temperature is 10°C to 30°C, for example 18°C ​​to 28°C or 18°C ​​to 25°C.

[0019] In some implementations, the temperature in ii) is 30°C to 80°C.

[0020] In some implementations, iii) below room temperature conditions is below 10°C, for example, -100°C to 10°C, -80°C to 0°C.

[0021] In some embodiments, the preparation method includes the following steps:

[0022] Cell expansion culture, dehydration treatment, isolation and purification of cytoplasmic vesicles.

[0023] In some embodiments, the preparation method includes the following steps:

[0024] Cell acquisition, cell expansion culture, dehydration treatment, culture (e.g., perfusion culture), and collection of cytoplasmic vesicles.

[0025] In some implementation schemes, the cells are derived from primary cells, genetically modified cells, cancer cells, stem cells, tissues, blood samples, etc.

[0026] In some embodiments, cell expansion culture specifically involves: seeding cells into a culture dish and culturing until the cells proliferate to 70%–90% of the total volume of the dish or until the cell density reaches 70%–90%. In some embodiments, when the dehydration treatment method is selected from I), the preparation method includes the following steps:

[0027] 1) Seed the cells into a culture dish and culture them until the cells proliferate to 70%–90% of the total volume of the culture dish or until the cell density is 70%–90%.

[0028] 2) Remove the culture medium; optionally, add buffer to wash and remove dead cells;

[0029] 3) Dehydration treatment: The cells are left to stand at a certain temperature for a period of time;

[0030] 4) After observing cell shrinkage, add buffer solution;

[0031] 5) Cultivation; optionally tap the culture dish (this can accelerate the vesicle detachment process);

[0032] 6) Collect the supernatant and centrifuge to purify it;

[0033] 7) The supernatant was further centrifuged and concentrated to obtain cytoplasmic vesicles.

[0034] In some implementations, the culture dish is a 10cm culture dish.

[0035] In some implementations, in step 1), the cell culture conditions are: cultured at 37°C and 5% CO2.

[0036] In some implementations, in step 1), the cells are passaged every 3 to 4 days, depending on the cell density.

[0037] In some implementations, in step 1), the cells are cultured until they proliferate to 80%–90% or 70%–80% of the entire culture dish.

[0038] In some implementations, in step 1), the cells are cultured to a density of 80%–90% or 70%–80%.

[0039] In some implementations, in step 1), the cell types include suspension cells and adherent cells.

[0040] In some implementations, in step 1), the cell type includes primary cells, such as primary ovarian cancer cells.

[0041] In some implementation schemes, in step 1), the cells are cancer cells, such as lung cancer cells (small cell lung cancer, non-small cell lung cancer), breast cancer cells, cervical cancer cells, gastric cancer cells, liver cancer cells, leukemia cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.

[0042] In some implementations, in step 1), the cells are selected from PC9, MCF7, A549, H460, HeLa, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780, and other cell types.

[0043] In some embodiments, in step 1), the culture medium is selected from DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1X penicillin-streptomycin (PS) or RPMI 1640 medium containing 10% FBS and 1% PS.

[0044] In some embodiments, in step 2) and / or step 4), the pH of the buffer is 7.4-8.4, preferably 7.4, and more preferably a DPBS buffer with a pH of 7.4.

[0045] In some embodiments, in step 2) and / or step 4), the ionic strength of the buffer solution is 10 mM to 10 M, preferably 10 mM to 3 M.

[0046] In some implementations, in step 2) and / or step 4), the buffer is selected from DPBS buffer, NaCl solution, complete culture medium, serum-free culture medium, etc.

[0047] In some implementations, in step 4), the amount of buffer solution added is 5 mL.

[0048] In some implementations, in step 5), the vesicles are cultured for 2 min to 60 min (e.g., 10 min to 60 min) before vesicle detachment, for example, for 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min.

[0049] In some implementations, in step 5), after culturing for 2 minutes, a large number of dehydration-induced vesicles can be observed detaching from the cells under a microscope.

[0050] In some implementations, in step 6) and / or step 7), the centrifugation conditions are 500g centrifugation for 15 min to 30 min.

[0051] In some embodiments, in dehydration treatment method II), the high-concentration sodium chloride solution is a sodium chloride solution with a mass concentration of 3%-10%, for example, an 8% sodium chloride solution;

[0052] And / or, a high concentration of sucrose solution is a sucrose solution with a mass concentration of 20%-50%, such as a 30% sucrose solution.

[0053] In some embodiments, when the dehydration treatment method is selected from II), the preparation method includes the following steps:

[0054] (1) Seed the cells into a culture dish and culture them until the cells proliferate to 70% to 90% of the entire culture dish or until the cell density is 70% to 90%.

[0055] (2) Remove the culture medium; optionally, add buffer solution to rinse and remove dead cells;

[0056] (3) Dehydration treatment: The cells are placed in a high concentration of sodium chloride and / or sucrose solution and left to stand for a period of time;

[0057] (4) After the cells shrink, remove the high concentration of sodium chloride and / or sucrose solution (optionally wash away the residual high concentration of sodium chloride and / or sucrose solution with buffer) and add buffer.

[0058] (5) Cultivation; optionally tap the culture dish (this can accelerate the vesicle detachment process);

[0059] (6) Collect the supernatant and centrifuge to purify it;

[0060] (7) The supernatant was further centrifuged and concentrated to obtain cytoplasmic vesicles.

[0061] In some implementations, the culture dish is a 10cm culture dish.

[0062] In some implementations, in step (1), the cell culture conditions are: cultured at 37°C and 5% CO2.

[0063] In some implementations, in step (1), the cells are passaged every 3 to 4 days, depending on the cell density.

[0064] In some implementations, in step (1), the cells are cultured until they proliferate to 80%–90% or 70%–80% (e.g., 80%) of the entire culture dish.

[0065] In some implementations, in step (1), the cells are cultured to a density of 80%–90% or 70%–80%.

[0066] In some implementations, in step (1), the cell types include suspension cells and adherent cells.

[0067] In some implementations, in step (1), the cell type includes primary cells, such as primary ovarian cancer cells.

[0068] In some implementation schemes, in step (1), the cells are cancer cells, such as lung cancer cells (small cell lung cancer, non-small cell lung cancer), breast cancer cells, cervical cancer cells, gastric cancer cells, liver cancer cells, leukemia cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.

[0069] In some implementations, in step (1), the cells are selected from PC9, MCF7, A549, H460, HeLa, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and other cells.

[0070] In some embodiments, in step (1), the culture medium is selected from DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1X penicillin-streptomycin (PS) or RPMI 1640 medium containing 10% FBS and 1% PS.

[0071] In some embodiments, in step (2) and / or step (4), the pH of the buffer is 7.4-8.4, preferably 7.4, and more preferably a DPBS buffer with a pH of 7.4.

[0072] In some implementations, in step (2) and / or step (4), the ionic strength of the buffer solution is 10 mM to 10 M, preferably 10 mM to 3 M.

[0073] In some implementations, in steps (2) and / or (4), the buffer is selected from DPBS buffer, NaCl solution, complete culture medium, serum-free culture medium, etc.

[0074] In some implementations, in step (4), the amount of buffer solution added is 5 mL.

[0075] In some implementations, in step 5), the vesicles are cultured for 2 min to 60 min (e.g., 10 min to 60 min) before vesicle detachment, for example, for 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min.

[0076] In some implementations, in steps (6) and / or (7), the centrifugation conditions are 500g centrifugation for 15 min to 30 min.

[0077] The present invention also provides extracellular cytoplasmic vesicles prepared by the above preparation method.

[0078] In some implementation schemes, the cells are cancer cells, such as lung cancer cells (small cell lung cancer, non-small cell lung cancer), breast cancer cells, cervical cancer cells, gastric cancer cells, liver cancer cells, leukemia cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.

[0079] In some implementations, the cells are selected from PC9, MCF7, A549, H460, HeLa, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780, etc.; PC9 cells and B16 cells are preferred.

[0080] In some implementations, extracellular cytoplasmic vesicles contain calnexin and / or β-actin proteins.

[0081] In some implementations, extracellular cytoplasmic vesicles do not contain a cell nucleus.

[0082] In some implementations, extracellular cytoplasmic vesicles do not contain apoptotic cell characteristic proteins.

[0083] In some implementations, the extracellular cytoplasmic vesicles contain one, two, or more of the following marker proteins: UTP20, TMEM109, ZW10, PVR, CD58, MRPS7, CHP1, RAB27B, STEAP4, JAGN1, DBT, SLC39A7, PACSIN3, SCAMP1, NDUFB4, MRPS17, PSMB6, ANO6, SYPL1, TRPC5 / 4, POLR2E, C7orf50, SEH1L, TM9SF4, MXRA7, BDH1, GTPBP1, MKNK1, MGST3, and ALG5.

[0084] In some embodiments, the size of the extracellular cytoplasmic vesicles is 0–40 μm, preferably greater than 5 μm, for example greater than 10 μm, for example 5 μm–20 μm, for example 5 μm–30 μm.

[0085] This invention also provides a method for preparing extracellular nanoscale cytoplasmic vesicles, comprising the following steps:

[0086] (i) Extracellular cytoplasmic vesicles were prepared using the above preparation method;

[0087] (ii) Extracellular nanoscale cytoplasmic vesicles were obtained using a liposome extruder and a nanopore filter.

[0088] In some implementations, step (ii) specifically includes:

[0089] (ii) Using a liposome extruder, the suspension of extracellular cytoplasmic vesicles is loaded into a syringe, and then the suspension is passed through a series of nanopore filters in sequence. The product of the 0.1 μm filter membrane extrusion is collected to obtain extracellular nanoscale cytoplasmic vesicles.

[0090] In some implementations, the filter is a polycarbonate membrane filter.

[0091] In some implementations, a range of nanopore sizes are 1 μm, 0.4 μm, and 0.1 μm.

[0092] This invention provides extracellular nanoscale cytoplasmic vesicles prepared by the above-described preparation method.

[0093] In some implementation schemes, the cells are cancer cells, such as lung cancer cells (small cell lung cancer, non-small cell lung cancer), breast cancer cells, ovarian cancer cells, esophageal cancer cells, cervical cancer cells, gastric cancer cells, liver cancer cells, and leukemia cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), etc.

[0094] In some implementations, the cells are selected from PC9, MCF7, A549, H460, HeLa, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780, and other cell types.

[0095] In some implementations, extracellular nanoscale cytoplasmic vesicles contain calnexin and / or β-actin proteins;

[0096] In some implementations, extracellular nanoscale cytoplasmic vesicles do not contain a cell nucleus;

[0097] In some implementations, extracellular nanoscale cytoplasmic vesicles do not contain apoptotic cell characteristic proteins;

[0098] In some implementations, the extracellular nanoscale cytoplasmic vesicles contain one, two, or more of the following marker proteins: UTP20, TMEM109, ZW10, PVR, CD58, MRPS7, CHP1, RAB27B, STEAP4, JAGN1, DBT, SLC39A7, PACSIN3, SCAMP1, NDUFB4, MRPS17, PSMB6, ANO6, SYPL1, TRPC5 / 4, POLR2E, C7orf50, SEH1L, TM9SF4, MXRA7, BDH1, GTPBP1, MKNK1, MGST3, and ALG5.

[0099] The present invention also provides the application of the above-mentioned extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles in the preparation of drug carriers.

[0100] In some implementations, the drug includes chemical drugs, aptamer drugs, DNA drugs, RNA drugs, protein drugs, etc.

[0101] The present invention also provides the application of the above-mentioned extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles in the preparation of tumor vaccines.

[0102] The present invention also provides a tumor vaccine comprising the above-mentioned extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles and an adjuvant.

[0103] The present invention also provides the application of the above-mentioned extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles in the preparation of disease diagnostic reagents, drugs (e.g., targeted drugs, anti-tumor drugs), biomedical imaging, etc.

[0104] The present invention also provides the above-mentioned method for preparing extracellular cytoplasmic vesicles and the application of the above-mentioned method for preparing extracellular nanoscale cytoplasmic vesicles in the preparation of drug carriers, preparation of tumor vaccines, preparation of disease diagnostic reagents, drugs (such as targeted drugs, anti-tumor drugs), biomedical imaging, etc.

[0105] Beneficial effects

[0106] The method for preparing extracellular cytoplasmic vesicles according to this invention is convenient, rapid, and not limited by cell type. It also boasts high yield, does not involve the use of toxic substances, and allows for modification of parental cells to alter the function of the cytoplasmic vesicles. The cytoplasmic vesicles prepared by this invention can be used for disease diagnosis and clinical medication, possessing high application value and suitable for widespread application.

[0107] The cytoplasmic vesicles prepared in this invention have similar properties to exosomes compared to cells, and can be used as exosome analogs for disease diagnosis and drug development. At the same time, cytoplasmic vesicles contain important organelles such as mitochondria and the cell nucleus, and can be used for organelle supplementation therapy or for mRNA expression or protein secretion.

[0108] This method is applicable to various cell types, and it yields good yields for both suspension cells and adherent cells. Attached Figure Description

[0109] Figure 1 This is a roadmap of the dehydration-induced preparation method of the present invention.

[0110] Figure 2 Cytoplasmic vesicles (PC9, A549, H460, MCF7, Hela, MKN74, H22, CEM) prepared from different tumor cells in the examples.

[0111] Figure 3The effect of different pH buffer conditions on the induced cytoplasmic vesicle production yield.

[0112] Figure 4 The effect of different cell drying times on the yield of induced cytoplasmic vesicle formation.

[0113] Figure 5 The effect of the duration of DPBS addition on the yield of induced cytoplasmic vesicle formation.

[0114] Figure 6 Comparative diagrams of cytoplasmic vesicles, cells, and EVs: 6a shows SDS-PAGE results of proteins in PC9 cells, PC9-derived cytoplasmic vesicles (DIMV), and exosomes (EV); 6b shows a heatmap of protein intensity z-scores in PC9 cells, PC9-derived cytoplasmic vesicles (DIMV), and exosomes (n=3 per sample) after unsupervised hierarchical clustering; 6c shows a Pearson correlation heatmap of PC9 cells, PC9-derived cytoplasmic vesicles, and exosomes, displaying a high correlation coefficient of protein intensity across three measurements; 6d shows a Venn diagram displaying the number of differentially expressed proteins in PC9 cells, DIMV, and EVs; 6e shows a heatmap comparing PC9 cells and PC9-derived cytoplasmic vesicles, assigning the top 40 proteins with low and high expression in cytoplasmic vesicles to different cellular components; 6f shows the relative expression levels of mitochondrial and nuclear-related proteins in PC9 cells, cytoplasmic vesicles, and exosomes analyzed by Western blot; 6g shows the Western blot analysis of... Blot analysis of exosome characteristic marker proteins in PC9 cells, PC9 cell cytoplasmic vesicles, and exosomes; 6h: Western blot analysis comparing PC9 cells, PC9 cell cytoplasmic vesicles, and exosomes with apoptotic bodies (Apo-); 6i: Comparison of fragment length and residual amount of RNA in PC9 cells, PC9 cell cytoplasmic vesicles, and exosomes; 6j: Comparison of genomic DNA fragment length and residual amount in PC9 cells, PC9 cell cytoplasmic vesicles, and exosomes. Figure 6 k represents the β-actin protein in B16 cell cells, B16 cell cytoplasmic vesicles (DIMV), and exosomes (Ex-DIMV) cells analyzed by Western blot.

[0115] Figure 7 Microscopic images of cytoplasmic vesicles prepared from different tumor cells in this example, including cytoplasmic vesicle size and flow cytometry images: Figure 7 a represents the cytoplasm formed by PC9 cells, with a scale bar of 100 μm; Figure 7 b represents the size of the PC9-derived cytoplasmic vesicles; Figure 7 c is a flow cytometry image of PC9-derived cytoplasmic vesicles and PC9 cells; Figure 7d represents the cytoplasm formed by MCF7 cells, with a scale bar of 100 μm; Figure 7 e represents the size of the cytoplasmic vesicles derived from PC9; Figure 7 f is a flow cytometry image comparing cytoplasmic vesicles derived from MCF7 with MCF cells.

[0116] Figure 8 Fluorescence microscopy and electron microscopy images of cytoplasmic vesicles prepared for PC9 cells: 8a is a route diagram for preparing fluorescently labeled cytoplasmic vesicle membranes, 8b is a confocal image of fluorescently labeled cytoplasmic vesicle membranes, 8c is a transmission electron microscopy image of cytoplasms, and 8d is a scanning electron microscopy image of cytoplasmic vesicles.

[0117] Figure 9 The following diagrams illustrate the preparation process and results of extracellular nanoscale cytoplasmic vesicles: 9a shows a schematic diagram of the process of producing exosome analogs by extruding cytoplasmic vesicles; 9b shows TEM images of the morphology and size of exosomes; 9c shows TEM images of the morphology and size of exosomes extruded from cytoplasmic vesicles; 9d shows the particle size distribution evaluated by NTA, indicating that EVs-like (Squeezed DIMV) and EVs are basically the same size (50–200 nm). Figure 9 e. Compare the number of exosomes and exosome-like cells produced from the same number of parent cells; Figure 9 f compares the protein content of exosomes and exosomes produced from the same number of maternal cells. Figure 9 g compares the relative protein content ratio of exosomes and cytoplasmic vesicles produced from the same number of parent cells.

[0118] Figure 10 Figures showing the results of using cytoplasmic vesicles as drug carriers: 10a is a schematic diagram of a nucleic acid delivery system in which cytoplasmic vesicles are loaded with DNA rich in different bases labeled with CY5.5 and cholesterol (CY5.5-chole-DNA); 10b, 10c, 10d, and 10e are confocal images of cytoplasmic vesicles enriched with A / T / C / G-cy5.5-chole-DNA and statistical analysis of the gray values ​​of the underlined portions; 10f is a flow cytometry analysis of cytoplasmic vesicles enriched with A / T / C / G-cy5.5-chole-DNA respectively; 10g is a schematic diagram of cytoplasmic vesicles loaded with aptamers containing random base sequences labeled with CY5; 10h is a confocal image of DIMV-CY5-aptamers loaded with confocal images and statistical analysis of the gray values ​​of the underlined positions; 10i is a flow cytometry analysis of cytoplasmic vesicles loaded with DIMV-CY5-aptamers.

[0119] Figure 11Figure 11a shows the protein expression of cytoplasmic vesicles prepared from three types of modified cells: 11a shows cytoplasmic vesicles containing GFP on the membrane prepared using gene editing technology; 11b shows flow cytometry analysis of cytoplasmic vesicles and cytoplasmic vesicles expressing GFP on the membrane; 11c shows a confocal image of cytoplasmic vesicles expressing GFP on the membrane; 11d shows... Figure 11 c. Gray value statistics of the underlined part; 11e. Process of preparing GFP-expressing cells from cytoplasm to express GFP inside cytoplasmic vesicles; 11f. Flow cytometry analysis of cytoplasmic vesicles and cytoplasmic vesicles expressing GFP on the membrane; 11g. Confocal image of GFP expression inside cytoplasmic vesicles; 11h. Figure 11 g shows the grayscale statistical values ​​of the underlined portion in the figure; 11i is a schematic diagram of the preparation of cytoplasmic vesicles expressing red fluorescent protein in the cytoplasm and GFP on the membrane; 11j shows the analysis of cytoplasmic vesicles expressing red fluorescent protein inside the cytoplasm and GFP on the membrane using flow cytometry; 11k is a confocal image of cytoplasmic vesicles expressing GFP on the membrane and red fluorescent protein inside; 11l is... Figure 11 The grayscale statistics of the underlined area in the k-graph.

[0120] Figure 12 Figures showing the results of using cytoplasmic vesicles as a tumor vaccine: 12a is a schematic diagram showing the immunotherapy steps used to evaluate B16 tumor-bearing C57BL mice; 12b is a representative optical image of tumor-bearing C57BL mice after three immunizations: injection of DPBS, subcutaneous injection of cytoplasmic vesicles, and tail vein injection of cytoplasmic vesicles; 12c is the tumor volume growth curve of C57BL tumor-bearing mice treated with different immunization methods; 12d is the survival rate of tumor-bearing C57BL mice after various treatments; 12e is the weight change of C57BL mice in different treatment groups.

[0121] Figure 13 A roadmap for the rapid, large-scale preparation of sterile cytoplasmic vesicles from cytoplasm.

[0122] Figure 14 To magnify the microscopic images of the cytoplasm in the preparation system. Detailed Implementation

[0123] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0124] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0125] Example 1: Construction of cytoplasmic vesicles (DIMV) using a dehydration induction method

[0126] HEK293T, MCF7, MKN74, PC9, A549, H460, and B16 cells were seeded into 10cm culture dishes. Once the cells reached a suitable density, the culture medium was removed, and the cells were washed 1-2 times with DPBS to remove dead cells and culture medium components. Subsequently, the cells were dehydrated and incubated in a clean bench for 10 minutes. After observing significant shrinkage of cancer cells under a microscope, 5mL of DPBS was slowly added along the wall of the culture dish. After incubation for approximately 2 minutes, numerous dehydration-induced vesicles were observed detaching from the cells under a microscope (Olympus, CKX53). The results are as follows: Figure 2 As shown, after dehydration at room temperature and re-addition of DPBS, numerous cytoplasmic vesicles produced by each cell type were quickly observed under a microscope. These experimental results demonstrate that the dehydration-induced method for preparing cytoplasmic vesicles is suitable for various cell types and exhibits good induction efficiency.

[0127] Example 2: Optimization of conditions for preparing cytoplasmic vesicles by dehydration induction method

[0128] PC9 cells were seeded into six-well plates. When the cell confluence reached 70%–80%, the culture medium was removed, and the cells were washed twice with DPBS. Subsequently, the cells were dehydrated and incubated in a clean bench for 10 minutes. After observing significant shrinkage of cancer cells under a microscope, 5 mL of phosphate-buffered saline at different pH values ​​(pH 7.1, pH 7.5, pH 8.0, pH 8.5) was added, and the cells were incubated for 30 minutes. Finally, the cells were observed under a microscope (Olympus, CKX53). Figure 3 As shown, cytoplasmic vesicles can be generated at pH 7.1-8.5. However, the number of cytoplasmic vesicles decreases as the pH of the incubation solution increases after dehydration.

[0129] PC9 cells were seeded into six-well plates. When the cell confluence reached 70%–80%, the culture medium was removed, and the cells were washed twice with DPBS. Subsequently, the cells were dehydrated and incubated in a clean bench for different times (10, 20, 30, 40 minutes). Then, 5 mL of DPBS was added, and the cells were incubated for 10 minutes. Finally, the cells were observed under a microscope (Olympus, CKX53). Figure 4 As shown, cytoplasmic vesicles can be generated during dehydration at room temperature for different times. The yield of cytoplasmic vesicles decreases with increasing cell drying time. Room temperature dehydration in the range of 10-30 minutes can achieve better results.

[0130] PC9 cells were seeded into six-well plates. When the cell confluence reached 70%–80%, the culture medium was removed, and the cells were washed twice with DPBS. Subsequently, the cells were dehydrated and incubated in a clean bench for 10 minutes. After observing significant shrinkage of cancer cells under a microscope, 5 mL of DPBS was added, and the cells were incubated for different times before observation under a microscope (Olympus, CKX53). Cytoplasmic vesicles were generated after 2 minutes of incubation with DPBS following dehydration at room temperature. Figure 5 As shown, the production of cytoplasmic vesicles increases with increasing DPBS incubation time.

[0131] Different cell types (see Table 1) were seeded into culture dishes. When the cell confluence reached 70%–80%, the culture medium was removed, and the cells were washed twice with DPBS. Subsequently, various cell types were dehydrated for different durations under different conditions (see Table 1). After each cell type was observed to shrink under a microscope, 5 mL of DPBS was added, and the cells were incubated for 2–10 minutes before observation and recording under a microscope (Olympus, CKX53). As shown in Table 1, different cell types could produce cytoplasmic vesicles after dehydration induction under different dehydration conditions. In Table 1, the heating temperatures ranged from 30°C to 80°C.

[0132] Table 1. Vesicle production in different cell types

[0133]

[0134] PC9 cells were seeded into culture dishes. When the cell confluence reached 70%–80%, the culture medium was removed, and the cells were washed twice with DPBS. Subsequently, the cells were dehydrated, and the shrinkage of each cell type was observed under a microscope. Then, 5 mL of different incubation solutions (see Table 2) were added, and the cells were incubated for 2 minutes before observation and recording under a microscope (Olympus, CKX53). As shown in Table 2, cytoplasmic vesicles were generated after dehydration induction using different incubation solutions. The heating temperatures in Table 2 ranged from 30°C to 80°C.

[0135] Table 2. Vesicle production in PC9 cells under different buffer solutions.

[0136]

[0137] Example 3: Cytoplasmic vesicles prepared by dehydration induction method are a novel type of cell-like vesicle.

[0138] Compared to exosomes (EVs), DIMVs have many advantages in preparation and extraction. To further determine their compositional differences, this invention compared the protein abundance of PC9 cells, DIMVs produced by PC9 cell dehydration induction, and EVs secreted by PC9 cells (prepared by conventional size exclusion chromatography, exhibiting good exosome purity). Various protein components of PC9 cells, DIMVs, and EVs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 6 a). The SDS-PAGE results show a very distinct band above 70 kDa for EVs, while the bands for DIMV and Cells show no significant difference. Further label-free quantitative mass spectrometry analysis yielded comprehensive differential protein profiles for PC9 cells, DIMV, and EVs. Consistency analysis indicated that DIMV is an intermediate product between EVs and Cells. Figure 6 (b, c) Of the 3088 proteins identified, 431 were PC9 cell-specific, 123 were DIMV cell-specific, and 96 were EV cell-specific, totaling 463 proteins. DIMV cells exhibited a greater diversity of proteins than EV cells. Figure 6 d). Next, Western blot analysis was performed on Cell, DIMV, and EV. Figure 6 g) The results showed that DIMV contains both characteristic proteins of EV and proteins that are generally not considered to be enriched in EV, such as calnexin; indicating that DIMV has rich protein characteristics and possesses characteristics of both cells and EVs. Therefore, the 123 enriched proteins unique to DIMV, especially the 30 organelle constituent proteins with the greatest differences, such as UTP20, TMEM109, ZW10, PVR, CD58, MRPS7, CHP1, RAB27B, STEAP4, JAGN1, DBT, SLC39A7, PACSIN3, SCAMP1, NDUFB4, MRPS17, PSMB6, ANO6, SYPL1, TRPC5 / 4, POLR2E, C7orf50, SEH1L, TM9SF4, MXRA7, BDH1, GTPBP1, MKNK1, MGST3, and ALG5, can serve as markers for DIMV.

[0139] Subsequently, statistical analysis was performed on the top 40 proteins with high and low expression in cells and DIMV to determine which cellular regions they belonged to. For example... Figure 6e. Compared to cells, the proteins most significantly underexpressed in DIMVs originate from the cell membrane and nucleus, while the proteins highly expressed originate from multiple organelles, indicating that multiple organelles are enriched in DIMVs. To confirm whether DIMVs contain important organelles such as mitochondria and the nucleus, further detection of marker proteins for each organelle was performed. Figure 6 f) The results showed that no obvious nuclear markers such as histone H3 were detected in the DIMV, but mitochondrial markers and important components of cytoskeleton proteins were detected, thus further confirming that the DIMV contains mitochondria and other related organelles and the cytoskeleton. Furthermore, to verify the difference between this cytoplasmic vesicle and the apoptotic body, Western blot analysis was subsequently performed on the apoptotic body, cell, DIMV, and EV. Figure 6 h), the results showed that DIMV, like cells and EVs, does not possess apoptotic cell characteristic proteins and is different from apoptotic bodies.

[0140] The total DNA and RNA quantity and length were analyzed in samples with a Cell, DIMV, and EV protein content of 85 μg. Figure 6 i, j). The results showed that DIMV and EV contained almost no large fragments of genomic DNA, while both DIMV and EV were enriched with a variety of RNAs, binding... Figure 6 e indicates that the cytoplasmic vesicles do not contain a nucleus.

[0141] In summary, the results indicate that DIMVs share similarities with cells and exosomes, but also possess unique properties. They contain several important non-nuclear organelles and can be considered as a novel type of cellular vesicle, without exhibiting the low production rates of other cellular vesicles such as exosomes.

[0142] Based on the differences between DIMVs prepared from PC9 cells, original cells, and EVs, differentially expressed proteins in DIMVs (including the aforementioned enriched proteins, markers of contained organelles, and cytoskeletal proteins) can serve as markers for rapid identification of DIMVs, such as β-actin protein. Therefore, Western blotting was performed on DIMVs induced by dehydration in B16 cells. After standardized quantitative loading using the BCA method, β-actin protein was easily detected in both DIMV and cell samples, distinguishing them from EVs (…). Figure 6 The results showed that B16 cell DIMV prepared by dehydration also contained the differentially expressed protein β-actin.

[0143] Example 4: Particle size analysis of cytoplasmic vesicles prepared by dehydration induction method

[0144] Cytoplasmic vesicles were prepared according to the method in Example 1. PC9 and MCF7 cells were seeded into culture dishes (10cm dish). When the cells proliferated to 80%–90% of the total volume of the culture dish, the culture medium was removed, and the cells were gently washed with DPBS to remove dead cells and culture medium components. Subsequently, the cells were dehydrated and incubated in a clean bench for 10 minutes. After observing obvious shrinkage of cancer cells under a microscope, 5mL of DPBS was added, followed by incubation. Afterward, a large number of cytoplasmic vesicles could be observed under a microscope (Olympus, CKX53). Figure 7 a) and 7d), collect the supernatant from the incubation, centrifuge at 500g for 15 minutes, and repeat 1-2 times to remove the precipitate and dead cells. Finally, further centrifuge and concentrate the supernatant to obtain a high concentration of cytoplasmic vesicles. Their size was determined using a nanoparticle tracking analyzer (Particle Metrix, ZetaView) and a nanoflow cytometer (Fuliu U30). The results showed that the obtained cytoplasmic vesicles were mainly micron-sized vesicles smaller than the original cells (e.g., ...). Figure 7 b, 7c, 7e, 7f).

[0145] PC9 cells were seeded into 10cm culture dishes, and the cell membranes were stained with the lipophilic dye Did. Cytoplasmic vesicles were then prepared according to the method described in Example 1. These vesicles were observed and analyzed using a single-photon confocal microscope (Nikon, A1HD25), a flow cytometer (Beckman Coulter, CytoFLEX LX), a transmission electron microscope (Jeol Ltd., Tokyo, Japan), and a scanning electron microscope (Jeol Ltd., Tokyo, Japan). The results are as follows: Figure 8 As shown, this further demonstrates that the membrane of cytoplasmic vesicles prepared by the dehydration-induced method originates from the cell membrane, and the particle size of cytoplasmic vesicles is smaller than that of cells, ranging from 0 to 40 μm. The large size characteristic of DIMV (especially sizes greater than 5 μm, such as greater than 10 μm) is different from the size of existing EVs (30-100 nm) and apoptotic bodies (50-5000 nm), and can be considered as one of the morphological characteristics of DIMV.

[0146] Example 5: Nanoparticle-sized cytoplasmic vesicles prepared by dehydration induction method

[0147] PC9 cell cytoplasmic vesicles were prepared according to the method in Example 1. After centrifugation, purification, and concentration, the prepared DIMV suspension was loaded into a syringe using a LiposoFast LF-50 (Avestin, York, UK). The suspension was then passed sequentially through a series of nanopores, specifically 1 μm, 0.4 μm, and 0.1 μm polycarbonate membrane filters (Whatman). Figure 9As shown in Figure a, cytoplasmic vesicles of corresponding sizes are obtained by passing the material through a polycarbonate membrane filter of a certain pore size. Finally, by collecting the extrusion product from a 0.1 μm filter membrane, exosome-like vesicles exceeding 100 nanometers in size are obtained. The morphology of the exosomes and the extracellular nanoscale cytoplasmic vesicles after extrusion through the filter membrane were observed and analyzed using transmission electron microscopy (Jeol Ltd., Tokyo, Japan). Figure 9 b) and c) They have uniform size. Exosomes and exosome analogues were analyzed using a nanoparticle tracking analyzer (Particle Metrix, ZetaView), such as... Figure 9 As shown in d, both have the same size.

[0148] like Figure 9 As shown in e, the number of extracellular nanoscale cytoplasmic vesicles prepared by this method, as determined by nanoflow cytometry, is 1.415E+11 per million cells, which is more than 50 times the yield of exosomes secreted by the same number of cells (2.7E+9) [International journal of molecular sciences 2020, 21(18), 6466]. Figure 9 As shown in f, the protein yield of extracellular nanoscale cytoplasmic vesicles and exosomes generated by extrusion in the same culture system was analyzed by Western blot. It was found that the total protein content of extracellular nanoscale cytoplasmic vesicles generated per million cells was 15.905 μg, while the total protein content of EVs generated by the same number of cells was only 0.098 μg. The protein yield of extracellular nanoscale cytoplasmic vesicles was more than 160 times that of exosomes for the same number of cells. Furthermore, considering the loss of proteins contained in DIMVs during the extrusion process, such as… Figure 9 As shown in g, the DIMV protein produced by the same cells was compared with that of exosomes again by Western blot. It was found that the total protein content of DIMV produced per million cells was 57.434 μg. In terms of total protein, the DIMV protein yield of the same number of cells was more than 580 times that of exosomes.

[0149] Example 6: Cytoplasmic vesicles prepared by dehydration induction method were used as drug carriers

[0150] DIMV has a membrane composition similar to that of a cell membrane, exhibiting excellent biocompatibility and targeting properties. To explore the drug-loading capacity of DIMV, this invention synthesized four cholesterol-CY55-labeled DNA strands enriched with different bases (ATCG) (see Table 3) to test whether DIMV has different loading capacities for DNA enriched with different bases. Then, DIMV was incubated with A-Chol-cy5.5, C-Chol-cy5.5, T-Chol-cy5.5, G-Chol-cy5.5, and Random DNA aptamer at room temperature for a specified duration, followed by two centrifugation and washing. The results were observed and photographed using a Nikon single-photon confocal microscope and analyzed by Beckman flow cytometry. The results are as follows: Figure 10 As shown in images a, 10b, 10c, 10d, 10e, 10g, and 10h, the confocal results demonstrate that cholesterol-CY5.5 modified DNA chains and Random DNA aptamer nucleic acid molecules can insert into the DIMV surface or cross the DIMV membrane to enter the cytoplasm, achieving DNA loading. Flow cytometry analysis of DIMVs loaded with CY5 aptamers (e.g., ...) was performed. Figure 10 The results (f, 10i) showed strong fluorescence intensity, indicating that DIMV can effectively load aptamers.

[0151] Table 3. Four DNA strands labeled with different bases (ATCG) of cholesterol-CY5.5

[0152]

[0153] Example 7: Dehydration-induced modification of cells to form DIMV with novel biological properties

[0154] Three cell types were constructed (three plasmids were constructed: GFP (green fluorescent protein) on the membrane, GFP in the cytoplasm, and GFP on the membrane with mScara-L (red fluorescent protein) in the cytoplasm; the plasmids were transfected into HEK293T cells using a lip3000 (ThermoFisher L3000075) to obtain the corresponding fluorescent protein expression proteins), namely: 1) GFP highly expressed on the membrane (cellular GFP... on ), 2) High expression of GFP in the cytoplasm (cellular GFP) in ), and 3) GFP is highly expressed on the membrane and mScara-L red fluorescent protein is present in the cytoplasm (mScara-L (cellular GFP) on -mScara-L in ), and cytoplasmic vesicles (DIMVs) were prepared according to the dehydration induction protocol. Analysis and observation were performed using a Beckman flow cytometer and a Nikon single-photon confocal microscope. The results are as follows: Figure 11As shown, the DIMV carries eGFP proteins expressed by the parent cells, and the localization of these proteins is completely consistent with that of the parent cells. This example demonstrates that, through bioengineering, DIMVs can possess many new properties and functional applications.

[0155] Example 8: Cytoplasmic vesicles prepared by dehydration induction method used as tumor vaccines

[0156] Cytoplasmic vesicles were prepared according to the method in Example 1. B16 cells were seeded into culture dishes. When the cells proliferated to 80%–90% of the total volume of the culture dish, the culture medium was removed, and the cells were gently washed with DPBS to remove dead cells and culture medium components. Subsequently, the cells were dehydrated and placed in a clean bench for 10 minutes. After observing obvious shrinkage of cancer cells under a microscope, 5 mL of DPBS was added, and the cells were incubated for 1 hour. After observing a large number of cytoplasmic vesicles under a microscope (Olympus, CKX53), the supernatant was collected, centrifuged at 500g for 15 minutes, and repeated 1–2 times to remove the precipitate of dead cells. Finally, the supernatant was further centrifuged and concentrated to obtain a high concentration of cytoplasmic vesicles.

[0157] A tumor vaccine prevention model was established in female C57 mice (4-6 weeks old) using tail vein and subcutaneous injection. Mice were randomly divided into three groups: (1) control group, injected with DPBS; (2) experimental group 1, injected subcutaneously with DIMV; (3) experimental group 2, injected via tail vein. B16 cell-derived DIMV immunovaccine (dose = protein 2 μg, volume = 0.1 mL) was injected via tail vein and subcutaneously on days -1, -3, and -5, respectively. The control group received intravenous injection of DPBS at the same time points. On day 0, B16 cells (10... 6 A subcutaneous tumor model of B16 mice was established, and tumor size (calculated using the formula: major diameter * minor diameter * minor diameter / 2) and body weight were recorded 7 days after B16 cell inoculation. Figure 12 a).

[0158] The experimental results showed that, on day 9 post-tumor implantation, compared with the DPBS control group, the groups treated with three doses of the B16 tumor-derived DIMV vaccine (Sub-immunity and IOCV-immunity) both exhibited significant inhibitory effects on B16 tumors. Figure 12 (b, 12c). Notably, the survival rate of the immunized mice was better than that of the control group ( Figure 12 d). Furthermore, throughout the entire in vivo experiment, the mouse body weight did not change significantly. Figure 12 e). This indicates that B16 tumor-derived DIMV has immunostimulatory capabilities and can serve as a candidate drug for inhibiting tumor growth and improving patient survival, with a good safety profile.

[0159] Example 9: A method for rapid large-scale preparation of sterile cytoplasmic vesicles based on dehydration induction.

[0160] like Figure 13 The obtained B16 cells were seeded into 150 times their volume of culture medium for expansion culture. When the cells proliferated to a high density (80%–90%), the culture medium was removed, and the cells were washed three times with DPBS. The cells were then dehydrated at room temperature, and then incubated with DPBS for about half an hour. The DPBS supernatant was collected, and a large number of cytoplasmic vesicles (such as…) were obtained by centrifugation and purification. Figure 14 The yield was increased by more than 100 times. These experimental results demonstrate that the dehydration-induced method for preparing cytoplasmic vesicles can achieve excellent linear scale-up and is suitable for large-scale production.

[0161] Example 10: Other dehydration methods used to induce the production of cytoplasmic vesicles

[0162] HEK293T and PC9 cells were proliferated to 80% in 10cm culture dishes. The culture medium was then discarded, and the cells were washed once with DPBS. Subsequently, they were dehydrated for 10 minutes each using three different methods: 30% sucrose, 8% sodium chloride, and 30% sucrose and 8% sodium chloride, respectively. After removing the supernatant, the cells were quickly washed once with DPBS, and then cultured in DPBS for 20 minutes. The production of cytoplasmic vesicles was observed under a microscope (Olympus, CKX53).

[0163] Table 4

[0164]

[0165] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing extracellular cytoplasmic vesicles, the method comprising the following steps: 1) Seed the cells into a culture dish and culture them until the cells have proliferated to 70%~90% of the total volume of the culture dish or until the cell density is 70%~90%; 2) Remove the culture medium; add buffer solution to rinse and remove dead cells; 3) Dehydration treatment: Without culture medium or buffer, incubate the cells at 10 to 40°C for 5 to 30 minutes; 4) After the cells shrink, add buffer solution; 5) Incubate for 2-60 minutes; 6) Collect the supernatant and centrifuge to purify it; 7) The supernatant was further centrifuged and concentrated to obtain cytoplasmic vesicles; The cells were selected from PC9 cells, MCF7 cells, A549 cells, H460 cells, HeLa cells, MKN74 cells, H22 cells, CEM cells, HEK293T cells, B16 cells, KYSE150 cells, and A2780 cells. The buffer solution is selected from DPBS buffer, the pH of the buffer solution is 7.4, and the ionic strength of the buffer solution is 10mM~3M; In steps 6) and 7), the centrifugation conditions are 500g for 15 to 30 minutes.

2. The production method according to claim 1, characterized by, In step 1), the cell culture conditions are: cultured at 37°C and 5% CO2. And / or, in step 1), passage once every 3 to 4 days, depending on cell density; And / or, in step 1), the culture medium is selected from DMEM medium supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin, or RPMI 1640 medium containing 10% FBS and 1% PS; And / or, in step 4), the amount of buffer solution added is 5 mL.

3. Extracellular cytoplasmic vesicles prepared by the preparation method according to claim 1 or 2; The extracellular cytoplasmic vesicles are micron-sized vesicles; The extracellular cytoplasmic vesicles are 5-30 μm in size; Extracellular cytoplasmic vesicles contain one, two, or more of the following marker proteins: UTP20, TMEM109, ZW10, PVR, CD58, MRPS7, CHP1, RAB27B, STEAP4, JAGN1, DBT, SLC39A7, PACSIN3, SCAMP1, NDUFB4, MRPS17, PSMB6, ANO6, SYPL1, TRPC5 / 4, POLR2E, C7orf50, SEH1L, TM9SF4, MXRA7, BDH1, GTPBP1, MKNK1, MGST3, and ALG5.

4. The extracellular cytosolic vesicle of claim 3, wherein, The cells were selected from B16 cells.

5. The extracellular cytosolic vesicle of claim 3, wherein, Extracellular cytoplasmic vesicles contain calnexin and / or β-actin proteins.

6. The extracellular cytosolic vesicle of claim 3, wherein, Extracellular cytoplasmic vesicles do not contain a nucleus.

7. The extracellular cytosolic vesicle of claim 3, wherein, Extracellular cytoplasmic vesicles do not contain apoptotic cell characteristic proteins.

8. A method for preparing extracellular nanoscale cytoplasmic vesicles, comprising the following steps: (i) Extracellular cytoplasmic vesicles are prepared using the preparation method described in claim 1 or 2; (ii) Using a liposome extruder, the suspension of extracellular cytoplasmic vesicles is loaded into a syringe, and then the suspension is passed through filters with nanopore sizes of 1 μm, 0.4 μm and 0.1 μm in sequence. The product of extrusion through the 0.1 μm filter membrane is collected to obtain extracellular nanoscale cytoplasmic vesicles.

9. The production method according to claim 8, characterized by, The filter is a polycarbonate membrane filter.

10. Extracellular nanoscale cytoplasmic vesicles prepared by the preparation method according to claim 8 or 9.

11. The use of the extracellular cytoplasmic vesicles according to any one of claims 3-7 in the preparation of drug carriers.

12. The use of the extracellular cytoplasmic vesicles according to claim 4 in the preparation of a tumor vaccine for the prevention of subcutaneous tumors.

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