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

Preparation method and application of cytoplasmic vesicles

[0001] This application claims priority to the prior application with patent application number 202311138823.6 filed with the State Intellectual Property Office of China on September 5, 2023, entitled “Method for preparing cytoplasmic vesicles and their application”, the full text of which is incorporated into this application by reference. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a preparation method of cytoplasmic vesicles and applications thereof. Background Art

[0003] Cellular vesicles are a biomimetic biotechnology that is mainly used to prepare micron or nano vesicles with cell membrane structures. Currently available cell-like vesicles mainly include vesicles prepared from cell membranes and extracellular vesicles secreted by cells. Cellular 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 under in vitro culture, 10 6 The amount of extracellular vesicles secreted by a cell 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 this type of induced secretion vesicles is relatively simple, 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]. In addition, there are physical methods, such as light-induced methods to make cells produce nano-vesicles [Nature Communications, 2022, 13 (1): 6534]. Through these means, a relatively sufficient number of vesicles with membrane components close to those of the mother cells can be obtained [ACS Appl. Mater. Interfaces 2021, 13, 55767-55779]. However, existing cell-secreted extracellular vesicle induction technologies 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.

[0004] In addition, the existing applications of cell-like vesicles are all based on the cell membrane structure they possess. By loading or modifying small molecule therapeutics or diagnostic agents in the vesicles or on the cell membrane, the small molecule therapeutics or diagnostic agents derived from cell-like vesicles can be given excellent biocompatibility and targeting. However, this type of cell-like vesicle loading method still has great difficulties for biomacromolecules (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 biomacromolecules is an important way for biotherapy and diagnosis. Although there are some other methods for the loading and in vivo delivery of biomacromolecules such as protein drugs and mRNA drugs, there are almost no reports on strategies for loading or delivering organelles of larger sizes (hundreds of nanometers to tens of microns) and fragile organelles. Therefore, the construction of cell-like vesicles containing biomacromolecules and structures (especially organelles) is of great significance to the development of biotherapy and diagnosis, and has potential application value.

[0005] Summary of the Invention

[0006] The present invention aims to provide an efficient preparation method of cytoplasmic vesicles and its application. The preparation method of the present invention is convenient, fast, inexpensive, does not involve toxic substances, and is suitable for promotion and application.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for preparing extracellular cytoplasmic vesicles, which comprises the step of inducing cells to generate vesicles through dehydration treatment.

[0009] 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.

[0010] 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.

[0011] In some embodiments, the dehydration treatment is selected from any one of the following methods:

[0012] I) incubating the cells at a certain temperature for a period of time without culture medium, buffer or solution; for example, at a temperature of -80°C to 80°C for 1 second to 60 minutes;

[0013] II) placing the cells in a high concentration sodium chloride solution and / or sucrose and allowing them to stand for a period of time; for example, for 1 minute to 60 minutes.

[0014] In some embodiments, in I), the buffer is selected from phosphate buffer solution, borate buffer solution, DPBS buffer, tris-hydrochloric acid buffer solution (Tris-HCl), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution, citric acid buffer solution, NaCl solution, complete culture medium, serum-free culture medium, etc.

[0015] A buffer solution refers to a mixed solution composed of a weak acid and its salt, or a weak base and its salt. It can offset and reduce the effects of external strong acids or strong bases on the pH of the solution to a certain extent, thereby keeping the pH value of the solution relatively stable.

[0016] In some embodiments, the preparation method can be carried out in a laboratory on a small scale or in a large-scale industrial production.

[0017] In some embodiments, in dehydration treatment method I), the resting time can be adjusted according to the resting temperature until cell shrinkage is observed under a microscope. For example, when the resting temperature is high, the resting time can be shortened; when the resting temperature is low, the resting time can be increased.

[0018] In some embodiments, the dehydration treatment method I) can specifically select any of the following treatment methods:

[0019] i) incubating the cells 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, or 10 minutes, in the absence of culture medium, buffer, or solution;

[0020] ii) incubating the cells at room temperature to 80° C. for 1 second to 20 minutes, e.g., 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, buffer, or solution;

[0021] iii) Incubating the cells at room temperature for 10 to 80 minutes, for example, 20 to 60 minutes, or 30 minutes, without culture medium, buffer, or solution.

[0022] In some embodiments, the room temperature in i) is 10°C to 30°C, eg, 18°C ​​to 28°C, 18°C ​​to 25°C.

[0023] In some embodiments, the temperature in ii) is from 30°C to 80°C.

[0024] In some embodiments, the temperature below room temperature in iii) is below 10° C., for example, -100° C. to 10° C., -80° C. to 0° C. In some embodiments, the preparation method comprises the following steps:

[0025] Cell expansion culture, dehydration treatment, isolation and purification of cytoplasmic vesicles.

[0026] In some embodiments, the preparation method comprises the following steps:

[0027] Cell acquisition, cell expansion culture, dehydration treatment, culture (such as perfusion culture), and collection of cytoplasmic vesicles.

[0028] In some embodiments, the cells are derived from primary cells, genetically modified cells, cancer cells, stem cells, tissues, blood samples, etc.

[0029] In some embodiments, the cell expansion culture is specifically: 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%. In some embodiments, when the dehydration treatment method is selected from I), the preparation method includes the following steps:

[0030] 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%;

[0031] 2) removing the culture medium; optionally adding a buffer to rinse and remove dead cells;

[0032] 3) Dehydration treatment: let the cells stand at a certain temperature for a period of time;

[0033] 4) After observing cell shrinkage, add buffer;

[0034] 5) Incubate; optionally tap the culture dish (to accelerate the detachment of vesicles);

[0035] 6) Collect the supernatant and purify by centrifugation;

[0036] 7) The supernatant is further concentrated by centrifugation to obtain cytoplasmic vesicles.

[0037] In some embodiments, the culture dish is a 10 cm culture dish.

[0038] In some embodiments, in step 1), the cell culture conditions are: culture at 37° C. and 5% CO 2 .

[0039] In some embodiments, in step 1), the cells are passaged once every 3 to 4 days depending on the cell density.

[0040] 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.

[0041] In some embodiments, in step 1), the cells are cultured to a cell density of 80% to 90% or 70% to 80%.

[0042] In some embodiments, in step 1), the types of cells include suspension cells and adherent cells.

[0043] In some embodiments, in step 1), the cell type includes primary cells, such as ovarian cancer primary cells.

[0044] In some embodiments, in step 1), the type of cells includes stem cells, such as mesenchymal stem cells.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In some embodiments, 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.

[0049] 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, for example, 0.01 M, 0.05 M, 0.1 M, 0.5 M, 1 M, 2 M, 3 M, or 5 M.

[0050] 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. In some embodiments, the ionic strength of the NaCl solution is 0.001M-10M, for example, 0.01M, 0.05M, 0.1M, 0.5M, 1M, 2M, 3M, 5M.

[0051] In some embodiments, in step 4), the amount of buffer added is 5 mL.

[0052] In some embodiments, in step 5), the vesicle detachment operation 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.

[0053] 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.

[0054] In some embodiments, in step 6) and / or step 7), the centrifugation condition is 500 g for 15 min to 30 min.

[0055] 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%, for example, an 8% sodium chloride solution;

[0056] And / or, the high concentration sucrose solution is a sucrose solution with a mass concentration of 20%-50%, such as a 30% sucrose solution.

[0057] In some embodiments, when the dehydration treatment method is selected from II), the preparation method comprises the following steps:

[0058] (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%;

[0059] (2) removing the culture medium; optionally adding a buffer to rinse and remove dead cells;

[0060] (3) Dehydration treatment: Place the cells in a high concentration of sodium chloride and / or sucrose solution for a period of time;

[0061] (4) After the cells have shrunk, the high concentration of sodium chloride and / or sucrose solution is removed (optionally, a buffer solution is used to wash the remaining high concentration of sodium chloride and / or sucrose solution), and a buffer solution is added;

[0062] (5) Incubation; optionally, tapping the culture dish (to accelerate the detachment of vesicles);

[0063] (6) Collect the supernatant and purify by centrifugation;

[0064] (7) The supernatant is further concentrated by centrifugation to obtain cytoplasmic vesicles.

[0065] In some embodiments, the culture dish is a 10 cm culture dish.

[0066] In some embodiments, in step (1), the cell culture conditions are: culture at 37° C. and 5% CO 2 .

[0067] In some embodiments, in step (1), the cells are passaged once every 3 to 4 days depending on the cell density.

[0068] 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.

[0069] In some embodiments, in step (1), the cells are cultured to a cell density of 80% to 90% or 70% to 80%.

[0070] In some embodiments, in step (1), the types of cells include suspension cells and adherent cells.

[0071] In some embodiments, in step (1), the type of cells includes primary cells, such as ovarian cancer primary cells.

[0072] In some embodiments, in step (1), the type of cells includes stem cells, such as mesenchymal stem cells.

[0073] In some embodiments, in step (1), the type of cells includes modified cells, such as plasmid-transfected cells and mRNA-transfected cells.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] In some embodiments, in step (2) and / or step (4), the pH of the buffer solution is 7.4-8.4, preferably 7.4, and more preferably DPBS buffer solution with a pH of 7.4.

[0078] 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.

[0079] 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. In some embodiments, the ionic strength of the NaCl solution is 0.001M-10M, for example, 0.01M, 0.05M, 0.1M, 0.5M, 1M, 2M, 3M, 5M.

[0080] In some embodiments, in step (4), the amount of buffer added is 5 mL.

[0081] In some embodiments, in step 5), the vesicle detachment operation 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.

[0082] In some embodiments, in step (6) and / or step (7), the centrifugation condition is 500 g for 15 min to 30 min.

[0083] The present invention also provides the extracellular cytoplasmic vesicles prepared by the above preparation method.

[0084] In some embodiments, the cell type includes primary cells, such as primary ovarian cancer cells.

[0085] In some embodiments, the type of cell includes stem cells, such as mesenchymal stem cells.

[0086] In some embodiments, the type of cell includes a modified cell, such as a plasmid-transfected cell, an mRNA-transfected cell.

[0087] In some embodiments, 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.

[0088] In some embodiments, the cells are selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and other cells; preferably PC9 cells and B16 cells.

[0089] In some embodiments, the extracellular cytoplasmic vesicles comprise calnexin protein and / or β-actin protein. In some embodiments, the extracellular cytoplasmic vesicles comprise Caprin-1 and / or Clathrin. In some embodiments, the extracellular cytoplasmic vesicles comprise (are enriched in) DNA binding proteins and / or RNA binding proteins.

[0090] In some embodiments, the extracellular cytoplasmic vesicles do not contain a nucleus. In some embodiments, the extracellular cytoplasmic vesicles contain mitochondria and cytoskeletal proteins. In some embodiments, the extracellular cytoplasmic vesicles contain almost no large fragments of genomic DNA. In some embodiments, the extracellular cytoplasmic vesicles are rich in RNA. In some embodiments, the extracellular cytoplasmic vesicles are rich in protein (high protein content).

[0091] In some embodiments, the extracellular cytoplasmic vesicles do not comprise proteins characteristic of apoptotic cells.

[0092] 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.

[0093] 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.

[0094] In some embodiments, the extracellular cytoplasmic vesicles have higher safety and lower immunogenicity.

[0095] The present invention also provides a method for preparing extracellular nanoscale cytoplasmic vesicles, comprising the following steps:

[0096] (i) preparing extracellular cytoplasmic vesicles using the above preparation method;

[0097] (ii) Extracellular nanoscale cytoplasmic vesicles were obtained using a liposome extruder and a nanopore filter.

[0098] In some embodiments, step (ii) is specifically:

[0099] (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.

[0100] In some embodiments, the filter is a polycarbonate membrane filter.

[0101] In some embodiments, the range of nanopore sizes is 1 μm, 0.4 μm, and 0.1 μm.

[0102] The present invention provides extracellular nanoscale cytoplasmic vesicles prepared by the above preparation method.

[0103] In some embodiments, the cell type includes primary cells, such as primary ovarian cancer cells.

[0104] In some embodiments, the type of cell includes stem cells, such as mesenchymal stem cells.

[0105] In some embodiments, the type of cell includes a modified cell, such as a plasmid-transfected cell, an mRNA-transfected cell.

[0106] 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.

[0107] In some embodiments, the cell is selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780, and the like.

[0108] In some embodiments, the extracellular nanoscale cytoplasmic vesicles comprise calnexin protein and / or β-actin protein. In some embodiments, the extracellular cytoplasmic vesicles comprise Caprin-1 and / or clathrin. In some embodiments, the extracellular cytoplasmic vesicles comprise (are enriched in) DNA binding proteins and / or RNA binding proteins.

[0109] In some embodiments, the extracellular nanoscale cytoplasmic vesicles do not contain a nucleus. In some embodiments, the extracellular nanoscale cytoplasmic vesicles contain mitochondria and cytoskeletal proteins. In some embodiments, the extracellular nanoscale cytoplasmic vesicles contain almost no large fragments of genomic DNA. In some embodiments, the extracellular nanoscale cytoplasmic vesicles are enriched for RNA. In some embodiments, the extracellular nanoscale cytoplasmic vesicles are rich in protein (high protein content).

[0110] In some embodiments, the extracellular nanoscale cytoplasmic vesicles do not comprise proteins characteristic of apoptotic cells.

[0111] 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.

[0112] In some embodiments, the extracellular nanoscale cytoplasmic vesicles have higher safety and lower immunogenicity.

[0113] The present invention also provides the use of the extracellular cytoplasmic vesicles and the extracellular nanoscale cytoplasmic vesicles in preparing drug carriers. In some embodiments, the drugs include chemical drugs, aptamer drugs, DNA drugs, RNA drugs, protein drugs, etc.

[0114] The present invention also provides the use of the above-mentioned extracellular cytoplasmic vesicles and extracellular nanoscale cytoplasmic vesicles in the preparation of delivery vectors. In some embodiments, the vectors are used to deliver small molecule active substances, nucleic acids, oligonucleotides, proteins, antibodies, etc. In some embodiments, the vectors are used to deliver chemical drugs, aptamer drugs, DNA drugs, RNA drugs, protein drugs, etc.

[0115] The present invention also provides the use of the extracellular cytoplasmic vesicles or the extracellular nanoscale cytoplasmic vesicles in preparing tumor vaccines.

[0116] The present invention also provides a tumor vaccine, comprising the above-mentioned extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles and an adjuvant. In some embodiments, the tumor vaccine further comprises other active ingredients, such as immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitors include one or more of PD-1 antibodies, PD-L1 antibodies, PD-L2 antibodies, CTLA-4 antibodies, LAG-3 antibodies, TIM-3 antibodies, VISTA antibodies, and A2aR antibodies, preferably PD-1 antibodies and / or PD-L1 antibodies.

[0117] The present invention also provides a combined tumor vaccine, comprising the above-mentioned extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles, and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises one or more of a PD-1 antibody, a PD-L1 antibody, a PD-L2 antibody, a CTLA-4 antibody, a LAG-3 antibody, a TIM-3 antibody, a VISTA antibody, and an A2aR antibody, preferably a PD-1 antibody and / or a PD-L1 antibody. In some embodiments, the combined tumor vaccine further comprises an adjuvant.

[0118] In some embodiments, tumors include solid tumors and non-solid tumors. In some embodiments, tumors include: lung cancer (including non-small cell lung cancer, small cell lung cancer, large cell lung cancer), cervical cancer, gastric cancer, liver cancer, blood cancer, melanoma, esophageal squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, etc.

[0119] The present invention also provides the use of the extracellular cytoplasmic vesicles or extracellular nanoscale cytoplasmic vesicles in the preparation of disease diagnostic reagents, the preparation of drugs (such as targeted drugs, anti-tumor drugs), the preparation of biomedical imaging reagents, etc.

[0120] 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 extracellular nanoscale cytoplasmic vesicles in the preparation of drug carriers, tumor vaccines, disease diagnostic reagents, drugs (such as targeted drugs, anti-tumor drugs), and biomedical imaging reagents. Beneficial effects

[0121] The method for preparing extracellular cytoplasmic vesicles of the present invention is convenient, rapid, and not restricted by cell type. It also has high yield and does not involve the use of toxic substances. The function of the cytoplasmic vesicles can also be modified by modifying the parent cells. The extracellular cytoplasmic vesicles prepared by the present invention have high safety and low immunogenicity. The cytoplasmic vesicles prepared by the present invention can be used for disease diagnosis and clinical medication, and have high application value and are suitable for widespread application.

[0122] Compared to cells, the cytoplasmic vesicles prepared by the present invention have similar properties to exosomes and can be used as exosome analogs for disease diagnosis and drug development. Furthermore, cytoplasmic vesicles contain important organelles such as mitochondria and can be used for organelle supplementation therapy, mRNA expression, or protein secretion.

[0123] The method for preparing extracellular cytoplasmic vesicles provided by the present invention is applicable to various types of cells, and has a good cytoplasmic vesicle yield for both suspended cells and adherent cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] FIG1 is a roadmap of the dehydration-induced preparation method of the present invention.

[0125] Figure 2 shows cytoplasmic vesicles prepared from different tumor cells in the examples (PC9, A549, H460, MCF7, Hela, MKN74, H22, CEM).

[0126] FIG3 shows the effect of different pH buffer conditions on the yield of induced cytoplasmic vesicles.

[0127] FIG4 shows the effect of different cell drying times on the yield of induced cytoplasmic vesicles.

[0128] FIG5 shows the effect of the time of adding DPBS on the yield of induced cytoplasmic vesicles.

[0129] Figure 6 is a comparison of the components of cytoplasmic vesicles, cells and EVs: 6a is the SDS-PAGE results of PC9 cell cells, PC9-derived cytoplasmic vesicles DIMV and exosomes EV; 6b is a z-score heat map of protein intensity of PC9 cell cells, PC9-derived cytoplasmic vesicles DIMV and exosomes EV 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 differential 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 cell component; 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; 6k is a Western blot analysis of β-actin protein in B16 cells Cell, B16 cell cytoplasmic vesicles DIMV and exosomes Ex-DIMV Cell.

[0130] Figure 7 shows microscopic images of cytoplasmic vesicles prepared from different tumor cells in the examples, the sizes of cytoplasmic vesicles and flow cytometric images of cytoplasmic vesicles: Figure 7a shows cytoplasmic bodies formed by PC9 cells, with a scale of 100 μm; Figure 7b shows the sizes of cytoplasmic vesicles derived from PC9; Figure 7c shows the flow cytometric images of cytoplasmic vesicles derived from PC9 and PC9 cells; Figure 7d shows cytoplasmic bodies formed by MCF7 cells, with a scale of 100 μm; Figure 7e shows the sizes of cytoplasmic vesicles derived from PC9; and Figure 7f shows the flow cytometric images of cytoplasmic vesicles derived from MCF7 compared with those of MCF cells.

[0131] Figure 8 shows fluorescence microscopy and electron microscopy 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 microscopy image of cytoplasmic bodies; and 8d is a scanning electron microscopy image of cytoplasmic vesicles.

[0132] Figure 9 shows the preparation process and results of extracellular nano-sized 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 exosome-like vesicles extruded from cytoplasmic vesicles; 9d is the particle size distribution assessed by NTA, showing that the size of EVs-like (Squeezed DIMV) and EVs is basically the same (50-200 nm); 9e compares the number of exosomes and exosome-like vesicles produced from the same number of maternal cells; 9f compares the protein content of exosomes and exosome-like vesicles produced from the same number of maternal cells. 9g compares the relative protein content ratio of exosomes and cytoplasmic vesicles produced from the same number of maternal cells.

[0133] Figure 10 shows the results of cytoplasmic vesicles as drug carriers: 10a is a schematic diagram of the nucleic acid delivery system in which cytoplasmic vesicles are loaded with DNA of different bases enriched in CY5.5 and cholesterol (CY5.5-chole-DNA); 10b, 10c, 10d and 10e are confocal images of cytoplasmic vesicles enriched in 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 in 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.

[0134] Figure 11 shows an analysis of proteins involved in the uptake of free oligonucleotides by DIMVs: Figure 11a shows the expression of proteins associated with free oligonucleotide uptake using proteomic data analysis; Figure 11b shows the expression of Caprin-1 and clathrin in cells, DIMV-1, DIMV-2, and residual cells using Western blot analysis; Figures 11c and 11d show DNA binding-related proteins (GO: 0003677) and RNA binding-related proteins (GO: 0003723) using proteomic data analysis. Figures 11a, 11c, and 11d show cells, DIMVs, and EVs (Exs) from left to right.

[0135] Figure 12 shows the protein expression of cytoplasmic vesicles prepared from three types of modified cells: 12a shows cytoplasmic vesicles containing GFP on the membrane prepared using gene editing technology; 12b shows flow cytometry analysis of cytoplasmic vesicles and cytoplasmic vesicles expressing GFP on the membrane; 12c shows confocal images of cytoplasmic vesicles expressing GFP on the membrane; 12d shows the grayscale value statistics of the underlined part of Figure 12c; 12e shows the process of preparing cytoplasmic vesicles expressing GFP inside the cells expressing GFP in the cytoplasm; 12f shows flow cytometry analysis of cytoplasmic vesicles and cytoplasmic vesicles expressing GFP on the membrane. 12g is a confocal image of cytoplasmic vesicles expressing GFP inside; 12h is the grayscale statistical value of the underlined part in Figure 12g; 12i is a schematic diagram of the preparation of cytoplasmic vesicles expressing red fluorescent protein in the cytoplasm and GFP on the membrane; 12j is a flow cytometry analysis of cytoplasmic vesicles expressing red fluorescent protein inside and GFP on the membrane; 12k is a confocal image of cytoplasmic vesicles expressing GFP on the membrane and red fluorescent protein inside; 12i is the grayscale statistical value of the underlined part in Figure 12k.

[0136] Figure 13 shows the safety evaluation of DIMVs: 13a shows histopathological analysis of important mouse organs, scale bar: 100 μm; 13b and 13c show immunogenicity analysis of mouse blood cells after DIMV treatment (n=5); 13d shows the percentage of various cell populations in the spleen of mice after DIMV treatment. Figures 13b and 13c show, from left to right, DPBS, LPS, DIMV (0.1 mg), and DIMV (1 mg); and Figure 13d shows, from left to right, DPBS, DIMV (0.1 mg), and DIMV (1 mg).

[0137] Figure 14 shows the results of cytoplasmic vesicles as tumor vaccines: 14a is a schematic diagram showing the steps used to evaluate immunotherapy in B16 tumor-bearing C57BL mice; 14b 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; 14c is a tumor volume growth curve of C57BL tumor-bearing mice treated with different immunization methods; 14d is the survival rate of tumor-bearing C57BL mice after various treatments; 14e is the weight change of C57BL mice in different treatment groups.

[0138] Figure 15 shows that DIMVs can induce specific T cell responses and enhance the anti-tumor effect of PD-1 antibodies: Figure 15a is a schematic diagram of the immunotherapy scheme; Figure 15b uses Western blot to analyze the OVA protein levels in cells, DIMV-1, DIMV-2, and the remaining cells after DIMV secretion; Figure 15c uses flow cytometry to analyze the CD8 +Expression of OVA antigen in T cells: 15d refers to CD8 + Figure 15e shows the average number of positive cells with OVA tetramers on the T cell surface; 15e shows the experimental scheme for evaluating the treatment effects of different groups in B16-OVA tumor-bearing mice; 15f shows the individual tumor growth trajectories of B16-OVA tumor-bearing mice under different treatment modes; 15g shows the average tumor growth curves of B16-OVA tumor-bearing mice after different treatment regimens; 15h shows the weight fluctuations of the tumor recurrence model of mice receiving different treatment regimens; 15i is the survival rate of B16-OVA tumor-bearing mice after various therapeutic interventions.

[0139] FIG16 is a roadmap for the rapid and large-scale preparation of sterile cytoplasmic vesicles.

[0140] FIG17 is a microscopic image of the cytoplasmic body of the magnified preparation system. DETAILED DESCRIPTION

[0141] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative 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 encompassed within the scope of protection that the present invention is intended to protect.

[0142] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0143] Example 1: Construction of a dehydration-induced method for preparing cytoplasmic vesicles (DIMVs)

[0144] HEK293T, MCF7, MKN74, PC9, A549, H460, and B16 cells were seeded into 10 cm culture dishes. When the cells reached a desired density, the culture medium was removed and the cells were washed 1-2 times with DPBS to remove dead cells and culture medium components. The cells were then dehydrated and allowed to stand in a clean bench for 10 minutes. After visible shrinkage of the cancer cells was observed under a microscope, 5 mL of DPBS was slowly added along the wall of the culture dish. The cells were then incubated for approximately 2 minutes. Under a microscope (Olympus, CKX53), a large number of dehydration-induced vesicles were observed to detach from the cells. As shown in Figure 2, after dehydration at room temperature and re-addition of DPBS, a large number of cytoplasmic vesicles were quickly observed under the microscope for each cell type. These experimental results demonstrate that the dehydration-induced method for preparing cytoplasmic vesicles is applicable to a variety of cell types and exhibits good induction efficiency.

[0145] Example 2: Optimization of conditions for preparing cytoplasmic vesicles by dehydration induction

[0146] PC9 cells were seeded 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 allowed to stand in a clean bench for 10 minutes. After observing obvious shrinkage of the cancer cells under a microscope, 5 mL of phosphate buffer with different pH values ​​(pH 7.1, pH 7.5, pH 8.0, pH 8.5) was added, incubated for 30 minutes, and finally observed under a microscope (Olympus, CKX53). As shown in Figure 3, cytoplasmic vesicles can be produced at pH values ​​of 7.1-8.5. However, as the pH of the incubation solution increases after dehydration, the number of cytoplasmic vesicles decreases.

[0147] PC9 cells were seeded 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 allowed to stand in a clean bench for different times (10, 20, 30, and 40 minutes). 5 mL of DPBS was then added and incubated for 10 minutes before observation under a microscope (Olympus, CKX53). As shown in Figure 4, cytoplasmic vesicles can be produced at different times of room temperature dehydration. The yield of cytoplasmic vesicles decreases with increasing cell drying time. Dehydration at room temperature within the range of 10-30 minutes can achieve relatively good results.

[0148] PC9 cells were seeded 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 allowed to stand in a clean bench for 10 minutes. After observing obvious shrinkage of the cancer cells under a microscope, 5 mL of DPBS was added. After incubation with DPBS for different times, the cells were observed under a microscope (Olympus, CKX53). After dehydration at room temperature, cytoplasmic vesicles were produced by incubation with DPBS for 2 minutes. As shown in Figure 5, the yield of cytoplasmic vesicles increased with increasing DPBS incubation time.

[0149] Different cells (see Table 1) were inoculated into culture dishes. When the cell confluence reached 70% to 80%, the culture medium was removed and the cells were washed twice with DPBS. Subsequently, the various cells were dehydrated under different conditions (see Table 1) for different times. After observing the dehydration shrinkage of each cell under a microscope, 5 mL of DPBS was added, incubated for 2-10 minutes, and then 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.

[0150] Table 1 Vesicle production in different cell types

[0151] PC9 cells were seeded into culture dishes. 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 shrink under a microscope. Then, N5mL of different incubation solutions (see Table 2) were added. After incubation for 2 minutes, the cells were observed and recorded under a microscope (Olympus, CKX53). As shown in Table 2, after dehydration induction, the cells were able to produce cytoplasmic vesicles using different incubation solutions. In Table 2, the heating temperature ranged from 30°C to 80°C.

[0152] Table 2 Vesicle production of PC9 cells in different buffers

[0153] PC9 cells: human lung cancer cells; MCF7 cells: human breast cancer cells; A549 cells: human non-small cell lung cancer cells; H460 cells: human large cell lung cancer cells; Hela cells: human cervical cancer cells; MKN74 cells: human gastric cancer cells; H22 cells: mouse hepatocellular carcinoma cells; CEM cells: human acute lymphoblastic leukemia cells; HEK293T cells: human embryonic kidney cells; B16 cells: mouse melanoma cells; KYSE150 cells: human esophageal squamous cell carcinoma recovery cells; A2780 cells: human ovarian cancer cells.

[0154] Example 3: Cytoplasmic vesicles prepared by dehydration induction method are a new type of cell-like vesicles

[0155] Compared with exosomes (EVs), DIMVs have many advantages in preparation and extraction. To further determine the differences in their composition, the present invention compared the protein abundance of PC9 cells, DIMVs produced by the dehydration induction method of PC9 cells (preparation method refers to Example 1) and EVs secreted by PC9 cells (prepared by conventional size exclusion chromatography, with good exosome purity). The various protein components of PC9 cells, DIMVs and EVs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 6a1. From the results of SDS-PAGE, it can be seen that the SDS-PAGE of EVs has a protein abundance of 70 kDa. There is a very obvious band above the position, while there is no obvious difference between the band of DIMV and the band of Cell. Further non-labeled quantitative mass spectrometry analysis was used to obtain a comprehensive differential protein spectrum of PC9 cells, DIMV and EV. The consistency analysis results showed that DIMV is a product between EV and Cell (Figure 6b, c). Among the 3088 proteins identified, 431 were PC9 cell-specific proteins, 123 were DIMV-specific proteins, and 96 were EV-specific proteins. The three had 463 proteins in common. Compared with EV, the protein types of DIMV are richer than those of EV (Figure 6d). Cell, DIMV and EV were then subjected to western blot. Blot analysis (Figure 6g) showed that DIMVs contained both characteristic EV proteins and proteins that are generally not considered to be enriched in EVs, such as calnexin, indicating that DIMVs have rich protein characteristics and share characteristics of both cells and EVs. Therefore, the 123 DIMV-specific enriched proteins, especially the most differentially expressed proteins of 30 organelles, including 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 be used as markers for DIMVs.

[0156] Subsequently, statistics were collected for the top 40 proteins with high and low expression in cells and DIMVs to analyze which parts of the cells they belonged to. As shown in Figure 6e, compared with cells, the proteins with the most significant low expression in DIMVs came from the cell membrane and nucleus, while the highly expressed proteins came from multiple organelles, indicating that multiple organelles were enriched in DIMVs. In order to confirm whether DIMVs contain important organelles such as mitochondria and nuclei, the marker proteins of each organelle were further detected (Figure 6f). The results showed that no obvious nuclear markers such as histone H3 were detected in DIMVs, but relevant markers of mitochondria and important components of cytoskeletal proteins could be detected, thus proving again that DIMVs contain relevant organelles such as mitochondria and the cytoskeleton. In addition, in order to verify the difference between the cytoplasmic vesicles and apoptotic bodies, western blot analysis was performed on apoptotic bodies, cells, DIMVs and EVs (Figure 6h). The results showed that DIMVs, like cells and EVs, did not have the characteristic proteins of apoptotic cells and were different from apoptotic bodies.

[0157] Samples containing 85 μg of cell, DIMV, and EV protein were analyzed for total DNA and RNA quantity and length (Figures 6i and 6j). The results showed that DIMV and EV contained almost no large fragments of genomic DNA, while both DIMV and EV were enriched for a wide range of RNA species. This, combined with Figure 6e, indicates that cytoplasmic vesicles do not contain nuclei.

[0158] The above results show that DIMV has both similar and different properties to 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.

[0159] 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 contained 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 both DIMV and cell samples, which was different from EVs (Figure 6k). The results showed that B16 cell DIMVs prepared by dehydration also contained differential protein β-actin.

[0160] Example 4: Particle size analysis of cytoplasmic vesicles prepared based on dehydration induction method

[0161] Cytoplasmic vesicles were prepared according to the method described in Example 1. PC9 and MCF7 cells were seeded into a 10 cm dish. When the cells had proliferated to 80% to 90% of the entire 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 allowed to stand in a clean bench for 10 minutes. After observing obvious shrinkage of the cancer cells under a microscope, 5 mL of DPBS was added and the cells were incubated. After a large number of cytoplasmic vesicles were observed under a microscope (Olympus, CKX53) (as shown in Figures 7a and 7d), the incubation supernatant was collected and centrifuged at 500g for 15 minutes. This process was repeated 1-2 times to remove precipitated dead cells. Finally, the supernatant was further centrifuged and concentrated to obtain a high concentration of cytoplasmic vesicles. The size of the vesicles was determined by a nanoparticle tracking analyzer (Particle Metrix, ZetaView) and a nanoflow cytometer (Flow U30). The results showed that the obtained cytoplasmic vesicles were mainly micron-sized vesicles that were smaller than the original cells (Figures 7b, 7c, 7e, and 7f).

[0162] PC9 cells were seeded into a 10 cm dish and the cell membrane was stained with the lipophilic dye Did. Cytoplasmic vesicles were then prepared according to the method in Example 1. They were observed and analyzed using a single-photon confocal microscope (Nikon, A1 HD25), 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, shown in Figure 8, further demonstrate that the membrane of the cytoplasmic vesicles prepared by the dehydration induction method is derived from the cell membrane. The particle size of the cytoplasmic vesicles is smaller than that of the cells, ranging from 0 to 40 μm. The large size of DIMVs (particularly larger than 5 μm, for example, larger than 10 μm) is different from the size of existing EVs (30-100 nm) and apoptotic bodies (50-5000 nm), and can be used as one of the morphological characteristics of DIMVs.

[0163] Example 5: Dehydration-induced cytoplasmic vesicle nanostructures

[0164] Cytoplasmic vesicles from PC9 cells were prepared according to the method described in Example 1. After centrifugation and concentration, the prepared DIMV suspension was loaded into a syringe using a LiposoFast LF-50 (Avestin, York, UK). The suspension was then sequentially passed through a series of nanopore-size polycarbonate membrane filters (Whatman), specifically 1 μm, 0.4 μm, and 0.1 μm. As shown in Figure 9a, cytoplasmic vesicles of corresponding sizes were obtained for each polycarbonate membrane filter. Finally, the extrusion product was collected through a 0.1 μm filter to obtain exosome-like vesicles measuring over 100 nanometers. The morphology of the exosomes and the extracellular nanoscale cytoplasmic vesicles after extrusion through the filter membrane was observed and analyzed using transmission electron microscopy (Jeol Ltd., Tokyo, Japan) (Figures 9b, c), which showed uniform size. Exosomes and exosome analogs were analyzed using a nanoparticle tracking analyzer (Particle Metrix, zetaView). As shown in Figure 9d, both were of uniform size.

[0165] As shown in Figure 9e, after nano-flow detection, the number of extracellular nanoscale cytoplasmic vesicles prepared by the method is 1.415E+11 per million cells, and the yield is more than 50 times that of exosomes (2.7E+9) [International journal of molecular sciences 2020, 21 (18), 6466] secreted by the same amount of cells. As shown in Figure 9f, the protein yield of extracellular nanoscale cytoplasmic vesicles and exosomes produced by the same culture system was analyzed by western blot. It was found that the total protein amount of the extracellular nanoscale cytoplasmic vesicles produced per million cells was 15.905 μg, while the total protein amount of EVs produced by the same amount of cells was only 0.098 μg, and the extracellular nanoscale cytoplasmic vesicle protein yield of the same amount of cells was more than 160 times that of exosomes. In addition, considering the loss of DIMV protein during the extrusion process, as shown in Figure 9g, the DIMV protein produced by the same cells was compared with exosomes by western blot. 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.

[0166] Example 6: Cytoplasmic vesicles prepared by dehydration induction method as drug carriers

[0167] The membrane composition of DIMV is similar to that of cell membrane and has good biocompatibility and targeting. In order to explore the drug loading capacity of DIMV, by testing whether DIMV has different loading capacities for DNA enriched with different bases, the present invention synthesized four cholesterol-CY5.5-labeled DNA chains enriched with different bases (ATCG) (see Table 3). Then, the DIMV prepared in Example 1 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. The cells were observed and photographed using a Nikon single-photon confocal microscope and detected by Beckman flow cytometry. The results are shown in Figures 10a, 10b, 10c, 10d, 10e, 10g, and 10h. It can be seen from the confocal results that the cholesterol-CY5.5-modified DNA chains and the 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. Flow cytometric analysis of DIMV loaded with CY5 aptamer (as shown in Figures 10f and 10i) showed strong fluorescence intensity, indicating that DIMV can effectively load the aptamer.

[0168] Table 3 Four cholesterol-CY5.5 labeled DNA chains with different bases (ATCG)

[0169] Example 7: Functional verification of nucleic acid endocytosis in cytoplasmic vesicles prepared by dehydration induction method

[0170] For cholesterol-modified DNA, loading is likely a passive process, primarily based on the natural affinity of cholesterol for the phospholipid bilayer. In contrast, the binding and uptake of non-cholesterol-modified nucleic acids is a complex process regulated by multiple proteins. Previous studies have reported the involvement of multiple cell membrane receptors in oligonucleotide recognition and uptake, including the Toll-like receptor family, Caprin-1, SERBP1, and clathrin- and caveolin-mediated endocytosis.

[0171] Proteomic data analysis was performed on PC9 cells, DIMVs produced by dehydration induction of PC9 cells (preparation method refers to Example 1), and EVs secreted by PC9 cells (prepared by conventional size exclusion chromatography, with good exosome purity). The results showed that DIMVs inherited more proteins related to naked oligonucleotide uptake than EVs, and showed similar expression levels between DIMVs and cells (Figure 11a). In addition, the results of WB experiments showed that DIMVs successfully enriched Caprin-1 and clathrin, and the results were consistent with the results of mass spectrometry analysis (Figure 11b). Therefore, DIMV may rely on these known nucleic acid receptors and endocytosis to achieve binding and loading of oligonucleotides.

[0172] Inside cells, studies have reported thousands of nucleic acid-binding proteins that are responsible for recognizing and binding to various nucleic acid sequences to perform diverse cellular biological functions. After in-depth analysis of proteomic data, it was found that DIMV inherited the vast majority of DNA-binding proteins (GO: 0003677, Figure 11c) and RNA-binding proteins (GO: 0003723, Figure 11d) from their parental cells. In contrast, the expression of these nucleic acid-binding proteins in EVs was significantly reduced. Therefore, once nucleic acids are taken up by DIMV, they may be recognized and bound by nucleic acid-binding proteins within the vesicles and remain stable therein. This finding shows that DIMV has great potential in nucleic acid delivery and stability maintenance.

[0173] Example 8: Dehydration induction method to induce the formation of DIMVs with new biological properties in transformed cells

[0174] Three cell lines were constructed (three plasmids were constructed, GFP green fluorescent protein on the membrane, GFP green fluorescent protein in the cytoplasm, GFP green fluorescent protein on the membrane and mScara-L red fluorescent protein in the cytoplasm; the plasmids were transferred into HEK293T cells using lip3000 (Thermo Fisher L3000075) to obtain proteins expressed by the corresponding fluorescent proteins), namely: 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 cytoplasmic vesicles (DIMVs) were prepared according to the dehydration induction protocol described in Example 1. Analysis and observation using a Beckman flow cytometer and a Nikon single-photon confocal microscope revealed, as shown in Figure 12, that the DIMVs carried the eGFP protein expressed by the parental cells, and the localization of these proteins was identical to that of the parental cells. This example demonstrates that through bioengineering, DIMVs can possess a variety of novel properties and functional applications.

[0175] Example 9: Safety Verification of Cytoplasmic Vesicles Prepared by Dehydration Induction Method

[0176] To further explore the fundamental properties of DIMVs and their potential advantages in bioengineering and drug delivery, we first conducted an in vivo safety assessment of DIMVs. DIMVs were prepared from the B16 melanoma cell line derived from C57 mice and injected into C57 mice to assess their safety and immunogenicity in a syngeneic setting.

[0177] For safety evaluation, mice were injected with DIMVs at doses 10-fold and 100-fold higher than the exosome dose (0.1 mg / dose and 1 mg / dose). Detailed blood biochemical parameters were then assessed, and histological observations of key organs were performed using H&E staining. Blood chemistry analysis revealed that B16 cell-derived DIMVs did not significantly affect blood alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), total bilirubin (T-BIL), indirect bilirubin (D-BIL), urea (Urea), creatinine (CR), and uric acid (UA) levels in C57 mice at either dose (see Table 4). Furthermore, histological analysis using H&E staining revealed that neither low- nor high-dose DIMVs induced significant pathological changes in major organs, including the heart, liver, spleen, lungs, and kidneys (Figure 13a). This finding demonstrates the safety of DIMVs as a therapeutic in a syngeneic model.

[0178] Table 4 Analysis of main blood chemical indicators of mice at different doses

[0179] To further explore the immunogenicity of DIMVs and their in vivo responses in mice, we first conducted a detailed analysis of the levels of 13 key inflammatory factors in mouse blood samples before and after DIMV treatment, and observed changes in the proportions of various lymphocytes in the spleen. Cytokine analysis revealed that after high-dose DIMV treatment, the concentrations of most inflammatory factors did not fluctuate significantly (<2-fold). Although high-dose DIMVs increased IL-6 and G-CSF levels, this immune response was far less severe than that induced by LPS, which is known to be a strong immunogenic agent. Specifically, LPS significantly increased TNF-α levels, while DIMVs had no significant effect on this cytokine. Similarly, LPS had a significantly greater effect on G-CSF levels than DIMVs (Figures 13b and 13c). High-dose DIMVs had minimal effect on splenic B cell numbers (Figure 13d, p-value > 0.05), indicating that high-dose DIMVs only slightly affected splenic B cell numbers.

[0180] The immunogenicity study results of DIMVs showed that at higher concentrations, DIMVs were able to activate the mouse immune system to a certain extent, and this activation was likely due to the high number of tumor-associated antigens carried by DIMVs. Therefore, it can be concluded that the immunogenicity induced by DIMVs is within an acceptable range and shows good biosafety.

[0181] Example 10: Cytoplasmic vesicles prepared by dehydration induction method are used as tumor vaccines

[0182] Cytoplasmic vesicles were prepared according to the method in Example 1. B16 cells were seeded into a culture dish. When the cells had 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 allowed to stand in a clean bench for 10 minutes. After observing obvious shrinkage of the cancer cells under a microscope, 5 mL of DPBS was added. The cells were then incubated for 1 hour. After a large number of cytoplasmic vesicles were observed under a microscope (Olympus, CKX53), the incubation supernatant was collected and centrifuged at 500g for 15 minutes. This was repeated 1-2 times to remove precipitated dead cells. Finally, the supernatant was further centrifuged and concentrated to obtain a high concentration of cytoplasmic vesicles.

[0183] 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: DIMV injected by tail vein. B16 cell-derived DIMV immune vaccine (dose = protein 2μg, volume = 0.1mL) was injected by tail vein and subcutaneous injection on days -1, -3, and -5, respectively. The control group was injected with DPBS intravenously at the same time points. On day 0, B16 cells (10 6 A B16 mouse subcutaneous tumor model was established using 444 mice (100 cells / mouse). Seven days after inoculation of B16 cells, the tumor size (calculated by the formula: long diameter*short diameter*short diameter / 2) and body weight were recorded ( FIG14 a ).

[0184] The experimental results showed that on the 9th day after tumor implantation, the groups (Sub-immunity and IOCV-immunity) that had undergone three B16 tumor-derived DIMV vaccine treatments showed significant inhibitory effects on B16 tumors compared with the DPBS control group (Figures 14b, 14c). It is worth noting that the survival rate of mice in the immunized group was better than that in the control group (Figure 14d). And during the entire in vivo experiment, there was no significant change in the weight of the mice (Figure 14e). This shows that B16 tumor-derived DIMV has the ability to stimulate immunity and can be used as a candidate drug to inhibit tumor growth and improve patient survival rate, and it has good safety.

[0185] Example 11: Cytoplasmic vesicles prepared by dehydration induction method specifically activate T cells and enhance the therapeutic effect of ICB

[0186] The B16-OVA melanoma model was used, which is widely used to study specific T cell immune responses because of its expression of OVA antigens. In in vitro experiments, DIMVs were successfully prepared and purified from B16-OVA cells, and Western blot (WB) analysis confirmed that these DIMVs were rich in OVA protein (Figure 15b); this also met the characteristics of DIMVs being rich in β-actin differential proteins. Next, C57 mice were inoculated with DPBS and DIMVs-OVA suspensions, and the purified OVA protein was combined with the immune adjuvant CPG as a positive control (Figure 15a). Subsequently, 14 days after the mice were immunized, periocular blood was collected from the mice, white blood cells in the blood were extracted, and the expression level of specific T cells was analyzed by flow cytometry.

[0187] Compared with the DPBS-treated group, the DIMVs-OVA-treated mice had significantly increased OVA antibody-specific CD8 +T cells (about 10 times) (Figure 15c, Figure 15d). The treatment effect of DIMVs-OVA is better than that of pure OVA protein without immune adjuvant reported in previous studies. Although the efficiency of OVA-specific T cells generated by DIMVs-OVA is slightly lower than that of the combined treatment of CPG and OVA, this may be related to the fact that the OVA protein content in DIMVs is much lower than that of purified OVA protein and the lack of CPG adjuvant. In summary, these results show that DIMVs-OVA can effectively induce specific T cell immune responses.

[0188] In addition, this application also introduces ICB therapy (i.e., programmed cell death protein 1 (PD-1) blocking therapy) to enhance the immunostimulatory effect of DIMVs, thereby evaluating the anti-tumor effect of DIMV in a clinical treatment model (Figure 15e). Compared with DPBS treatment, DIMV-OVAs alone or in combination with PD-1 antibodies can slow tumor growth. In particular, the combined treatment group of DIMV-OVA and PD-1 antibodies showed a more significant anti-tumor effect, with smaller tumor volume and longer simulated survival time (Figures 15f-15h). It is worth noting that the body weight of mice in different treatment groups did not change much, which further confirmed the safety of DIMVs (Figure 15i). In summary, DIMVs can be combined with PD-1 antibodies as an effective therapeutic tumor vaccine, showing significant therapeutic potential in the process of tumor treatment.

[0189] Example 12: A method for rapid and large-scale preparation of sterile cytoplasmic vesicles based on dehydration induction

[0190] As shown in Figure 16, 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 three times with DPBS. The cells were dehydrated at room temperature and then re-incubated with DPBS for about half an hour. The DPBS supernatant was collected and purified by centrifugation to obtain a large number of cytoplasmic vesicles (as shown in Figure 17). The yield was also 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.

[0191] Example 13: Other dehydration methods for inducing the production of cytoplasmic vesicles

[0192] 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. Subsequently, 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 incubated with DPBS for 20 minutes. The production of cytoplasmic vesicles was observed under a microscope (Olympus, CKX53).

[0193] Table 5 Vesicle production under other dehydration methods

[0194] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection 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 cells until they shrink, thereby inducing vesicle formation; Preferably, the dehydration treatment is selected from any one of the following methods: 1) leaving the cells at a certain temperature for a period of time without culture medium, buffer or solution; II) placing the cells in a high concentration of sucrose and / or sodium chloride solution for a period of time; Preferably, the preparation method comprises the following steps: cell expansion culture, dehydration treatment, and separation and purification of cytoplasmic vesicles.

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 for 1 minute to 60 minutes without culture medium, buffer or solution; ii) incubating the cells at room temperature to 80° C. for 1 second to 20 minutes without culture medium, buffer or solution; iii) Incubate the cells at room temperature for 10 to 80 minutes without culture medium, buffer or solution.

3. 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%; And / or, the high concentration sodium chloride solution is a sodium chloride solution with a mass concentration of 3%-10%.

4. The extracellular cytoplasmic vesicles prepared by the preparation method according to any one of claims 1 to 3; Preferably, the types of cells include primary cells; Preferably, the cell types include stem cells; Preferably, the cell is a cancer cell; preferably, the cell is selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like; Preferably, the extracellular cytoplasmic vesicles contain calnexin protein and / or β-actin protein; preferably, the extracellular cytoplasmic vesicles contain Caprin-1 and / or clathrin; preferably, the extracellular cytoplasmic vesicles contain (are enriched in) DNA binding protein and / or RNA binding protein; Preferably, the extracellular cytoplasmic vesicles do not contain a nucleus; preferably, the extracellular cytoplasmic vesicles contain mitochondria and cytoskeletal proteins; 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.

5. 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 3; (ii) Using a liposome extruder and a nanopore filter, extracellular nanosized cytoplasmic vesicles are obtained. Preferably, step (ii) is specifically as follows: 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.

6. The extracellular nanoscale cytoplasmic vesicles prepared by the preparation method according to claim 5; Preferably, the types of cells include primary cells; Preferably, the cell types include stem cells; Preferably, the cell is a cancer cell; preferably, the cell is selected from PC9, MCF7, A549, H460, Hela, MKN74, H22, CEM, HEK293T, B16, KYSE150, A2780 and the like; Preferably, the extracellular nanoscale cytoplasmic vesicles contain calnexin protein and / or β-actin protein; preferably, the extracellular cytoplasmic vesicles contain Caprin-1 and / or clathrin; preferably, the extracellular cytoplasmic vesicles contain (are enriched in) DNA binding protein and / or RNA binding protein; Preferably, the extracellular nanoscale cytoplasmic vesicles do not contain a nucleus; preferably, the extracellular cytoplasmic vesicles contain mitochondria and cytoskeletal proteins; 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.

7. A tumor vaccine, comprising the extracellular cytoplasmic vesicles according to claim 4, or the extracellular nanoscale cytoplasmic vesicles according to claim 5, and an adjuvant.

8. Use of the extracellular cytoplasmic vesicles according to claim 4 or the extracellular nanoscale cytoplasmic vesicles according to claim 5, wherein the use is selected from the following: (1) Application in the preparation of drug carriers; (2) Application in the preparation of delivery vectors; (3) Application in the preparation of tumor vaccines; (4) Application in the preparation of disease diagnostic reagents; (5) Application in the preparation of medicines; (6) Application in the preparation of biomedical imaging reagents.

9. The use of the method for preparing the extracellular cytoplasmic vesicles according to any one of claims 1 to 3, or the method for preparing the extracellular nanoscale cytoplasmic vesicles according to claim 5, wherein the use is selected from the following: (1) Application in the preparation of drug carriers; (2) Application in the preparation of delivery vectors; (3) Application in the preparation of tumor vaccines; (4) Application in the preparation of disease diagnostic reagents; (5) Application in the preparation of medicines; (6) Application in the preparation of biomedical imaging reagents.

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