A method for preparing extracellular vesicles in mammals

Abstract

This invention relates to a method for preparing mammalian extracellular vesicles, comprising the step of isolating mammalian extracellular vesicles from a culture medium expressing the FREE1 protein. By expressing the FREE1 protein, which plays a role in plant vesicle transport, in mammalian cells, this invention can significantly increase the yield of mammalian extracellular vesicles. This beneficial effect is not limited to ordinary unloaded mammalian extracellular vesicles; it also significantly increases the yield of mammalian extracellular vesicles loaded with therapeutic drugs (such as interleukins and anti-inflammatory proteins).

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a method for preparing extracellular vesicles in mammalian cells. Background Technology

[0002] Extracellular vesicles are a group of vesicles with a lipid bilayer membrane structure secreted by living cells. Based on their size, they can be classified into exosomes, microvesicles, and apoptotic bodies. Originating from endocytosis, extracellular vesicles are released extracellularly through the fusion of intracellular multivesicles with the plasma membrane. They can mediate the intercellular transfer of intracellular substances and thus serve as drug delivery carriers in vivo. Among them, exosomes, as natural nanoparticles, have great potential as a drug delivery platform. Considering biocompatibility, immunogenicity, safety and toxicity, and the difficulty of extraction and purification, mammalian extracellular vesicles are the more recommended choice.

[0003] Extracellular vesicle drugs (drugs using extracellular vesicles as carriers) require extremely high dosages, while the high cost of preparing mammalian extracellular vesicles hinders their drug development. The mechanism of vesicle production in mammalian cells relies on the ESCRT / ATP pathway. This involves assembling the ESCRT protein complex and the COP vesicle transporter complex, consuming a significant amount of ATP. Due to these rate-limiting steps, the yield of extracellular vesicles secreted by mammalian cells is normally limited, exhibiting a physiological limit. Furthermore, considering the extremely high loss rate and low yield during extraction and purification, the cost of mammalian extracellular vesicles remains high at present, making their drug development prospects less than optimistic. Therefore, effectively increasing the yield of mammalian extracellular vesicles has always been a key bottleneck in the field of innovative extracellular vesicle drugs.

[0004] Currently, some attempts have been made in this field to improve the yield of extracellular vesicles, including the optimization of culture conditions and the optimization of extraction and purification methods. The latter has a limited effect on improving the yield of extracellular vesicles. The former mainly involves adding some small molecules, such as calcium ion carriers and ceramides, during cell culture to affect cell growth and metabolism, hoping to improve the efficiency of single cells in secreting extracellular vesicles. However, the results of these attempts are not satisfactory. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new method for preparing mammalian extracellular vesicles that can more stably and effectively increase the yield of mammalian extracellular vesicles.

[0006] The first aspect of the present invention provides a method for preparing extracellular vesicles of mammalian cells, comprising the step of isolating the extracellular vesicles of mammalian cells from a culture medium expressing the FREE1 protein.

[0007] FREE1 protein is a protein involved in the transport of plant vesicles. In this invention, the FREE1 protein can be any FREE1 protein that functions in the transport of plant vesicles. The FREE1 protein can be derived from plants or microorganisms. Different FREE1 protein sequences from different plant or microbial sources have shown considerable effectiveness in promoting extracellular vesicle production in mammals. In specific embodiments of this invention, the FREE1 protein can be one or more combinations of FREE1 proteins from plants or microorganisms, such as Arabidopsis thaliana FREE1 protein or FREE1 proteins with more than 85% homology, wheat FREE1 protein, rice FREE1 protein, liverwort FREE1 protein, and *Hemiberlesia lataniae* FREE1 protein.

[0008] According to some specific embodiments of the present invention, the amino acid sequence of the FREE1 protein is shown in SEQ ID NO: 3.

[0009] According to the present invention, when referring to an amino acid sequence, unless otherwise specified, the amino acid sequence referred to includes not only sequences that are exactly the same as the sequence, but also those sequences that are readily conceived by a person skilled in the art based on the sequence and that can be expected to have equivalent effects (for example, a person skilled in the art should know that sequences with high consistency (e.g., more than 85%, especially more than 90%, especially more than 95%) generally have equivalent effects).

[0010] According to a specific embodiment of the present invention, the method includes the following steps:

[0011] (1) Construct and prepare a recombinant expression vector for expressing FREE1 protein;

[0012] (2) Recombinant mammalian cells were obtained by transfecting mammalian cells with the recombinant expression vector described above;

[0013] (3) The recombinant mammalian cells were cultured to obtain a recombinant mammalian cell culture medium;

[0014] (4) The animal extracellular vesicles were isolated from the recombinant mammalian cell culture medium.

[0015] According to the present invention, the mammalian cells involved include, but are not limited to, cells of mammals commonly found in the art, such as those of cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice; cells of non-human primates, such as monkeys, such as cynomolgus monkeys or rhesus monkeys, and chimpanzees; and cells of humans, including but not limited to those of humans. The mammalian cells involved include cells of transgenic mammals.

[0016] In embodiments of the present invention, the host cells of the recombinant mammalian cells include one or more of the following: ovarian cell lines, fibroblast cell lines, embryonic kidney cell lines, kidney cell lines, mesenchymal stem cell lines, induced cellular stem cell lines, megakaryocyte cell lines, platelet cell lines, or lymphocyte cell lines. Exemplary host cells include hamster ovary (CHO cells), human embryonic kidney 293 cells (e.g., HEK293, HEK293E, HEK293T), COS cells, BHK cells, NIH3T3 cells, lymphocyte cell lines (e.g., NSO myeloma cells and Sp2 / O cells), mammary epithelial cells, induced cellular stem cells, adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, or placental mesenchymal stem cells, etc.

[0017] In this embodiment of the invention, the recombinant expression vector may be an expression vector conventionally used in the art, including viral plasmid vectors and eukaryotic expression plasmid vectors, with eukaryotic expression plasmid vectors being preferred.

[0018] In some specific embodiments of the present invention, the recombinant expression vector used is the pAAVS1 plasmid, the pIRES plasmid, or the pLENTI plasmid.

[0019] In this embodiment of the invention, the separation can be performed using extracellular vesicle separation and purification methods known in the art, including one or more of ultracentrifugation, density gradient centrifugation, size exclusion chromatography, affinity chromatography, adsorption chromatography, bonded phase chromatography, immunoassay, or sieving.

[0020] According to some embodiments of the present invention, the recombinant animal cell culture medium is first centrifuged at a centrifugal force of less than 1000g, and the supernatant is collected. Then, the supernatant is centrifuged at a centrifugal force of 100000g to 200000g, and the precipitate is collected, which is the extracellular vesicle of the mammalian cell.

[0021] Further, the cell culture medium of the recombinant cells is first centrifuged at a centrifugal force of 200g to 500g, and the supernatant is collected. Then, the supernatant is centrifuged at a centrifugal force of 120000g to 150000g, and the precipitate is collected, which is the extracellular vesicle of the cargo.

[0022] In this embodiment of the invention, the recombinant mammalian cell culture medium is a serum-free culture medium.

[0023] In this embodiment of the invention, the recombinant mammalian cells are cultured at 34°C to 38°C and 5% to 10% CO2 to obtain the recombinant mammalian cell culture medium.

[0024] In this embodiment of the invention, the transfection operation involves mixing the recombinant expression vector, the host cell, and the transfection reagent in a serum-free culture medium and incubating at 20°C to 30°C.

[0025] A second aspect of the present invention provides a method for preparing extracellular vesicles of mammalian cells loaded with drug-active molecules, the method comprising the following steps:

[0026] 1) Obtain recombinant mammalian cell culture medium, wherein the recombinant mammalian cells not only express a fusion protein based on extracellular vesicle scaffold protein, but also express FREE1 protein, wherein the fusion protein based on extracellular vesicle scaffold protein is a fusion protein formed by the extracellular vesicle scaffold protein and a drug active molecule or a linker for linking the drug active molecule.

[0027] 2) Separating the recombinant mammalian cell culture medium; and,

[0028] When the fusion protein based on extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and a linker for connecting drug active molecules, the preparation method further includes:

[0029] 3) After the separation step, the separated extracellular vesicles are mixed with the active drug molecules, so that the active drug molecules bind to the extracellular vesicles through the linker.

[0030] In this embodiment of the invention, step 1) includes: constructing and preparing a first recombinant expression vector for expressing FREE1 protein and a second recombinant expression vector for expressing a fusion protein formed by extracellular vesicle scaffold protein and drug-active molecules or linkers; transfecting mammalian cells with the first recombinant expression vector and the second recombinant expression vector sequentially or simultaneously to obtain recombinant mammalian cells; and culturing the recombinant mammalian cells to obtain a recombinant mammalian cell culture medium.

[0031] In this embodiment of the invention, the FREE1 protein includes one or more of Arabidopsis thaliana FREE1 protein, wheat FREE1 protein, rice FREE1 protein, liverwort FREE1 protein, and spirea FREE1 protein.

[0032] In this embodiment of the invention, the host cell of the recombinant mammalian cell includes one or more of the following: ovarian cell line, fibroblast cell line, embryonic kidney cell line, kidney cell line, or lymphocyte cell line.

[0033] In this embodiment of the invention, the expression vectors used for the first recombinant expression vector and the second recombinant expression vector are eukaryotic expression plasmid vectors or viral plasmid vectors, respectively.

[0034] In embodiments of the present invention, the separation includes one or more of the following: ultracentrifugation, density gradient centrifugation, size exclusion chromatography, affinity chromatography, adsorption chromatography, bonded phase chromatography, immunoassay, or sieving.

[0035] In this embodiment of the invention, the recombinant mammalian cell culture medium is a serum-free culture medium.

[0036] In this embodiment of the invention, the recombinant mammalian cells were cultured at 34℃~38℃ and 5%~10% CO2 to obtain the recombinant mammalian cell culture medium.

[0037] In this embodiment of the invention, all other unmentioned operating methods or conditions in the method for preparing mammalian extracellular vesicles loaded with drug-active molecules are consistent with the operating methods or conditions in the method for preparing mammalian extracellular vesicles without drug-active molecules described above.

[0038] The active pharmaceutical molecules in this invention include active molecules of preventive drugs, active molecules of therapeutic drugs, and active molecules of diagnostic drugs.

[0039] In embodiments of the present invention, the active molecules include one or more of protein molecules and their fragments, polypeptide molecules and their fragments, nucleic acids and their fragments, or small molecule compounds.

[0040] In this invention, the connection between the active pharmaceutical molecule and the extracellular vesicle scaffold protein or the active pharmaceutical molecule and the linker can be achieved using linking techniques such as gene fusion, chemical synthesis, enzyme-catalyzed linking, self-assembly into a complex, click chemistry, protein engineering, and nanotechnology.

[0041] In some embodiments of the present invention, when the active pharmaceutical molecule is a protein molecule and its fragments and / or a polypeptide molecule and its fragments, the active pharmaceutical molecule is directly or indirectly linked to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein via a linker. Whether a linker is needed can be determined based on the structure, function, and stability of the extracellular vesicle scaffold protein and the active ingredient.

[0042] In other embodiments of the present invention, when the active pharmaceutical molecule is a nucleic acid or its fragment (e.g., RNA), the nucleic acid or its fragment can exist in the form of a protein-nucleic acid complex with a linker. The linker is linked to an extracellular vesicle scaffold protein, thereby indirectly linking the nucleic acid or its fragment to the extracellular vesicle scaffold protein, and then loading it into extracellular vesicles. For example, a fusion protein is formed by the extracellular vesicle scaffold protein and the protein linker, while the protein linker and the nucleic acid or its fragment form a complex through non-covalent interactions.

[0043] For example, when sgRNA needs to be delivered, Cas9 protein can be used as a protein linker. Cas9 protein is fused to the N-terminus and / or C-terminus of an extracellular vesicle scaffold protein. The sgRNA then forms an RNP complex with the Cas9 protein, thereby loading the sgRNA into the extracellular vesicle for delivery.

[0044] For example, when siRNA needs to be delivered, AGO2 protein can be used as a protein linker. AGO2 protein is fused to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein. siRNA can then form an RNP complex with AGO2 protein, thereby loading the siRNA into the extracellular vesicle for delivery.

[0045] In some other embodiments of the present invention, the active pharmaceutical molecule is a small molecule compound. The small molecule compound can be directly mixed with the extracellular vesicle that simultaneously expresses the FREE1 protein and the fusion protein formed by the extracellular vesicle scaffold protein and the linker. The small molecule compound binds to the linker to form a complex, and the linker is linked to the extracellular vesicle scaffold protein, thereby indirectly linking the small molecule compound to the extracellular vesicle scaffold protein and loading it into the extracellular vesicle.

[0046] The binding of nucleic acids and their fragments and / or small molecule compounds to linkers is achieved using conventional techniques in the art, such as binding small molecule compounds to linkers through affinity adsorption between the small molecule compounds and the linkers.

[0047] In embodiments of the present invention, the protein molecules include, but are not limited to, various therapeutic proteins, immunogenic proteins, or functional proteins (such as proteins used for gene editing, reporter gene proteins), enzyme proteins, etc.

[0048] In this embodiment of the invention, the proteins used for gene editing include various proteins used in gene editing technology / engineering, including but not limited to various recombinases capable of promoting recombination or rearrangement of specific sequences between DNA molecules, as well as gene editing system-related proteins (i.e., proteins used in various gene editing systems). Commonly known gene editing-related proteins include, but are not limited to, Cre recombinase, Dre recombinase, Flp recombinase, RadA protein, RAD51 protein, RecA protein, Cas protein, transposon proteins, zinc finger proteins, and TALE protein.

[0049] In embodiments of the present invention, the polypeptide molecule includes, but is not limited to, various therapeutic polypeptides, targeted polypeptides, or partial polypeptide fragments of the aforementioned proteins. For example, it may be an antibody, cytokine, anti-inflammatory protein or polypeptide, serum albumin, lectin, coagulation factor, etc.

[0050] In embodiments of the present invention, the nucleic acid includes, but is not limited to, various functional nucleic acid molecules. For example, it may be DNA or mRNA molecules encoding proteins or polypeptides, RNA molecules with regulatory functions such as miRNA, antisense oligonucleotides ASO or siRNA, sgRNA of the CRISPR system, or even plasmid DNA.

[0051] In this embodiment of the invention, the active pharmaceutical molecule is a cytokine receptor protein, a protein used for gene editing, a cytokine, or RNA.

[0052] In some embodiments of the present invention, the active pharmaceutical molecule is Cre recombinase, Dre recombinase, Flp recombinase, Cas protein, cGAS, srIκB, IL4, IL5, IL10, TGF-β, STAT6, A20, SIRPα, IL-1Ra, SGK1, KDM5B, 4-1BBL, TMPRSS2, CD20, CD133, CCR4, CCR7, CCR8, CXCR4, CXCR5, GIPR, GPRC5D, NaPi2b, sgRNA, or siRNA.

[0053] In this embodiment of the invention, the extracellular vesicle scaffold protein is any scaffold protein capable of enriching and loading cargo within extracellular vesicles, including but not limited to scaffold proteins known in the art. For example, the extracellular vesicle scaffold protein includes one or more of the following: BASP1 protein or a fragment thereof, PTGFRN protein or a fragment thereof, TSPAN2 protein or a fragment thereof, TSPAN3 protein or a fragment thereof, CD63 protein or a fragment thereof, CD9 protein or a fragment thereof, CD81 protein or a fragment thereof, LEAP protein or a fragment thereof, and PVR protein or a fragment thereof.

[0054] In this embodiment of the invention, a first recombinant expression vector is used to introduce a foreign nucleic acid fragment encoding the FREE1 protein into mammalian cells for expression. A second recombinant expression vector is used to introduce a foreign nucleic acid fragment encoding a fusion protein of "extracellular vesicle scaffold protein" or "extracellular vesicle scaffold protein-linker" or "extracellular vesicle scaffold protein-drug active molecule" into mammalian cells for expression, thereby obtaining recombinant mammalian cells that simultaneously overexpress the FREE1 protein and the extracellular vesicle scaffold protein, or recombinant mammalian cells that simultaneously overexpress the FREE1 protein and the fusion protein formed by the extracellular vesicle scaffold protein and the linker, or recombinant mammalian cells that simultaneously overexpress the FREE1 protein and the fusion protein formed by the extracellular vesicle scaffold protein and the drug active molecule.

[0055] A third aspect of the present invention also provides extracellular vesicles of mammals prepared by the above-described preparation method.

[0056] In this embodiment of the invention, the extracellular vesicles of mammalian cells are exosomes, microvesicles, or apoptotic bodies.

[0057] A fourth aspect of the present invention also provides mammalian extracellular vesicles loaded with drug-active molecules prepared by the above preparation method, wherein the mammalian extracellular vesicles are exosomes, microvesicles or apoptotic bodies.

[0058] A fifth aspect of the invention also provides the use of the above-described mammalian extracellular vesicles or the above-described mammalian extracellular vesicles loaded with pharmaceutically active molecules in the preparation of pharmaceutical compositions.

[0059] The sixth aspect of the present invention also provides a recombinant mammalian cell for preparing extracellular vesicles of mammalian cells, which is any of the recombinant mammalian cells mentioned above, including recombinant mammalian cells expressing FREE1 protein, recombinant mammalian cells simultaneously expressing FREE1 protein and a fusion protein formed by extracellular vesicle scaffold protein and pharmaceutically active molecules, and recombinant mammalian cells simultaneously expressing FREE1 protein and a fusion protein formed by extracellular vesicle scaffold protein and linkers.

[0060] A seventh aspect of the present invention also provides a recombinant expression vector system for preparing extracellular vesicles of mammals, comprising one or both of the first and second recombinant expression vectors described above.

[0061] An eighth aspect of the present invention also provides a pharmaceutical composition comprising any of the mammalian extracellular vesicles mentioned above, and optionally comprising pharmaceutically acceptable excipients.

[0062] In embodiments of the present invention, specific excipients may be selected according to the intended administration mode and therapeutic application, and the pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically, nutritionally, or physiologically acceptable carriers.

[0063] Furthermore, the pharmaceutical composition includes, but is not limited to, tablets, granules, pills, capsules, emulsions, ointments, gels, suspensions, solutions, powders, transdermal patches, sprays, suppositories, or implants.

[0064] A ninth aspect of the present invention provides a kit comprising the above-described mammalian extracellular vesicles or the above-described pharmaceutical composition.

[0065] The tenth aspect of the present invention provides a method for diagnosing, preventing or treating a disease, the method comprising administering to a subject the above-described mammalian extracellular vesicles (preferably mammalian extracellular vesicles loaded with pharmaceutically active molecules) or the above-described pharmaceutical composition.

[0066] In this embodiment of the invention, the specific disease to be detected, prevented or treated by the mammalian extracellular vesicles is preferably determined based on the drug active molecules loaded in the mammalian extracellular vesicles, including but not limited to various tumor diseases or infectious diseases.

[0067] By adopting the above technical solution, the present invention has the following advantages compared with the prior art:

[0068] This invention significantly increases the yield of extracellular vesicles (EVs) in mammalian cells by overexpressing the plant-derived FREE1 protein, compared to existing methods. This beneficial effect extends beyond ordinary, unloaded EVs; it also significantly increases the yield of EVs loaded with therapeutic drugs (such as interleukins and anti-inflammatory proteins). Furthermore, compared to existing methods that add exogenous small molecule compounds to the culture medium, affecting the growth and metabolism of mammalian cells, the preparation method of this invention stably increases the yield of extracellular vesicles while avoiding the introduction of exogenous substances into the cell culture medium, thereby reducing impurities in the cell culture medium and resulting in higher purity of the extracted EVs. Attached Figure Description

[0069] Figure 1 This is a particle size distribution diagram of exosomes from Example 1;

[0070] Figure 2 This is a particle size distribution diagram of exosomes in Comparative Example 1;

[0071] Figure 3 This is a particle size distribution diagram of exosomes from Comparative Example 2;

[0072] Figure 4This is a particle size distribution diagram of exosomes in Comparative Example 3;

[0073] Figure 5 This is a particle size distribution diagram of exosomes from Example 2;

[0074] Figure 6 This is a particle size distribution diagram of exosomes in Comparative Example 4;

[0075] Figure 7 This is a particle size distribution diagram of exosomes from Example 3;

[0076] Figure 8 This is a particle size distribution diagram of exosomes from Comparative Example 5;

[0077] Figure 9 Transmission electron micrograph of exosomes from Example 1;

[0078] Figure 10 This is a transmission electron microscope image of exosomes from Comparative Example 1.

[0079] Figure 11 Transmission electron microscopy image of exosomes in Comparative Example 2;

[0080] Figure 12 This is a transmission electron microscope image of the exosomes in Comparative Example 3;

[0081] Figure 13 This is a comparison chart showing the yield of exosomes loaded with IL15 interleukin in the supernatant of Example 2 and Comparative Example 4;

[0082] Figure 14 This is a comparison chart showing the yield of exosomes loaded with srIkB anti-inflammatory protein in the supernatant of Example 3 and Comparative Example 5. Detailed Implementation

[0083] The mechanism by which mammalian cells produce extracellular vesicles relies solely on the ESCRT / ATP pathway, and the production of extracellular vesicles secreted through this pathway is very limited. FREE1 protein, a protein involved in plant vesicle transport and expressed only in plant cells, was overexpressed in mammalian cells in this invention. Unexpectedly, it was found that overexpressing plant-derived FREE1 protein in mammalian cells effectively promoted the production of more extracellular vesicles and also had a stable effect on increasing the production of exosomes carrying these vesicles.

[0084] The technical solution of the present invention will be further described below with reference to specific embodiments and comparative examples.

[0085] In this invention, it should be noted that, unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in conjunction with the entire text and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0086] "FREE1 protein" is a protein involved in the process of plant vesicle transport. The FREE1 protein in this invention includes any FREE1 protein in the art that has the function of participating in the process of plant vesicle transport, including FREE1 proteins from different plant sources.

[0087] Extracellular vesicles are a group of vesicles with a lipid bilayer membrane structure secreted by living cells. Based on their size, they can be divided into: exosomes, microvesicles, and apoptotic bodies.

[0088] "Exosomes" are cell-derived extracellular vesicles (EVs) with a diameter of 30-150 nm. They include a membrane that encloses the internal space. The membrane is a phospholipid bilayer structure. Exosomes can mediate the intercellular transfer of intracellular substances.

[0089] "Loading" refers to attaching and enriching cargo into extracellular vesicles, including various forms such as cargo inside the cavity of extracellular vesicles, on the membrane of extracellular vesicles, or attached to the outside of the membrane of extracellular vesicles.

[0090] "Extracellular vesicle scaffold protein" refers to a connector used to attach cargo to extracellular vesicles, which is used to anchor any cargo of interest to extracellular vesicles.

[0091] "Cargo" primarily refers to "pharmaceutical active molecules," that is, any functional (e.g., preventative, therapeutic, diagnostic) proteins and their fragments, polypeptides and their fragments, nucleic acids and their fragments, or small molecule compounds of interest. "Cargo" is also often referred to as "functional cargo" or "active substance."

[0092] "Small molecule compounds" refer to compounds with a molecular weight of less than 1000, including conventional organic and inorganic compounds.

[0093] "Polypeptide" and "oligopeptide" are defined according to the length of the peptide chain. Peptides with a length of 10 to 100 amino acids are called "polypeptides", while those with a length of less than 10 amino acids are called "oligopeptides".

[0094] "Recombinase" is a class of enzymes that can promote the recombination or rearrangement of modified sequences between DNA molecules. Common recombinases can be loaded and delivered using the method of this invention. Common recombinases include, but are not limited to, Cre recombinase, Dre recombinase, Flp recombinase, RadA protein, RAD51 protein, and RecA protein.

[0095] Cytokines are a class of small-molecule proteins with broad biological activity synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, and NK cells) and certain non-immune cells (endothelial cells, epidermal cells, and fibroblasts) upon stimulation. Cytokines generally regulate cell growth, differentiation, and effector functions by binding to corresponding receptors, modulating immune responses and possessing multiple functions including regulating innate and adaptive immunity, hematopoiesis, cell growth, and repair of damaged tissues. Common cytokines include, but are not limited to, pro-inflammatory cytokines, anti-inflammatory cytokines, interleukins, interferons, growth factors, and chemokines.

[0096] "Gene editing proteins" are a class of proteins used in gene editing systems. Common gene editing proteins include Cas proteins (such as Cas9, Cas12, Cas13, Cas2, Cas3, Cas10, Cas14, Cas12f, CasX, CasY, or Csm), transposon proteins, zinc finger proteins, and TALE proteins.

[0097] "Exogenous nucleic acid fragments" include any gene or fragment thereof of interest, such as a gene or fragment encoding a scaffold protein. Exogenous nucleic acid fragments have a different origin from the host cell; for example, nucleic acid sequences isolated from an organism different from the host cell are exogenous nucleic acid fragments relative to the host cell.

[0098] "Plasmid" refers to a circular double-stranded DNA that can accept exogenous nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0099] A promoter is a nucleic acid sequence that controls the transcription of a coding sequence. Promoter sequences include modified sequences that enable RNA polymerase to recognize, bind to, and initiate transcription. Promoters can affect the transcription of genes located on the same nucleic acid molecule as themselves or genes located on different nucleic acid molecules.

[0100] "Host cell" is also called recipient cell, with an example host cell including human embryonic kidney cell HEK293. It should be understood that, due to natural, accidental, or intentional mutations, the offspring of a single parent cell may not be completely identical to the original parent in terms of morphology or in terms of genome or total DNA complement.

[0101] The technical solution of the present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0102] The implementation conditions used in the following examples and comparative examples can be further adjusted according to specific requirements. Unless otherwise specified, the implementation conditions are generally those used in routine experiments. Instruments, materials, and reagents, unless otherwise specified, are commercially available.

[0103] Example 1

[0104] This embodiment provides a method for preparing exosomes, as detailed below:

[0105] 1. Constructing plasmids

[0106] Using pIRES plasmid as the expression vector, a recombinant expression vector for expressing the FREE1 protein was constructed. The expression cassette of the first recombinant expression vector was designed as follows: the CMV promoter (sequence shown in SEQ ID NO:1) was used as the transcription start point, and SV40 polyA (sequence shown in SEQ ID NO:2) was used as the transcription end point to express the FREE1 protein (sequence shown in SEQ ID NO:3).

[0107] The recombinant expression vector was prepared according to the following specific steps:

[0108] (1) PCR cloning: Using conventional methods in this field, upstream and downstream primers were designed according to the expression cassette, and upstream, downstream primers and template DNA were synthesized. In a 0.2 mL EP tube, 25 μL of DNA polymerase (2×Phanta MaxMaster Mix, Vazyme, P515-01), 2 μL of upstream primer, 2 μL of downstream primer, and 10 g of template DNA were added, and the volume was brought up to 50 μL with enzyme-free water (RNase-free dd H2O, Vazyme, P071-01-AA). After mixing, the tube was placed in a PCR instrument, and the amplification program was designed according to the primer annealing temperature.

[0109] (2) Purification of PCR products: Purify PCR products according to the kit instructions (Vazyme, DC301-01).

[0110] (3) Enzyme digestion: Add 5 μL rCutSmart to a 0.2 mL EP tube. TM Buffer (BioLabs, 136004s), 0.5 μL Pac I enzyme (BioLabs, R0547L), 0.5 μL Not I enzyme (BioLabs, R3189L), 2 μg of PCR purified fragment, and 5 μg pAAVS1 vector plasmid were prepared. The mixture was brought to a final volume of 10 μL with enzyme-free water (RNase-free ddH2O, Vazyme, P071-01-AA), mixed thoroughly, and then incubated in a PCR instrument at 37°C for 16 h for enzyme digestion.

[0111] (4) Ligation transformation: In a 0.2 mL EP tube, add 4 μL of the enzyme digestion solution obtained in step (3), 1 μL of T4 DNA ligase (Novizan Vazyme, N103-01-AA) and 1 μL of T4 DNA ligase buffer (Novizan Vazyme, N103-01-AC). After incubating at room temperature for 10 min, 5 μL was added to competent cells (DH5α, Vazyme, C502-03). The cells were incubated on ice for 30 min, 42℃ for 90 s, and on ice for 2 min. Then, 1 mL of LB medium (BeyoPure™, 04.05.ST156) was added and the cells were placed in a shaking incubator for 1 h. After incubation, the cells were centrifuged at 12000g for 1 min, the supernatant was discarded, and 100 μL was resuspended and added to a bacterial culture dish (Biosharp, BS-90-D). The mixture was then spread evenly with a disposable plastic spreader (Biosharp, BS-PS-A) and stored in a 37℃ incubator for later use.

[0112] In step (1), the upstream primer sequence designed according to the expression cassette of the recombinant expression vector for expressing the FREE1 protein is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:5, and the template DNA sequence is shown in SEQ ID NO:6.

[0113] 2. Cell transfection

[0114] The specific steps are as follows:

[0115] (1) Preparation of transfected cells

[0116] EXPI293F cells were seeded at a concentration of 1.5E+6 / mL, with a viability of over 95%, in 125mL shake flasks with a total volume of 25mL serum-free cell culture medium (Opmai medium, CD05). The flasks were incubated overnight at 37°C and 100rpm in an 8% CO2 constant-temperature shaker. Transfection began the following day.

[0117] (2) PEI transfection

[0118] The transfection process using TA293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001) is as follows:

[0119] The target plasmid was extracted from the transformed competent cells preserved by the above-mentioned plating culture using conventional techniques in the art, thus obtaining the constructed recombinant expression vector for expressing the FREE1 protein. Two 15 mL centrifuge tubes were prepared. In one tube, 1.25 mL of serum-free cell culture medium and 25 μg of the target plasmid were added, and the mixture was vortexed. In the other tube, 1.25 mL of serum-free cell culture medium and 125 μL of TA293 transfection reagent were added, and the mixture was vortexed. All liquid in the centrifuge tube containing the transfection reagent was transferred to the centrifuge tube containing the target plasmid, and the mixture was vortexed. The cells were incubated at room temperature for 10 minutes to prepare the plasmid-vector complex. EXPI293F cells were removed from the constant-temperature shaker, and the prepared plasmid-vector complex was added dropwise while shaking. The cells were then returned to the 8% CO2 constant-temperature shaker and cultured at 37°C and 100 rpm for 72 hours.

[0120] 3. Isolation and purification of exosomes

[0121] After 72 hours of shaking culture, the cell culture medium was first centrifuged at 300g, 4℃, and low speed for 5 min, and the supernatant was collected. Then, the supernatant was ultracentrifuged at 133900g, 4℃ for 1 h, and the precipitate was collected. The precipitate (i.e., the isolated exosomes) was resuspended in 100μL PBS and stored at 4℃ for later use.

[0122] Comparative Example 1

[0123] This comparative example directly used EXPI293F cells to prepare exosomes, as detailed below:

[0124] 1. EXPI293F cells were seeded at a concentration of 1.5E+6 / mL, with a viability of over 95%, into 125mL shake flasks containing 25mL of serum-free cell culture medium (Opmai medium, CD05). The flasks were then incubated in an 8% CO2 constant-temperature shaker at 37°C and 100rpm for 72 hours.

[0125] 2. After 72 hours of shaking culture, centrifuge the cell culture medium at 300g, 4℃, low speed for 5 min, collect the supernatant, and then centrifuge the supernatant at 133900g, 4℃ for 1 h, collect the precipitate. Resuspend the precipitate (i.e., the isolated exosomes) in 100μL PBS and store at 4℃ for later use.

[0126] Comparative Example 2

[0127] This comparative example provides another method for exosome preparation. In this comparative example, a calcium ion carrier (calcimycin A23187) was added to a serum-free culture medium at a final concentration of 10 μM, as follows:

[0128] 1. EXPI293F cells were seeded at a concentration of 1.5E+6 / mL, with a viability of over 95%, into 125mL shake flasks containing 25mL of serum-free cell culture medium (Opmai medium, CD05) supplemented with 10μM catecholamine. The flasks were then incubated in an 8% CO2 constant-temperature shaker at 37°C and 100rpm for 72 hours.

[0129] 2. After 72 hours of shaking culture, centrifuge the cell culture medium at 300g, 4℃, low speed for 5 min, collect the supernatant, and then centrifuge the supernatant at 133900g, 4℃ for 1 h, collect the precipitate. Resuspend the precipitate (i.e., the isolated exosomes) in 100μL PBS and store at 4℃ for later use.

[0130] Comparative Example 3

[0131] This comparative example provides another method for exosome preparation, in which ceramide is added to a serum-free culture medium to a final concentration of 100 μg / mL, as follows:

[0132] 1. EXPI293F cells were seeded at a concentration of 1.5E+6 / mL, with a viability of over 95%, in 125mL shake flasks. The total volume of serum-free cell culture medium (Opmai medium, CD05) was 25mL, with ceramide added to a final concentration of 100μg / mL. The flasks were then incubated in an 8% CO2 constant-temperature shaker at 37°C and 100rpm for 72 hours.

[0133] 2. After 72 hours of shaking culture, centrifuge the cell culture medium at 300g, 4℃, low speed for 5 min, collect the supernatant, and then centrifuge the supernatant at 133900g, 4℃ for 1 h, collect the precipitate. Resuspend the precipitate (i.e., the isolated exosomes) in 100μL PBS and store at 4℃ for later use.

[0134] Example 2

[0135] This embodiment provides a method for preparing exosomes loaded with IL15 interleukin, as detailed below:

[0136] 1. Construction of recombinant expression plasmid vector system

[0137] A recombinant expression vector for expressing the FREE1 protein was constructed and prepared according to the method in Example 1.

[0138] Using pIRES plasmid as the expression vector and PTGFRN protein (sequence shown in SEQ ID NO:7) as the exosome scaffold protein, a recombinant expression vector for expressing cargo (IL15 interleukin, sequence shown in SEQ ID NO:8) was constructed. The expression cassette was designed as follows: using the CMV promoter (sequence shown in SEQ ID NO:1) as the transcription start point and SV40 polyA (sequence shown in SEQ ID NO:2) as the transcription end point, to express the fusion protein of IL15 interleukin and PTGFRN, with IL15 interleukin linked to the N-terminus of the PTGFRN protein. The recombinant expression vector for expressing the IL15 interleukin and PTGFRN fusion protein was prepared according to the method in Example 1. The upstream primer sequence designed according to the expression cassette of the recombinant expression vector for expressing the IL15 interleukin and PTGFRN fusion protein is shown in SEQ ID NO:9, the downstream primer sequence is shown in SEQ ID NO:10, and the template DNA sequence is shown in SEQ ID NO:11.

[0139] 2. Cell transfection

[0140] The specific steps are as follows:

[0141] (1) Preparation of transfected cells

[0142] EXPI293F cells were seeded at a concentration of 1.5E+6 / mL, with a viability of over 95%, in 125mL shake flasks with a total volume of 25mL serum-free cell culture medium (Opmai medium, CD05). The flasks were incubated overnight at 37°C and 100rpm in an 8% CO2 constant-temperature shaker. Transfection began the following day.

[0143] (2) PEI transfection

[0144] The transfection process using TA293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001) is as follows:

[0145] The target plasmid was extracted from the transformed competent cells preserved by the above-mentioned plating culture using conventional techniques in the art, thus obtaining the constructed recombinant expression vector for expressing FREE1 protein and the recombinant expression vector for expressing the fusion protein of IL15 interleukin and PTGFRN. Two 15 mL centrifuge tubes were prepared. In one tube, 1.25 mL of serum-free cell culture medium and 25 μg of the target plasmid (a 1:1 mass ratio mixture of the recombinant expression vector for FREE1 protein and the recombinant expression vector for expressing the fusion protein of IL15 interleukin and PTGFRN) were added, and the mixture was vortexed. In the other tube, 1.25 mL of serum-free cell culture medium and 125 μL of TA293 transfection reagent were added, and the mixture was vortexed. All liquid in the centrifuge tube containing the transfection reagent was transferred to the centrifuge tube containing the target plasmid, and the mixture was vortexed. The mixture was incubated at room temperature for 10 minutes to prepare the plasmid-vector complex. EXPI293F cells were removed from the constant temperature shaker, and the prepared plasmid-vector complex was added dropwise while shaking. The cells were then returned to the 8% CO2 constant temperature shaker and cultured at 37°C and 100 rpm for 72 hours.

[0146] 3. Isolation and purification of exosomes

[0147] After 72 hours of shaking culture, the cell culture medium was first centrifuged at 300g, 4℃, and low speed for 5 min, and the supernatant was collected. Then, the supernatant was ultracentrifuged at 133900g, 4℃ for 1 h, and the precipitate was collected. The precipitate (i.e., the isolated exosomes loaded with IL15 interleukin) was resuspended in 100μL PBS and stored at 4℃ for later use.

[0148] Comparative Example 4

[0149] This comparative example provides another method for preparing exosomes loaded with IL15 interleukin. The host cells (EXPI293F cells) in this comparative example do not express the FREE1 protein. The specific steps of this comparative example are as follows:

[0150] 1. EXPI293F cells were seeded at a concentration of 1.5E+6 / mL, with a viability of over 95%, in 125mL shake flasks with a total volume of 25mL serum-free cell culture medium (Opmai medium, CD05). The flasks were incubated overnight at 37°C and 100rpm in an 8% CO2 constant-temperature shaker. Transfection began the following day.

[0151] 2. Prepare two 15mL centrifuge tubes. Add 1.25mL of serum-free cell culture medium and 25μg of the target plasmid (the recombinant expression vector for the fusion protein of IL15 interleukin and PTGFRN prepared in Example 2) to one tube, and vortex to mix. Add 1.25mL of serum-free cell culture medium and 125μL of TA293 transfection reagent to the other tube, and vortex to mix. Transfer all liquid from the centrifuge tube containing the transfection reagent to the centrifuge tube containing the target plasmid, and vortex to mix. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove EXPI293F cells from the constant-temperature shaker, add the prepared plasmid-vector complex dropwise while shaking, and then return the cell to the 8% CO2 constant-temperature shaker and incubate at 37°C and 100rpm for 72 hours.

[0152] 3. After 72 hours of shaking culture, centrifuge the cell culture medium at 300g, 4℃, low speed for 5 min, collect the supernatant, and then centrifuge the supernatant at 133900g, 4℃ for 1 h, collect the precipitate. Resuspend the precipitate (i.e., the isolated exosomes loaded with IL15 interleukin) in 100μL PBS and store at 4℃ for later use.

[0153] Example 3

[0154] This embodiment provides an exosome loaded with the srIkB anti-inflammatory protein.

[0155] In this embodiment, pIRES plasmid was used as the expression vector, and Basp1 protein (sequence shown in SEQ ID NO:12) was used as the exosome scaffold protein to construct a recombinant expression vector for expressing cargo (srIkB anti-inflammatory protein, sequence shown in SEQ ID NO:13). The expression cassette was designed as follows: the CMV promoter (sequence shown in SEQ ID NO:1) was used as the transcription start point, and SV40 polyA (sequence shown in SEQ ID NO:2) was used as the transcription end point to express the fusion protein of srIkB anti-inflammatory protein and Basp1 protein. The srIkB anti-inflammatory protein was linked to the C-terminus of the Basp1 protein. The recombinant expression vector for expressing the fusion protein of srIkB anti-inflammatory protein and Basp1 protein was prepared according to the method of Example 1. The upstream primer sequence designed according to the expression cassette of the recombinant expression vector for expressing the fusion protein of srIkB anti-inflammatory protein and Basp1 protein is shown in SEQ ID NO:14, the downstream primer sequence is shown in SEQ ID NO:15, and the template DNA sequence is shown in SEQ ID NO:16.

[0156] Following the method in Example 2, EXPI293F cells were transfected with a mixture of a recombinant expression vector for expressing FREE1 protein and a recombinant expression vector for expressing srIkB anti-inflammatory protein and Basp1 protein at a mass ratio of 1:1. After the cells were cultured, exosomes loaded with srIkB anti-inflammatory protein were isolated.

[0157] Comparative Example 5

[0158] This comparative example provides another method for preparing exosomes loaded with the srIkB anti-inflammatory protein. The host cells (EXPI293F cells) in this comparative example do not express the FREE1 protein. Except for replacing the recombinant expression vector used in Comparative Example 4 to express the fusion protein of IL15 interleukin and PTGFRN with the recombinant expression vector prepared in Example 3 for expressing the srIkB anti-inflammatory protein and Basp1 protein, all other operations are the same as in Comparative Example 4.

[0159] Example 4

[0160] This embodiment provides a method for preparing exosomes loaded with sgRNA, the sequence of which is shown in SEQ ID NO:17. In this embodiment, following the method of Example 3, exosomes loaded with Cas9 protein (sequence shown in SEQ ID NO:18) are first prepared using Basp1 protein as the exosome scaffold protein. Then, the sgRNA is mixed with the exosomes loaded with Cas9 protein, using Cas9 protein as a protein linker. The sgRNA and Cas9 protein form an RNP complex, thereby loading the sgRNA onto the exosomes to obtain exosomes loaded with sgRNA.

[0161] Example 5

[0162] This embodiment provides a method for preparing exosomes loaded with a small molecule compound (rapamycin), using the FKBP12 protein (sequence shown in SEQ ID NO:19) as a protein linker. In this embodiment, following the method of Example 3, exosomes loaded with FKBP12 protein are first prepared using Basp1 protein as an exosome scaffold protein. Then, rapamycin is mixed with the exosomes loaded with FKBP12 protein, forming a complex with the FKBP12 protein, thereby loading rapamycin onto the exosomes to obtain exosomes loaded with the small molecule compound.

[0163] (1) The exosome resuspensions of the above examples and comparative examples were subjected to NTA analysis using a ZetaView instrument (Fuliu Biotechnology Particle Metrix, catalog number N30E). The particle size distribution of exosomes in Example 1 is shown in [Figure 1]. Figure 1 The particle size distribution of exosomes in Comparative Example 1 is shown in [reference needed]. Figure 2 The particle size distribution of exosomes in Comparative Example 2 is shown in [reference needed]. Figure 3 The particle size distribution of exosomes in Comparative Example 3 is shown in [reference needed]. Figure 4 The particle size distribution of the exosomes loaded with IL15 interleukin in Example 2 is shown in [Figure 2]. Figure 5 The particle size distribution of exosomes loaded with IL15 interleukin in Comparative Example 4 is shown in [Figure 4]. Figure 6 The particle size distribution of exosomes loaded with srIkB anti-inflammatory protein in Example 3 is shown in [reference needed]. Figure 7 The particle size distribution of exosomes loaded with the srIkB anti-inflammatory protein in Comparative Example 5 is shown in [Figure 5]. Figure 8 The average particle size and median particle size are shown in Table 1.

[0164]

[0165] Table 1 shows that the particle size of the exosomes in the examples and comparative examples is between 110 and 170 nm. IL15 interleukin is loaded on the membrane surface of the exosomes, while srIkB anti-inflammatory protein is loaded inside the lumen of the exosomes. Therefore, the particle size of the exosomes loaded with IL15 interleukin in Example 2 is larger than that of the exosomes loaded with srIkB anti-inflammatory protein in Example 3.

[0166] (2) The structures of the exosomes prepared in Example 1 and Comparative Examples 1-3 were observed under a transmission electron microscope (TEM). Figures 9-12 As shown, the exosomes of Examples 1 and Comparative Examples 1-3 all exhibited typical exosome vesicle structures (cup-shaped or saucer-shaped) under electron microscopy, and the exosomes of Example 1 had a cleaner background and higher purity.

[0167] (3) Comparison of exosome production of cargo

[0168] Equal volumes of the supernatant from Example 2 and Comparative Example 4 were analyzed by Western blotting (WB). The electrophoresis results are as follows: Figure 13 As shown, the band of IL15 interleukin in Example 2 is brighter, indicating that the yield of exosomes loaded with IL15 interleukin in the supernatant of Example 2 is higher than that in Comparative Example 4.

[0169] Equal volumes of the supernatant from Example 3 and Comparative Example 5 were subjected to Western blotting (WB) analysis. The electrophoresis results are as follows: Figure 14 As shown, the band of the srIkB anti-inflammatory protein in Example 3 is brighter, indicating that the yield of exosomes loaded with the srIkB anti-inflammatory protein in the supernatant of Example 3 is higher than that in Comparative Example 5.

[0170] (4) The above examples and comparative examples were repeated three times. The exosome particle number concentration of the three batches of exosome resuspension was analyzed by NTA. The exosome yield per unit volume of cell supernatant and the exosome yield per single cell were calculated. The average exosome yield per unit volume of cell supernatant and the average exosome yield per single cell of the three batches of each example and comparative example are shown in Table 2.

[0171]

[0172] The method for calculating the exosome yield per unit volume of cell supernatant is: exosome particle number concentration obtained from NTA analysis × total volume of exosomes after PBS resuspension ÷ total volume of cell supernatant collected by low-speed centrifugation.

[0173] The yield of exosomes per cell was calculated as follows: exosome particle count concentration obtained from NTA analysis × total volume of exosomes after resuspending in PBS ÷ total number of cells in the cell culture medium at the time of exosome harvesting. The total cell count was determined using a cell counter and trypan blue staining.

[0174] Table 2 shows that, compared with Comparative Example 1, Comparative Examples 2 and 3 did not increase exosome production, while Example 1 significantly increased exosome production, with a 91% increase in exosome production per mL of cell supernatant and a 142% increase in exosome production per cell. Compared with Comparative Example 4, Example 2 increased the exosome production per mL of cell supernatant loaded with IL15 interleukin by 33% and the exosome production per cell by 68%. Compared with Comparative Example 5, Example 3 increased the exosome production per mL of cell supernatant loaded with srIkB anti-inflammatory protein by 118% and the exosome production per cell by 187%.

[0175] The results above show that this invention can significantly increase exosome production by overexpressing the plant-derived FREE1 protein in mammalian cells. This beneficial effect is not limited to ordinary exosomes; it also significantly increases the production of exosomes loaded with therapeutic drugs such as interleukins and anti-inflammatory proteins. Furthermore, exosomes extracted using the method of this invention have a cleaner background under electron microscopy, indicating higher purity.

[0176] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand the content of the present invention and implement it. However, this description should not be construed as limiting the scope of protection of the present invention. Furthermore, the present invention is not limited to the above-described embodiments. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing extracellular vesicles in mammalian cells, characterized in that, The step includes isolating the extracellular vesicles of the mammalian cells from a culture medium expressing the FREE1 protein. The host cell for the recombinant mammalian cells is the embryonic kidney cell line. The amino acid sequence of the FREE1 protein is shown in SEQ ID NO:

3.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: (1) Construct and prepare a recombinant expression vector for expressing FREE1 protein; (2) The recombinant mammalian cells were obtained by transfecting mammalian cells with the recombinant expression vector; (3) The recombinant mammalian cells were cultured to obtain a recombinant mammalian cell culture medium; (4) The extracellular vesicles of the mammalian cells were isolated from the recombinant mammalian cell culture medium.

3. The preparation method according to claim 2, characterized in that, The recombinant expression vector used is a eukaryotic expression plasmid vector or a viral plasmid vector; And / or, the separation includes one or more of ultracentrifugation, adsorption chromatography, immunoassay, or sieving. And / or, the recombinant mammalian cell culture medium is a serum-free medium; And / or, the recombinant mammalian cells are cultured at 34°C to 38°C under conditions of 5% to 10% CO2 to obtain the recombinant mammalian cell culture medium.

4. The preparation method according to claim 2, characterized in that, The host cell for the recombinant mammalian cells was HEK293 cells; And / or, the recombinant expression vector used is a pAAVS1 plasmid, a pIRES plasmid, or a pLENTI plasmid.

5. A method for preparing extracellular vesicles of mammalian cells loaded with drug-active molecules, characterized in that, The preparation method includes the following steps: 1) Obtain recombinant mammalian cell culture medium, wherein the recombinant mammalian cells express not only a fusion protein based on extracellular vesicle scaffold protein, but also FREE1 protein. The fusion protein based on extracellular vesicle scaffold protein is a fusion protein formed by the extracellular vesicle scaffold protein and a drug active molecule or a linker for linking the drug active molecule. The host cell of the recombinant mammalian cells is the embryonic kidney cell line. The amino acid sequence of the FREE1 protein is shown in SEQ ID NO:

3. 2) The extracellular vesicles of the mammalian cells were isolated from the recombinant mammalian cell culture medium; and, When the fusion protein based on extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and a linker for connecting drug active molecules, the preparation method further includes: 3) After the separation step, the separated mammalian extracellular vesicles are mixed with the active pharmaceutical molecule, so that the active pharmaceutical molecule binds to the mammalian extracellular vesicle through the linker.

6. The preparation method according to claim 5, characterized in that, Step 1) includes: constructing and preparing a first recombinant expression vector for expressing FREE1 protein and a second recombinant expression vector for expressing a fusion protein formed by extracellular vesicle scaffold protein and drug-active molecules or linkers; transfecting mammalian cells with the first recombinant expression vector and the second recombinant expression vector sequentially or simultaneously to obtain recombinant mammalian cells; and culturing the recombinant mammalian cells to obtain a recombinant mammalian cell culture medium.

7. The preparation method according to claim 6, characterized in that, The expression vectors used in the first recombinant expression vector and the second recombinant expression vector are eukaryotic expression plasmid vectors or viral plasmid vectors, respectively. And / or, the separation includes one or more of ultracentrifugation, adsorption chromatography, immunoassay, or sieving separation; And / or, the culture is performed using a serum-free culture medium; And / or, the culture is carried out at 34℃~38℃ and 5%~10% CO2. And / or, the active pharmaceutical molecule includes one or more of protein molecules and their fragments, polypeptide molecules and their fragments, nucleic acids and their fragments, or small molecule compounds; And / or, when the active pharmaceutical molecule is a protein molecule and its fragments and / or a polypeptide molecule and its fragments, the fusion protein based on extracellular vesicle scaffold protein is a fusion protein formed by the protein serving as the extracellular vesicle scaffold protein and the active pharmaceutical molecule. And / or, when the active pharmaceutical molecule is a nucleic acid and its fragments and / or a small molecule compound, the fusion protein based on extracellular vesicle scaffold protein is a fusion protein formed by a protein that serves as an extracellular vesicle scaffold protein and a linker. And / or, the extracellular vesicle scaffold proteins include one or more of the following: BASP1 protein or a fragment thereof, PTGFRN protein or a fragment thereof, TSPAN2 protein or a fragment thereof, TSPAN3 protein or a fragment thereof, CD63 protein or a fragment thereof, CD9 protein or a fragment thereof, CD81 protein or a fragment thereof, LEAP protein or a fragment thereof, and PVR protein or a fragment thereof.

8. A recombinant mammalian cell, characterized in that, The recombinant mammalian cell is the same as the recombinant mammalian cell described in any one of claims 1 to 4, or the same as the recombinant mammalian cell described in any one of claims 5 to 7.

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