Preparation method and application of cytoplast vesicles

Abstract

The invention discloses a preparation method and application of cytoplast vesicles. The preparation method is realized through a dehydration method. After the cells are dehydrated and shrink, a culture medium is added for culture, a large number of vesicles with organelles such as ribosomes and mitochondria and cell matrixes can be rapidly secreted in a short time, and the vesicles can be centrifugally collected and further used for later processing. The method is convenient and rapid, is not limited by cell types, is high in yield, does not involve the use of toxic substances, and can change the functions of the cytoplast vesicles by transforming parent cells. The prepared cytoplast vesicles can be used for disease diagnosis, clinical medication and the like, have high application value and are suitable for application and popularization.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method of cytoplasmic vesicles and applications thereof. Background Art

[0002] Cell-like vesicles are a biomimetic biotechnology that is mainly used to prepare micro or nano vesicles with cell membrane-like structures. Currently available cell-like vesicles mainly include vesicles prepared from cell membranes and extracellular vesicles secreted by cells. Cell-like vesicles have been shown to have many advantages in the preparation of drugs and diagnosis of diseases, such as good biosafety, targeting of membrane proteins, and multiple drug loading, which gives cell-like vesicles broad application prospects. However, there are still some challenges in the large-scale preparation and multifunctionalization of cell-like vesicles. In particular, the number of extracellular vesicles secreted by cells is extremely small, far less than the actual demand. Studies have shown that in the case of in vitro culture, 10 6 The amount of extracellular vesicles secreted by cells per day is less than the microgram level, which has caused a research dilemma for extracellular vesicles (exosomes) secreted by cells in the fields of disease diagnosis and treatment. To overcome this shortcoming, the demand for large-scale production of cell-derived micron and nano vesicles has emerged. While the induced secretion vesicles have similar functions to exosomes, the preparation process of such induced secretion vesicles has relatively simple steps, and the amount of vesicle extraction is also relatively large. The most commonly used 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], and also includes physical methods, such as light-induced methods to make cells produce nano vesicles [Nature Communications, 2022, 13 (1): 6534]. These means can obtain a relatively sufficient number of vesicles with membrane components close to those of mother cells [ACS Appl. Mater. Interfaces 2021, 13, 55767-55779]. However, existing technologies for inducing cell-secreted extracellular vesicles are either time-consuming and have little improvement in yield, or introduce reagents with biosafety risks. Therefore, the development of new, efficient and safe methods for preparing cell-like vesicles is urgent.

[0003] In addition, the existing applications of cell-like vesicles are all based on the cell membrane structure. By loading or modifying small molecule therapeutic agents or diagnostic agents in the vesicles or on the cell membrane, the cell-like vesicle-derived small molecule therapeutic agents or diagnostic agents can be given excellent biocompatibility and targeting. However, such a cell-like vesicle loading method is still very difficult for biological macromolecules (proteins, mRNA, organelles), and the loading efficiency is very low, which also indirectly reflects the limitations of the expandable functions of cell-like vesicles. The delivery of biological macromolecules is an important way for biological treatment and diagnosis. Although there are some other ways to load and deliver biological macromolecules such as protein drugs and mRNA drugs in vivo, for organelles of larger size (hundreds of nanometers to more than ten microns) and fragility, the strategy of loading or delivering organelles has hardly been reported. Therefore, the construction of cell-like vesicles containing biological macromolecules and structures (especially organelles) is of great significance to the development of biological treatment and diagnosis, and has potential application value. Summary of the invention

[0004] The purpose of the present invention is to provide an efficient preparation method of cytoplasmic vesicles and its application. The preparation method of the present invention is convenient, fast, low-cost, does not involve toxic substances, and is suitable for popularization and application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

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

[0007] In some embodiments, 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 comprises the following steps: dehydrating the cells until the cells shrink (for example, cell shrinkage is observed under a microscope), thereby inducing the cells to generate vesicles.

[0009] In some embodiments, the dehydration treatment is selected from any one of the following methods:

[0010] I) incubating the cells at a certain temperature for a period of time without culture medium or buffer; for example, incubating the cells at a temperature of -80°C to 80°C for 1 second to 60 minutes;

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

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

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

[0014] In some embodiments, the dehydration treatment method I) can specifically select any of the following treatment methods:

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

[0016] ii) incubating 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, or 30 seconds, without culture medium or buffer;

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

[0018] In some embodiments, the room temperature in i) is 10°C to 30°C, such as 18°C ​​to 28°C, 18°C ​​to 25°C.

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

[0020] In some embodiments, the below room temperature condition in iii) is below 10°C, such as -100°C to 10°C, -80°C to 0°C.

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

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

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

[0024] Obtaining cells, expanding cell culture, dehydrating cells, culturing (e.g., perfusion culture), and collecting cytoplasmic vesicles.

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

[0026] In some embodiments, the cell expansion culture is specifically: inoculating the cells into a culture dish, culturing until the cells proliferate to 70% to 90% of the entire culture dish or culturing until the cell density is 70% to 90%. In some embodiments, when the dehydration treatment method is selected from I), the preparation method comprises the following steps:

[0027] 1) Inoculating the cells into a culture dish and culturing them until the cells proliferate to 70% to 90% of the entire culture dish or until the cell density reaches 70% to 90%;

[0028] 2) removing the culture medium; optionally adding a buffer to wash and remove dead cells;

[0029] 3) Dehydration treatment: leaving the cells at a certain temperature for a period of time;

[0030] 4) After the cells shrink, add buffer;

[0031] 5) Incubate; optionally tap the culture dish (to accelerate the detachment process of the vesicles);

[0032] 6) Collect the supernatant and purify by centrifugation;

[0033] 7) The supernatant is further concentrated by centrifugation to obtain cytoplasmic vesicles.

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

[0035] In some embodiments, in step 1), the cell culture conditions are: culture at 37° C. and 5% CO 2 .

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

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

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

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

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

[0041] In some embodiments, 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, blood cancer cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.

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

[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), and 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 is 10 mM to 10 M, preferably 10 mM to 3 M.

[0046] In some embodiments, 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 embodiments, in step 4), the amount of buffer added is 5 mL.

[0048] In some embodiments, in step 5), the detachment operation of the vesicles is performed after culturing for 2 min-60 min (e.g., 10 min-60 min), for example, culturing for 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min.

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

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

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

[0052] And / or, the high concentration 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 comprises the following steps:

[0054] (1) inoculating cells into a culture dish and culturing until the cells proliferate to 70% to 90% of the entire culture dish or until the cell density reaches 70% to 90%;

[0055] (2) removing the culture medium; optionally adding a buffer to wash and remove dead cells;

[0056] (3) Dehydration treatment: placing the cells in a high concentration of sodium chloride and / or sucrose solution for a period of time;

[0057] (4) after the cells shrink, remove the high concentration sodium chloride and / or sucrose solution (optionally use a buffer to wash the residual high concentration sodium chloride and / or sucrose solution), and add a buffer;

[0058] (5) Cultivating; optionally tapping the culture dish (which can accelerate the detachment process of the vesicles);

[0059] (6) collecting the supernatant and centrifuging for purification;

[0060] (7) The supernatant is further concentrated by centrifugation to obtain cytoplasmic vesicles.

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

[0062] In some embodiments, in step (1), the cell culture conditions are: culture at 37° C. and 5% CO 2 .

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

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

[0065] In some embodiments, in step (1), the cell density is cultured to 80% to 90% or 70% to 80%.

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

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

[0068] In some embodiments, in step (1), the cell is a cancer cell, 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, blood cancer cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.

[0069] In some embodiments, 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), and 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 DPBS buffer with a pH of 7.4.

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

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

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

[0075] In some embodiments, in step 5), the detachment operation of the vesicles is performed after culturing for 2 min-60 min (e.g., 10 min-60 min), for example, culturing for 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min.

[0076] In some embodiments, in step (6) and / or step (7), the centrifugation condition is 500 g for 15 min to 30 min.

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

[0078] In some embodiments, the cell is a cancer cell, such as a lung cancer cell (small cell lung cancer, non-small cell lung cancer), a breast cancer cell, a cervical cancer cell, a gastric cancer cell, a liver cancer cell, a blood cancer cell (leukemia cell, acute myeloid leukemia cell, acute lymphoblastic leukemia cell), an esophageal cancer cell, an ovarian cancer cell, etc.

[0079] In some embodiments, the cells are selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like; preferably PC9 cells and B16 cells.

[0080] In some embodiments, the extracellular cytoplasmic vesicles comprise calnexin protein and / or β-actin protein.

[0081] In some embodiments, the extracellular cytoplasmic vesicle does not contain a nucleus.

[0082] In some embodiments, the extracellular cytoplasmic vesicles do not comprise proteins characteristic of apoptotic cells.

[0083] In some embodiments, the extracellular cytoplasmic vesicles comprise 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, ALG5;

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

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

[0086] (i) preparing extracellular cytoplasmic vesicles using the above preparation method;

[0087] (ii) Using a liposome extruder and a nanopore filter, extracellular nanoscale cytoplasmic vesicles are obtained.

[0088] In some embodiments, step (ii) is specifically:

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

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

[0091] In some embodiments, the series of nanopore sizes is 1 μm, 0.4 μm, and 0.1 μm.

[0092] The present invention provides extracellular nanoscale cytoplasmic vesicles prepared by the above preparation method.

[0093] In some embodiments, the cell is a cancer cell, such as a lung cancer cell (small cell lung cancer, non-small cell lung cancer), a breast cancer cell, an ovarian cancer cell, an esophageal cancer cell, a cervical cancer cell, a gastric cancer cell, a liver cancer cell, a blood cancer cell (leukemia cell, acute myeloid leukemia cell, acute lymphoblastic leukemia cell), etc.

[0094] In some embodiments, the cell is selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like.

[0095] In some embodiments, the extracellular nanoscale cytoplasmic vesicles comprise calnexin protein and / or β-actin protein;

[0096] In some embodiments, the extracellular nanoscale cytoplasmic vesicle does not contain a nucleus;

[0097] In some embodiments, the extracellular nanoscale cytoplasmic vesicles do not contain apoptotic cell signature proteins;

[0098] In some embodiments, the extracellular nanoscale cytoplasmic vesicles comprise 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, ALG5.

[0099] The present invention also provides the use of the extracellular cytoplasmic vesicles or the extracellular nanoscale cytoplasmic vesicles in preparing drug carriers.

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

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

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

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

[0104] The present invention also provides a method for preparing the above-mentioned extracellular cytoplasmic vesicles, and the application of the above-mentioned method for preparing the extracellular nanoscale cytoplasmic vesicles in the preparation of drug carriers, tumor vaccines, 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 of the present invention is convenient and fast, is not limited by cell types, has high yield, does not involve the use of toxic substances, and can also change the function of cytoplasmic vesicles by modifying parent cells. The cytoplasmic vesicles prepared by the present invention can be used for disease diagnosis and clinical medication, etc., have high application value, and are suitable for promotion and application.

[0107] Compared with cells, the cytoplasmic vesicles prepared by the present invention have similar properties to exosomes and can be used as an exosome analog for disease diagnosis and drug preparation. At the same time, the cytoplasmic vesicles contain important organelles such as mitochondria and cell nuclei, which can be used for organelle supplementation therapy or for mRNA expression or protein secretion.

[0108] This method is applicable to a variety of cell types, and both suspension cells and adherent cells have good yields. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0112] Figure 4 The effect of different cell drying time on the yield of induced cytoplasmic vesicles.

[0113] Figure 5 This is the effect of the length of time of adding DPBS on the yield of induced cytoplasmic vesicles.

[0114] Figure 6 Figure 6 is a comparison of the components of cytoplasmic vesicles, cells and EVs: 6a is the SDS-PAGE results of PC9 cell cells, and PC9-derived cytoplasmic vesicles DIMV and exosomes EV proteins; 6b is a heat map of protein intensity z-scores of PC9 cell cells, PC9-derived cytoplasmic vesicles DIMV and exosomes EVs after unsupervised hierarchical clustering (n=3 for each sample); 6c is a Pearson correlation heat map of PC9 cells, PC9-derived cytoplasmic vesicles and exosomes, showing the high correlation coefficient of protein intensity measured three times; 6d is a Venn diagram showing the number of differentially expressed proteins in PC9 cells, DIMV and EVs; 6e is a comparison of PC9 cells and PC9 cell-derived cytoplasmic vesicles, and a heat map of the top 40 low- and high-expressed proteins in cytoplasmic vesicles belonging to each component of the cell; 6f is a Western blot analysis of the relative expression levels of mitochondrial and nuclear-related proteins in PC9 cells, cytoplasmic vesicles and exosomes; 6g is a Western blot analysis of the relative expression levels of mitochondrial and nuclear-related proteins in PC9 cells, cytoplasmic vesicles and exosomes; Blot analysis of exosome characteristic marker proteins in PC9 cells, PC9 cell cytoplasmic vesicles and exosomes; 6h is a Western blot analysis comparing the differences between PC9 cells, PC9 cell cytoplasmic vesicles and exosomes and apoptotic bodies Apo-; 6i is a comparison of the fragment length and residual amount of RNA in PC9 cells, PC9 cell cytoplasmic vesicles and exosomes; 6j is a comparison of the fragment length and residual amount of genomic DNA in PC9 cells, PC9 cell cytoplasmic vesicles and exosomes; Figure 6 k is Western blot analysis of β-actin protein in B16 cell Cell, B16 cell cytoplasmic vesicles DIMV and exosomes Ex-DIMV Cell.

[0115] Figure 7 The microscopic pictures of cytoplasmic vesicles prepared from different tumor cells in the examples, the size of cytoplasmic vesicles and the flow cytometric pictures of cytoplasmic vesicles: Figure 7 a shows the cytoplasmic bodies formed in PC9 cells, the scale bar is 100 μm; Figure 7 b is the size of PC9-derived cytoplasmic vesicles; Figure 7 c is the flow cytometry image of PC9-derived cytoplasmic vesicles and PC9 cells; Figure 7d shows the cytoplasmic bodies formed in MCF7 cells, the scale bar is 100 μm; Figure 7 e is the size of PC9-derived cytoplasmic vesicles; Figure 7 f is a flow cytometry image comparing cytoplasmic vesicles derived from MCF7 and MCF cells.

[0116] Figure 8 Fluorescence display and electron microscope images of cytoplasmic vesicles prepared from PC9 cells: 8a is a roadmap for preparing fluorescently labeled cytoplasmic vesicle membranes, 8b is a confocal image of fluorescently labeled cytoplasmic vesicle membranes, 8c is a transmission electron microscopic image of cytoplasmic bodies, and 8d is a scanning electron microscopic image of cytoplasmic vesicles.

[0117] Fig. 9 Figure 9 is a schematic diagram of the preparation process and results of extracellular nanoscale cytoplasmic vesicles: 9a is a schematic diagram of the process of producing exosome analogs by extruding cytoplasmic vesicles, 9b is a TEM image of the morphology and size of exosomes; 9c is a TEM image of the morphology and size of exosomes squeezed from cytoplasmic vesicles; 9d is the particle size distribution evaluated by NTA, indicating that the size of EVs-like (SqueezedDIMV) and EVs is basically the same (50-200nm); Fig. 9 e Comparison of the amount of exosomes and exosome-like vesicles produced from the same number of maternal cells; Fig. 9 f Comparison of the protein amounts contained in exosomes and exosome-like vesicles produced from the same number of maternal cells. Fig. 9 g Comparison of the relative protein amounts contained in exosomes and cytoplasmic vesicles produced from the same number of maternal cells.

[0118] Fig.10 The results of cytoplasmic vesicles as drug carriers are as follows: 10a is a schematic diagram of the nucleic acid delivery system in which cytoplasmic vesicles are loaded with DNA rich in CY5.5 and cholesterol-labeled different bases (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 the grayscale value statistics of the underlined parts; 10f is the flow cytometric analysis of cytoplasmic vesicles enriched with bases 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-aptamer loaded and the grayscale value statistics of the underlined position; 10i is the flow cytometric analysis of cytoplasmic vesicles loaded with DIMV-CY5-aptamer.

[0119] Fig.11The protein expression of cytoplasmic vesicles prepared from three types of modified cells: 11a is a cytoplasmic vesicle containing GFP on the membrane prepared using gene editing technology, 11b is a flow cytometry analysis of cytoplasmic vesicles and cytoplasmic vesicles expressing GFP on the membrane; 11c is a confocal image of cytoplasmic vesicles expressing GFP on the membrane; 11d is Fig.11 c is the gray value statistics of the underlined part; 11e is the process of preparing cytoplasmic vesicles expressing GFP in cells expressing GFP in the cytoplasm; 11f is the flow cytometry analysis of cytoplasmic vesicles and cytoplasmic vesicles expressing GFP on the membrane; 11g is the confocal image of cytoplasmic vesicles expressing GFP; 11h is Fig.11 g is the grayscale statistical value of the underlined part in the figure; 11i is a schematic diagram of preparing cytoplasmic vesicles expressing red fluorescent protein in the cytoplasm and GFP on the membrane; 11j is the analysis of cytoplasmic vesicles expressing red fluorescent protein inside cytoplasmic vesicles and GFP on the membrane by flow cytometry; 11k is a confocal image of cytoplasmic vesicles expressing GFP on the membrane and red fluorescent protein inside; 11l is Fig.11 The grayscale statistics of the underlined part in the k graph.

[0120] Fig.12 Figures 12a are the results of cytoplasmic vesicles as tumor vaccines: 12a is a schematic diagram showing the steps used to evaluate immunotherapy in B16 tumor-bearing C57BL mice; 12b is a representative optical image of tumor-bearing C57BL mice after three immunizations with 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] Fig.13 Roadmap for rapid and large-scale preparation of sterile cytosolic vesicles for cytosol.

[0122] Fig.14 The figure is a microscopic picture of the cytoplasmic body of the enlarged preparation system. DETAILED DESCRIPTION

[0123] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations 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 included in the scope that the present invention is intended to protect.

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

[0125] Example 1: Construction of dehydration-induced method for preparing cytoplasmic vesicles (DIMV)

[0126] HEK293T, MCF7, MKN74, PC9, A549, H460, B16 and other cells were inoculated into culture dishes (10 cm dish) respectively. When the cells proliferated to a good 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 left to stand in a clean bench for 10 minutes. After the cancer cells were observed to have obvious shrinkage under a microscope, 5 mL of DPBS was slowly added along the wall of the culture dish. After incubation for about 2 minutes, a large number of dehydration-induced vesicles were observed to detach from the cells under a microscope (Olympus, CKX53). The results are shown in FIG. Figure 2 As shown in the figure, after dehydration at room temperature and re-addition of DPBS, a large number of cytoplasmic vesicles produced by each cell type can be quickly seen under a microscope. The experimental results show that the dehydration induction method for preparing cytoplasmic vesicles is suitable for a variety of cells and has good induction efficiency.

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

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

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

[0130] PC9 cells were inoculated into a six-well plate. When the cell confluence reached 70% to 80%, the culture medium was removed and the cells were washed twice with DPBS. Subsequently, the cells were dehydrated and left to stand in a clean bench for 10 minutes. After the cancer cells were observed to have obvious shrinkage under a microscope, 5 mL of DPBS was added. After incubation with DPBS for different time periods, the cells were observed under a microscope (Olympus, CKX53). Cytoplasmic vesicles were generated after dehydration at room temperature and incubation with DPBS for 2 minutes. Figure 5 As shown, the yield of cytoplasmic vesicles increased with increasing DPBS incubation time.

[0131] Different cells (see Table 1) were inoculated into a culture dish. When the cell confluence reached 70% to 80%, the culture medium was removed and the cells were washed twice with DPBS. Subsequently, various cells were dehydrated for different periods of time under different conditions (see Table 1), and after each cell was observed to be dehydrated and shrunken under a microscope, 5 mL of DPBS was added, and after incubation for 2-10 minutes, it was observed and recorded under a microscope (Olympus, CKX53). As shown in Table 1, different cells can produce cytoplasmic vesicles after dehydration induction under different dehydration conditions. In Table 1, the heating temperature is 30°C to 80°C.

[0132] Table 1 Vesicle production in different cell types

[0133]

[0134] PC9 cells were inoculated into a culture dish. When the cell confluence reached 70% to 80%, the culture medium was removed and the cells were washed twice with DPBS. Subsequently, the cells were dehydrated and each cell was observed to be dehydrated and shrunken under a microscope. 5 mL of different incubation solutions (see Table 2) were added respectively. After incubation for 2 minutes, the cells were observed and recorded under a microscope (Olympus, CKX53). As shown in Table 2, after dehydration induction, cells can produce cytoplasmic vesicles using different incubation solutions. In Table 2, the heating temperature is 30°C to 80°C.

[0135] Table 2 Vesicle production of PC9 cells in different buffers

[0136]

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

[0138] Compared with exosomes (EVs), DIMVs have many advantages in preparation and extraction. To further determine their compositional differences, the present invention compared the protein abundance of PC9 cells, DIMVs produced by PC9 cell dehydration induction method, and EVs secreted by PC9 cells (prepared by conventional size exclusion chromatography, with better exosome purity). The various protein components of PC9 cells, DIMVs, and EVs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 6 a). From the results of SDS-PAGE, it can be seen that there is a very obvious band above the 70KDa position in the SDS-PAGE of EV, while there is no obvious difference between the bands of DIMV and Cell. Further, the comprehensive differential protein spectrum of PC9 cells, DIMV and EV was obtained by non-labeled quantitative mass spectrometry analysis. The consistency analysis results showed that DIMV is a product between EV and Cell ( Figure 6 b, c). Among the 3088 proteins identified, 431 were PC9 cell-specific proteins, 123 were DIMV-specific proteins, 96 were EV-specific proteins, and 463 proteins were shared by the three. Compared with EV, DIMV has more abundant protein types than EV ( Figure 6 d). Then, western blot analysis was performed on Cell, DIMV and EV ( Figure 6 g), the results showed that DIMV contained both characteristic proteins of EV and proteins that are generally considered not to be enriched in EV, such as calnexin; this indicates that DIMV has rich protein characteristics and has the characteristics of both cells and EVs. Therefore, the 123 DIMV-specific enriched proteins, especially the most different 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, ALG5 and other 30 organelle constituent proteins, can be used as markers for DIMV.

[0139] Then, the top 40 proteins with high and low expression in cells and DIMV were counted to analyze which part of the cell they belonged to. Figure 6e, Compared with cells, the proteins with significantly lower expression in DIMV come from the cell membrane and nucleus, while the proteins with higher expression come from multiple organelles, indicating that multiple organelles are enriched in DIMV. In order to confirm whether DIMV contains important organelles such as mitochondria and nucleus, the marker proteins of each organelle were further detected ( Figure 6 f), the results showed that no obvious nuclear markers such as histone H3 could be detected in DIMV, but mitochondrial markers and important components of cytoskeletal proteins could be detected, which once again proved that DIMV contained mitochondria and other related organelles and cytoskeleton. In addition, in order to verify the difference between the cytoplasmic vesicles and apoptotic bodies, western blot analysis was performed on apoptotic bodies, Cell, DIMV and EV ( Figure 6 h), the results showed that DIMV, like cells and EVs, did not possess the characteristic proteins of apoptotic cells and were different from apoptotic bodies.

[0140] The total DNA and RNA quantity and length were tested for samples containing 85 μg of Cell, DIMV and EV protein ( 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 large number of types of RNA. Figure 6 e shows that the cytoplasmic vesicles do not contain nuclei.

[0141] The above results show that DIMV has both similar and different properties with cells and exosomes. It has multiple important non-nuclear organelles and can be used as a new type of cellular vesicle. It does not have the problem of low production of cellular vesicles such as exosomes.

[0142] Based on the differences between DIMVs prepared from PC9 cells and original cells and EVs, differential proteins in DIMVs (including the above-mentioned enriched proteins, markers of internal organelles, and cytoskeletal proteins) can be used as markers for rapid identification of DIMVs, such as β-actin protein. Therefore, WB verification of β-actin in DIMVs induced by dehydration of B16 cells was performed. After unified quantitative loading by the BCA method, β-actin protein was easily detected in samples of DIMVs and cells, which is different from EVs ( Figure 6 k). The results showed that B16 cell DIMVs prepared by dehydration method also contained differentially expressed protein β-actin.

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

[0144] Cytoplasmic vesicles were prepared according to the method in Example 1. PC9 and MCF7 cells were inoculated into a culture dish (10 cm dish). When the cells proliferated to 80% to 90% of the entire 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 left to stand in a clean bench for 10 minutes. After the cancer cells were observed to have obvious shrinkage under a microscope, 5 mL of DPBS was added. After subsequent incubation, a large number of cytoplasmic vesicles were observed under a microscope (Olympus, CKX53) (e.g. Figure 7 a, 7d), collect the incubation supernatant, centrifuge at 500g for 15 minutes, and repeat 1-2 times to remove the precipitated dead cells, and finally further centrifuge and concentrate the supernatant to obtain high-concentration cytoplasmic vesicles. The size was measured by nanoparticle tracking analyzer (Particle Metrix, ZetaView) and nanoflow cytometry (Flow U30), and the results showed that the obtained cytoplasmic vesicles were mainly micron-sized vesicles smaller than the original cells (such as Figure 7 b, 7c, 7e, 7f).

[0145] PC9 cells were inoculated into a culture dish (10 cm dish) and the cell membrane was stained with the lipophilic dye Did. Cytoplasmic vesicles were prepared according to the method in Example 1. Single-photon confocal microscope (Nikon, A1HD25), flow cytometer (Beckman Coulter, CytoFLEX LX), transmission electron microscope (Jeol Ltd., Tokyo, Japan) and scanning electron microscope (Jeol Ltd., Tokyo, Japan) were used to observe and analyze them. The results are shown in Figure 2. Figure 8 As shown, it is further shown that the membrane of the cytoplasmic vesicles prepared by the dehydration induction method is derived from the cell membrane, and the particle size of the cytoplasmic vesicles is smaller than that of the cells, ranging from 0 to 40 μm. The large size of DIMV (especially the size greater than 5 μm, such as greater than 10 μm) is different from the existing EV (30-100 nm) and apoptotic bodies (50-5000 nm), which can be used as one of the morphological characteristics of DIMV.

[0146] Example 5: Dehydration-induced preparation of cytoplasmic vesicles nanostructured

[0147] According to the method in Example 1, the cytoplasmic vesicles of PC9 cells were prepared, and after centrifugation purification and concentration, the prepared DIMV suspension was loaded into a syringe using LiposoFast LF-50 (Avestin, York, UK), and then the suspension was sequentially passed through a series of nanopore sizes, specifically 1 μm, 0.4 μm and 0.1 μm polycarbonate membrane filters (Whatman), such as Fig. 9As shown in a, each time a polycarbonate membrane filter with a pore size is passed, cytoplasmic vesicles of the corresponding size will be obtained. Finally, by collecting the 0.1μm filter membrane extrusion product, exosome-like vesicles of more than 100 nanometers can be obtained. The morphology of exosomes and extracellular nanoscale cytoplasmic vesicles after filter membrane extrusion was observed and analyzed using a transmission electron microscope (Jeol Ltd., Tokyo, Japan) ( Fig. 9 b, c), they have uniform size. Exosomes and exosome analogs were analyzed using a nanoparticle tracking analyzer (Particle Metrix, ZetaView), such as Fig. 9 As shown in d, the two have the same size.

[0148] like Fig. 9 As shown in Figure e, the number of extracellular nanoscale cytoplasmic vesicles prepared by this method was 1.415E+11 per million cells, and the yield was more than 50 times that of exosomes secreted by the same amount of cells (2.7E+9) [International journal of molecular sciences 2020, 21(18), 6466]. Fig. 9 As shown in Figure f, the protein yield of extracellular nanoscale cytoplasmic vesicles and exosomes produced by extrusion in the same culture system was analyzed by western blot. It was found that the total protein amount of extracellular nanoscale cytoplasmic vesicles produced per million cells was 15.905μg, while the total protein amount of EVs produced by the same amount of cells was only 0.098μg. The protein yield of extracellular nanoscale cytoplasmic vesicles of the same amount of cells was more than 160 times that of exosomes. In addition, considering the loss of proteins contained in DIMVs during the extrusion process, as shown in Figure 5, the protein yield of EVs produced by the same amount of cells was more than 0.098μg. Fig. 9 As shown in g, the DIMV protein produced by the same cells was compared with exosomes by western blot, and it was found that the total amount of protein contained in DIMV produced per million cells was 57.434 μg. From the perspective of total protein, the DIMV protein yield of the same amount of cells was more than 580 times that of exosomes.

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

[0150] The membrane composition of DIMV is similar to that of cell membrane, and it has good biocompatibility and targeting. In order to explore the drug loading capacity of DIMV, the present invention synthesized four cholesterol-CY5.5-labeled DNA chains enriched with different bases (ATCG) (see Table 3) by testing whether DIMV has different loading capacities for DNA enriched with different bases. Then, DIMV was stirred and 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 certain period of time, and then washed twice by centrifugation, and then observed and photographed by Nikon single-photon confocal microscope and tested by Beckman flow cytometer. The results are shown in Figure 3. Fig.10 As shown in Figures 10a, 10b, 10c, 10d, 10e, 10g, and 10h, it can be seen from the confocal results that cholesterol-CY5.5 modified DNA chains and random DNA aptamer nucleic acid molecules can be inserted into the DIMV surface or pass through the DIMV membrane into the cytoplasm to achieve DNA loading. DIMV loaded with CY5 aptamer was analyzed by flow cytometry (e.g. Fig.10 f, 10i) The results showed strong fluorescence intensity, indicating that DIMV can effectively load the aptamer.

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

[0152]

[0153] Example 7: Dehydration induction method to induce the transformation of cells to form DIMV with new biological properties

[0154] Three types of cells were constructed (three plasmids were constructed, GFP green fluorescent protein was on the membrane, GFP green fluorescent protein was in the cytoplasm, GFP green fluorescent protein was on the membrane and mScara-L red fluorescent protein was in the cytoplasm; the plasmids were transferred into HEK293T cells using lip3000 (ThermoFisher L3000075) to obtain proteins expressed by corresponding fluorescent proteins), respectively: 1) high expression of GFP on the membrane (cell GFP on ), 2) High expression of GFP in the cytoplasm (cellular GFP in ), and 3) high expression of GFP on the membrane and mScara-L red fluorescent protein in the cytoplasm (mScara-L (cellular GFP on -mScara-L in ), and prepared cytoplasmic vesicles DIMV according to the dehydration induction scheme. The results were analyzed and observed by Beckman flow cytometer and Nikon single-photon confocal microscope. Fig.11As shown, DIMV carries the eGFP protein expressed by the parental cells, and the localization of these proteins is completely consistent with that of the parental cells. This example shows that through bioengineering, DIMV can have more new properties and functional applications.

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

[0156] According to the method in Example 1, cytoplasmic vesicles were prepared, B16 cells were inoculated into a culture dish, and when the cells proliferated to 80% to 90% of the entire 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, left to stand in a clean bench for 10 minutes, and after observing that the cancer cells had obvious shrinkage under a microscope, 5 mL of DPBS was added, and then cultured and incubated for 1 hour. After a large number of cytoplasmic vesicles were observed under a microscope (Olympus, CKX53), the incubated supernatant was collected, centrifuged at 500g for 15 minutes, and repeated 1-2 times to remove the precipitated dead cells, and 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 C57 female mice (4-6 weeks old) by tail vein and subcutaneous injection. The mice were randomly divided into 3 groups: (1) control group, injected with DPBS; (2) experimental group 1: subcutaneous injection of DIMV; (3) experimental group 2: tail vein injection of DIMV. B16 cell-derived DIMV immune vaccine (dose = protein 2μg, volume = 0.1mL) was injected via tail vein and subcutaneous injection on days -1, -3, and -5, respectively, and DPBS was injected intravenously in the control group at the same time points. On day 0, B16 cells (10 6 The B16 mouse subcutaneous tumor model was established by inoculating B16 cells. The tumor size (calculated by the formula: long diameter*short diameter*short diameter / 2) and body weight ( Fig.12 a).

[0158] The experimental results showed that on the 9th day after tumor implantation, compared with the DPBS control group, the groups (Sub-immunity and IOCV-immunity) treated with three B16 tumor-derived DIMV vaccines showed significant inhibitory effects on B16 tumors ( Fig.12 b, 12c). It is worth noting that the survival rate of mice in the immunized group was better than that in the control group ( Fig.12 d). And during the entire in vivo experiment, there was no significant change in the weight of the mice ( Fig.12 e). This indicates that DIMV derived from B16 tumor has the ability of immune stimulation and can be used as a candidate drug for inhibiting tumor growth and improving patient survival rate with good safety.

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

[0160] like Fig.13 The obtained B16 cells were inoculated into 150 times the volume of culture medium for expansion culture. When the cells proliferated to a high density (80% to 90%), the culture medium was removed, and the cells were washed with DPBS for 3 times, and then the cells were dehydrated at room temperature. Then, DPBS was added again and cultured for about half an hour. The DPBS supernatant was collected and purified by centrifugation to obtain a large number of cytoplasmic vesicles (such as Fig.14 ), and the yield was increased by more than 100 times. The experimental results show that the dehydration-induced method for preparing cytoplasmic vesicles can achieve good linear amplification and is suitable for large-scale production.

[0161] Example 10: Other dehydration methods for inducing the production of cytoplasmic vesicles

[0162] After HEK293T and PC9 cells proliferated to 80% in a 10 cm culture dish, the culture medium was discarded, and the cells were washed once with DPBS. Then, they were dehydrated for 10 minutes using 30% sucrose, 8% sodium chloride, and 30% sucrose and 8% sodium chloride, respectively. The upper solution was then removed, the cells were quickly washed once with DPBS, and then DPBS was added for 20 minutes of culture. The production of cytoplasmic vesicles was observed under a microscope (Olympus, CKX53).

[0163] Table 4

[0164]

[0165] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing extracellular cytoplasmic vesicles, the method comprising the step of inducing cells to generate vesicles by dehydration treatment; Preferably, the dehydration treatment time is 1 second to 60 minutes; Preferably, the preparation method comprises the following steps: Dehydrating the cells until cell shrinkage is observed (e.g., cell shrinkage is observed under a microscope), thereby inducing vesicle formation; Preferably, the dehydration treatment is selected from any one of the following methods: I) incubating the cells at a certain temperature for a period of time without culture medium or buffer; for example, incubating the cells at a temperature of -80°C to 80°C for 1 second to 60 minutes; II) placing the cells in a high concentration sucrose and / or sodium chloride solution and letting them stand for a period of time; for example, for 1 minute to 60 minutes; Preferably, the preparation method comprises the following steps: cell expansion culture, dehydration treatment, separation and purification of cytoplasmic vesicles; Preferably, the preparation method comprises the following steps: obtaining cells, expanding cell culture, dehydrating, culturing (e.g., perfusion culture), and collecting cytoplasmic vesicles; Preferably, the cells are derived from primary cells, genetically modified cells, cancer cells, stem cells, tissues, blood samples, etc.; Preferably, the cell expansion culture specifically includes: inoculating the cells into a culture dish, and culturing until the cells proliferate to 70% to 90% of the entire culture dish or culturing until the cell density is 70% to 90%.

2. The preparation method according to claim 1, characterized in that: Dehydration treatment method I) can specifically select any of the following treatment methods: i) incubating the cells at room temperature without culture medium or buffer for 1 minute to 60 minutes, for example, 5 minutes to 30 minutes, 10 minutes to 30 minutes, 5 minutes to 20 minutes, or 10 minutes; ii) incubating 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, or 1 second to 10 seconds, without culture medium or buffer; iii) incubating the cells at a temperature below room temperature for 10 minutes to 80 minutes, for example, 20 minutes to 60 minutes, or 30 minutes, without culture medium or buffer; Preferably, the room temperature in i) is 10°C to 30°C, such as 18°C ​​to 28°C, 18°C ​​to 25°C; Preferably, the temperature in ii) is from 30°C to 80°C. Preferably, the below-room temperature condition in iii) is below 10°C, such as -100°C to 10°C, -80°C to 0°C.

3. The preparation method according to claim 1 or 2, characterized in that: When the dehydration treatment method is selected from I), the preparation method comprises the following steps: 1) Inoculating the cells into a culture dish and culturing them until the cells proliferate to 70% to 90% of the entire culture dish or until the cell density reaches 70% to 90%; 2) removing the culture medium; optionally adding a buffer to wash and remove dead cells; 3) Dehydration treatment: leaving the cells at a certain temperature for a period of time; 4) After the cells shrink, add buffer; 5) Cultivation; 6) Collect the supernatant and purify by centrifugation; 7) The supernatant is further concentrated by centrifugation to obtain cytoplasmic vesicles; Preferably, in step 1), the cell culture conditions are: culture at 37° C. and 5% CO 2 ; Preferably, in step 1), the cells are passaged once every 3 to 4 days depending on the cell density; Preferably, in step 1), the cells are cultured until they proliferate to 80% to 90% or 70% to 80% of the entire culture dish; Preferably, in step 1), the cells are cultured to a cell density of 80% to 90% or 70% to 80%; Preferably, in step 1), the types of cells include suspension cells and adherent cells; Preferably, in step 1), the cell type includes primary cells, such as ovarian cancer primary cells; Preferably, 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, blood cancer cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.; Preferably, in step 1), the cells are selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like; Preferably, in step 1), the culture medium is selected from DMEM culture medium supplemented with 10% fetal bovine serum (FBS) and 1X penicillin-streptomycin (PS), and RPMI 1640 culture medium containing 10% FBS and 1% PS; Preferably, in step 2) and / or step 4), the pH of the buffer is 7.4-8.4, preferably 7.4, more preferably DPBS buffer with a pH of 7.4; Preferably, in step 2) and / or step 4), the ionic strength of the buffer is 10 mM to 10 M, preferably 10 mM to 3 M; Preferably, in step 2) and / or step 4), the buffer is selected from DPBS buffer, NaCl solution, complete culture medium, serum-free culture medium, etc.; Preferably, in step 4), the amount of buffer added is 5 mL; Preferably, in step 5), the vesicles are separated after culturing for 2 min-60 min (e.g., 10 min-60 min), for example, culturing for 10 min, 20 min, 30 min, 40 min, 50 min, 60 min; Preferably, in step 6) and / or step 7), the centrifugation condition is 500 g for 15 min to 30 min.

4. The preparation method according to claim 1, characterized in that: In dehydration treatment method II), The high concentration sucrose solution is a sucrose solution with a mass concentration of 20%-50%, such as a 30% sucrose solution; And / or, the high concentration sodium chloride solution is a sodium chloride solution with a mass concentration of 3%-10%, such as an 8% sodium chloride solution.

5. The preparation method according to claim 1 or 4, characterized in that: When the dehydration treatment method is selected from II), the preparation method comprises the following steps: (1) inoculating cells into a culture dish and culturing until the cells proliferate to 70% to 90% of the entire culture dish or until the cell density reaches 70% to 90%; (2) removing the culture medium; optionally adding a buffer to wash and remove dead cells; (3) Dehydration treatment: placing the cells in a high concentration of sucrose and / or sodium chloride solution for a period of time; (4) after the cells shrink, remove the high concentration sucrose and / or sodium chloride solution (optionally use a buffer to wash the residual high concentration sucrose and / or sodium chloride solution), and add a buffer; (5) cultivation; (6) collecting the supernatant and centrifuging for purification; (7) The supernatant is further concentrated by centrifugation to obtain cytoplasmic vesicles; Preferably, in step (1), the cell culture conditions are: culture at 37°C and 5% CO2; Preferably, in step (1), the cells are passaged once every 3 to 4 days depending on the cell density; Preferably, in step (1), the cells are cultured until they proliferate to 80% to 90% or 70% to 80% (e.g., 80%) of the entire culture dish; Preferably, in step (1), the cell density is cultured to 80% to 90% or 70% to 80%; Preferably, in step (1), the types of cells include suspension cells and adherent cells; Preferably, in step (1), the cell type includes primary cells, such as ovarian cancer primary cells; Preferably, in step (1), the cell is a cancer cell, 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, blood cancer cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc.; Preferably, in step (1), the cells are selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like; Preferably, in step (1), the culture medium is selected from DMEM culture medium supplemented with 10% fetal bovine serum (FBS) and 1X penicillin-streptomycin (PS), and RPMI 1640 culture medium containing 10% FBS and 1% PS; Preferably, in step (2) and / or step (4), the pH of the buffer is 7.4-8.4, preferably 7.4, more preferably DPBS buffer with a pH of 7.4; Preferably, in step (2) and / or step (4), the ionic strength of the buffer is 10 mM to 10 M, preferably 10 mM to 3 M; Preferably, in step (2) and / or step (4), the buffer is selected from DPBS buffer, NaCl solution, complete culture medium, serum-free culture medium, etc.; Preferably, in step (4), the amount of buffer added is 5 mL; Preferably, in step 5), the vesicles are separated after culturing for 2 min-60 min (e.g., 10 min-60 min), for example, culturing for 10 min, 20 min, 30 min, 40 min, 50 min, 60 min; Preferably, in step (6) and / or step (7), the centrifugation condition is 500 g for 15 min to 30 min.

6. The extracellular cytoplasmic vesicles prepared by the preparation method according to any one of claims 1 to 5; Preferably, the cell is a cancer cell, 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, blood cancer cells (leukemia cells, acute myeloid leukemia cells, acute lymphoblastic leukemia cells), esophageal cancer cells, ovarian cancer cells, etc. Preferably, the cells are selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like; for example, PC9 cells, B16 cells; Preferably, the extracellular cytoplasmic vesicles contain calnexin protein and / or β-actin protein; Preferably, the extracellular cytoplasmic vesicle does not contain a nucleus; Preferably, the extracellular cytoplasmic vesicles do not contain proteins characteristic of apoptotic cells; Preferably, 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, ALG5; Preferably, the size of the extracellular cytoplasmic vesicles is 0-40 μm, preferably larger than 5 μm, such as larger than 10 μm.

7. A method for preparing extracellular nanoscale cytoplasmic vesicles, comprising the following steps: (i) preparing extracellular cytoplasmic vesicles using the preparation method according to any one of claims 1 to 5; (ii) Using a liposome extruder and a nanopore filter, extracellular nanosized cytoplasmic vesicles are obtained. Preferably, step (ii) specifically comprises: using a liposome extruder, loading a suspension of extracellular cytoplasmic vesicles into a syringe, and then sequentially passing the suspension through a series of nanopore filters, collecting the 0.1 μm filter membrane extrusion product, and obtaining extracellular nanoscale cytoplasmic vesicles; Preferably, the filter is a polycarbonate membrane filter; Preferably, the series of nanopore sizes are 1 μm, 0.4 μm and 0.1 μm.

8. The extracellular nanoscale cytoplasmic vesicles prepared by the preparation method according to claim 7; Preferably, the cell is a cancer cell, such as a lung cancer cell (small cell lung cancer, non-small cell lung cancer), a breast cancer cell, an ovarian cancer cell, an esophageal cancer cell, a cervical cancer cell, a gastric cancer cell, a liver cancer cell, a blood cancer cell (leukemia cell, acute myeloid leukemia cell, acute lymphoblastic leukemia cell), etc.; Preferably, the cells are selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and other cells. Preferably, the extracellular nanoscale cytoplasmic vesicles contain calnexin protein and / or β-actin protein; Preferably, the extracellular nanoscale cytoplasmic vesicles do not contain a nucleus; Preferably, the extracellular nanoscale cytoplasmic vesicles do not contain proteins characteristic of apoptotic cells; Preferably, 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, ALG5.

9. Use of the extracellular cytoplasmic vesicles according to claim 6 or the extracellular nanoscale cytoplasmic vesicles according to claim 8 in the preparation of drug carriers, in the preparation of tumor vaccines, and in the preparation of disease diagnostic reagents, drugs (such as targeted drugs, anti-tumor drugs), biomedical imaging, etc.

10. Use of the method for preparing the extracellular cytoplasmic vesicles according to any one of claims 1 to 5 and the method for preparing the extracellular nanoscale cytoplasmic vesicles according to claim 7 in the preparation of drug carriers, tumor vaccines, disease diagnostic reagents, drugs (such as targeted drugs, anti-tumor drugs), biomedical imaging, etc.

Images