WO2023093780A1 - 工程化迁移体及其制备方法和用途 - Google Patents

工程化迁移体及其制备方法和用途 Download PDF

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WO2023093780A1
WO2023093780A1 PCT/CN2022/133863 CN2022133863W WO2023093780A1 WO 2023093780 A1 WO2023093780 A1 WO 2023093780A1 CN 2022133863 W CN2022133863 W CN 2022133863W WO 2023093780 A1 WO2023093780 A1 WO 2023093780A1
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migratory
cells
membrane
cell
protein
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PCT/CN2022/133863
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English (en)
French (fr)
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俞立
王东锔
戴宸
贾漠野
王硕
丁天伦
钟丘实
郑羿
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北京迈格松生物科技有限公司
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Priority to CN202280007395.0A priority Critical patent/CN116964192A/zh
Publication of WO2023093780A1 publication Critical patent/WO2023093780A1/zh
Priority to US18/351,696 priority patent/US20240084264A1/en

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Definitions

  • the invention relates to the field of biomedicine, and specifically relates to an engineered migratory body, a method for preparing the engineered migratory body, a delivery system containing the engineered migratory body and a preparation method thereof.
  • the present application finds that engineered migratory bodies that are structurally similar to natural migratory bodies can be induced by various methods, and the engineered migratory bodies are different from "hypoosmotic pressure-induced vesicles" known in the art.
  • the engineered migratory body discovered in this application is of biological origin, which has the advantages of low toxicity and low immunogenicity of biological origin; etc. have unique advantages; at the same time, the preparation method of the engineered migrator is simple and the yield is high. Therefore, the engineered migratory body provided by this application has extremely high application value in biomedical fields such as drug delivery and vaccine preparation.
  • the present application provides an engineered migratory body, a method for preparing the engineered migratory body, a delivery system comprising the engineered migratory body and a preparation method thereof.
  • the engineered migratory body includes, but is not limited to, the following advantages: low toxicity, low immunogenicity, ability to transport a variety of exogenous load molecules, suitable biodistribution in vivo, simple preparation method, and/or high yield.
  • the engineered migration body provided by this application can be used for drug delivery and vaccine preparation, etc., and has excellent effects.
  • the application provides a method for preparing a migratory body, the method comprising causing cells to generate relative displacements and then producing a migratory body derived from the cells.
  • the method further comprises isolating migratory bodies produced by said cells.
  • the method comprises subjecting said cells to hypotonic treatment.
  • hypotonic treatment comprises placing said cells in a hypotonic buffer solution.
  • the method wherein the hypotonic treatment comprises placing the cells in a buffer solution, and reducing the osmotic pressure of the buffer solution into a hypotonic buffer.
  • said reduction comprises linear reduction and/or stepwise reduction.
  • the method wherein the osmolarity of the hypotonic buffer solution is less than about 305 mOsmol/L.
  • the osmotic pressure of the hypotonic buffer solution is from about 10 mOsmol/L to about 274.5 mOsmol/L.
  • the method comprises disrupting the cytoskeleton of said cell.
  • disrupting the cytoskeleton of the cell comprises contacting the cell with a cytoskeleton disrupting agent.
  • said cytoskeleton disrupting agent comprises a microfilament and/or microtubule depolymerizing agent.
  • said cytoskeleton disrupting agent comprises Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D and/or Cytochalasin E .
  • the method comprises inhibiting the cell volume regulating function of said cells.
  • inhibiting the cell volume regulating function of the cell comprises reducing the quantity and/or function of the volume regulating protein in the cell.
  • the method wherein the volume-regulated protein comprises a volume-regulated ion channel and/or a transporter.
  • volume-regulated ion channel comprises a volume-regulated anion channel VRAC and/or a volume-regulated cation channel VRCC.
  • volume-regulating anion channel VRAC comprises SWELL1 or a functionally active fragment thereof.
  • the method wherein the volume-regulated cation channel VRCC comprises TRPV4, TRPM3 and/or functionally active fragments thereof.
  • said transporter comprises a cotransporter.
  • the method wherein the co-transporter comprises KCC1, KCC3 and/or KCC4.
  • inhibiting the cell volume regulating function of the cells comprises placing the cells in a buffered solution having reduced volume regulating capabilities.
  • the cations include K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , Ba 2+ , Mn 2+ , Fe 2+ , Ni 2+ , Zn 2+ , Al 3+ , Fe 3+ , CH 3 NH 3 + , C 2 H 5 NH 3 + , (CH 3 ) 2 NH 2 + , (C 2 H 5 ) 2 NH 2 + , (C 2 H 5 ) 3 N + , ammonia ion and/or choline ion.
  • the anions include Br - , Cl - , I - , F - , OH - , HCO 3 - , H 2 PO 4 - , NO 2 - , NO 3 - , CN - , HPO 4 2- , CO 3 2- , SO 4 2- and/or PO 4 3- .
  • said method comprises detaching said cell from its adhesion surface.
  • the method comprises causing a relative displacement between the cell membrane of the cell and its adhesion surface.
  • it further comprises increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cells.
  • the method comprises allowing said cells to overexpress said tetraspanin protein, functional fragments and/or functional variants thereof.
  • tetraspanin protein is selected from: Tspan1, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, Tspan10, Tspan11, Tspan12, Tspan13, Tspan14, Tspan15, Tspan16, Tspan17 , Tspan18, Tspan19, Tspan20(UPK1B), Tspan21(UPK1A), Tspan22(PRPH2), Tspan23(ROM1), Tspan24(CD151), Tspan25(CD53), Tspan26(CD37), Tspan27(CD82), Tspan28(CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32 and Tspan33.
  • the method further comprises reducing the size of the migratory body.
  • said reducing the size of said migratory bodies comprises squeezing said migratory bodies using a filter or a squeezer.
  • the method wherein the filter or squeezer has a pore size of about 30 nm to about 10000 nm.
  • the size of the migrator is from about 50 nm to about 8000 nm.
  • said migratory bodies are produced from contractile filaments of said cells.
  • the membrane of the migratory body is enriched with sodium/potassium ATPase and/or its functional fragments.
  • the membrane of the migratory body is enriched with integrin and/or its functional fragments.
  • the membrane of the migratory body is enriched with tetraspanin protein, its functional variant and/or its functional fragment.
  • the membrane of the migratory body is enriched with cholesterol.
  • said mobilizer is enriched with membrane microdomains.
  • the method wherein the content of said migratory body is at least partially reduced or absent compared to a native migratory body produced by the corresponding cell.
  • said at least partially reduced contents comprise intraluminal vesicles.
  • the method is an in vitro or ex vivo method.
  • the method wherein the cells are cultured cells in vitro.
  • the method wherein the cells are suspension-cultured or adherent-cultured cells.
  • said cells comprise primary cells.
  • said primary cells comprise tissue cells derived from organisms including humans, monkeys, mice, rats, rabbits, chickens and/or insects.
  • said primary cells include hepatocytes, spleen cells, kidney cells, tissue macrophages, brain glial cells, osteoclasts, bone marrow cells, leukocytes, fibroblasts and/or fat cells.
  • said leukocytes comprise B cells, T cells, NK cells, dendritic cells, neutrophils and/or macrophages.
  • said cells comprise tumor cells.
  • the method wherein the tumor cells comprise tumor cell lines, primary or limited passage tumor cells derived from patients, tumor stromal cells, and/or tumor organoids.
  • the cells include CHO cells, CHO-K1 cells, HEK293 cells, HEK293T cells, HEK293FT cells, HEK293F cells, Vero cells, NRK cells, L929 cells, MC38 cells, 4T1 cells, DC2 .4 cells, MGC803 cells, Jurkat cells, NK-92MI cells, BJ cells and/or HepG2 cells.
  • said cells comprise leukocytes, stem cells and/or fibroblasts.
  • said stem cells comprise mesenchymal stem cells.
  • Migratory bodies prepared in a method of one embodiment are prepared in a method of one embodiment.
  • the present application provides a migration body prepared in vitro or ex vivo, wherein the size of the migration body is from about 50 nm to about 8000 nm.
  • the migratory body is produced in vitro from contractile filaments of cells.
  • the membrane of the migratory is enriched with sodium/potassium ATPase and/or its functional fragments.
  • the membrane of the migratory body is enriched with integrin and/or its functional fragments.
  • the membrane of the migratory body is enriched with tetraspanin protein, its functional variant and/or its functional fragment.
  • the membrane of the migratory body is enriched with cholesterol.
  • transsome wherein said transsome is enriched with membrane microdomains.
  • the transsome wherein the content of said transsome is at least partially reduced or absent compared to a native transsome produced by the corresponding cell.
  • the migratory body wherein said at least partially reduced contents comprise luminal vesicles.
  • the present application provides a use of a migratory body for delivering exogenous loads.
  • the migration body comprises the migration body described in this application.
  • the present application provides a delivery system comprising a migratory body and one or more exogenous payloads.
  • the exogenous cargo is directly or indirectly bound, linked or embedded into the membrane and/or inside of the migratory body.
  • the migration body comprises the migration body described in this application.
  • said migratory body is derived from cells.
  • the exogenous load comprises one or more targeting substances and/or therapeutically active substances.
  • the exogenous load comprises proteins, lipids, polynucleotides, small molecular compounds, complexes, polysaccharides, polymers, nanoparticles, microparticles and/or organelles.
  • the exogenous cargo includes membrane protein, soluble protein and/or polypeptide.
  • said exogenous cargo comprises DNA and/or RNA.
  • the exogenous load comprises an antibody or an antigen-binding antibody fragment thereof, an integrin or a fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or a fragment thereof, Enzymes, tumor suppressor gene products, siRNA, microRNA, antisense oligonucleotides ASO, mRNA, DNA, gene editing tools and/or cytotoxic agents.
  • the exogenous load includes PAMP, DAMP, CD47, CD24, IL-12, IL-15, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX and/or their functions active fragment.
  • the exogenous load is directly or indirectly connected to the mobilizer through gene editing, exogenous expression, liquid-to-solid conversion, membrane fusion, charge adsorption, physical adsorption and/or chemical linkage combined.
  • the exogenous cargo is bound or embedded into the migrating body through direct or indirect connection with a membrane component of the migrating body.
  • the membrane component of the migratory body comprises membrane proteins, cholesterol, phospholipids, sugar chains on glycoproteins and/or polysaccharides.
  • said indirect linking comprises linking by click chemistry.
  • said indirect linking comprises providing said exogenous cargo linked to a first member of a binding pair, and bringing it into contact with said migrator, said migrator has a membrane comprising A second member of said binding pair, wherein said first member is capable of binding to said second member.
  • the first and second members of the binding pair are selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; HaloTag and its ligand; CP05 and CD63.
  • the exogenous cargo is expressed as a membrane protein on the inner or outer surface of the membrane of the migratory body.
  • the exogenous cargo is expressed on the inner or outer surface of the membrane of the migratory body as a fusion protein fused with a membrane protein or a part thereof.
  • the exogenous load is expressed on the inner or outer surface of the migratory membrane as a fusion protein fused with a membrane protein or a part thereof by gene editing and/or exogenous expression .
  • the present application provides a method for preparing a delivery system, the method comprising providing a migratory body, and allowing the migratory body to carry an exogenous load.
  • the mobilization body is an isolated or purified mobilization body.
  • the making the migrating body carry the exogenous load comprises directly or indirectly connecting or embedding the exogenous load into the membrane of the migrating body and/or the interior of the migrating body.
  • the method further comprises isolating or purifying the migratory body from the cells.
  • the method comprises providing a complex of said exogenous cargo and a first member of a binding pair; causing a cell to produce a migratory body comprising a second member of a binding pair; and causing said A migratory body is contacted with the complex to form the delivery system.
  • the migration body comprises the migration body described in this application.
  • said migratory body is derived from a cell.
  • the exogenous load comprises one or more targeting substances and/or therapeutically active substances.
  • the exogenous load comprises proteins, lipids, polynucleotides, small molecular compounds, complexes, polysaccharides, polymers, nanoparticles, microparticles and/or organelles.
  • said exogenous load comprises membrane protein, soluble protein and/or polypeptide.
  • said exogenous cargo comprises DNA and/or RNA.
  • the exogenous load comprises an antibody or an antigen-binding antibody fragment thereof, an integrin or a fragment thereof, an immunogenic protein, a cytokine, a chemokine, a receptor protein or a fragment thereof, an enzyme , tumor suppressor gene products, siRNA, microRNA, antisense oligonucleotides ASO, mRNA, DNA, gene editing tools and/or cytotoxic agents.
  • said exogenous load comprises PAMP, DAMP, CD47, CD24, IL-12, IL-15, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX and/or their functional activity fragment.
  • the exogenous load is directly or indirectly combined with the mobilizer through gene editing, exogenous expression, liquid-to-solid conversion, membrane fusion, charge adsorption, physical adsorption and/or chemical linkage .
  • the exogenous load is attached or embedded in the migrating body by directly or indirectly binding to a membrane component of the migrating body.
  • the membrane components of the migratory body comprise membrane proteins, cholesterol, phospholipids, sugar chains on glycoproteins and/or polysaccharides.
  • said indirect binding comprises ligation by click chemistry.
  • said indirect binding comprises providing said exogenous load attached to a first member of a binding pair and bringing it into contact with said migrating body, said migrating body having a membrane comprising said A second member of said binding pair, wherein said first member is capable of binding to said second member.
  • the first and second members of the binding pair are selected from the group consisting of an antigen and its antibody; a receptor and its ligand; biotin and avidin; HaloTag and its ligand; and CD63.
  • the exogenous cargo is expressed as a membrane protein on the inner or outer surface of the membrane of the migratory body.
  • the exogenous cargo is expressed on the inner or outer surface of the migratory membrane as a fusion protein fused with a membrane protein or a part thereof.
  • the present application provides a method for preparing a delivery system, the method comprising: allowing cells to express mRNA; causing the cells to produce migratory bodies, the migratory bodies comprising mRNA binding proteins, and the mRNA passing through the An mRNA binding protein is attached to the migratory body.
  • the present application provides a method for preparing a delivery system, the method comprising: allowing cells to express an exogenous load on the cell membrane; and causing the cell to produce a migratory body containing the exogenous load .
  • said exogenous cargo is a protein
  • said protein is a membrane protein.
  • the protein is a soluble protein, and it is fused to a membrane protein or a portion thereof to form a fusion protein.
  • the present application provides a composition comprising the migratory body described in the present application or the delivery system described in the present application.
  • Figure 1a-c Effect of hypoosmolarity stimulation on the formation of engineered migratory bodies.
  • Figure 2a-b Effects of different concentrations of latrunculin A on the formation of engineered migratory bodies.
  • Figure 3a-c Effect of knockdown of the SWELL1-encoding gene Lrrc8a in cells on the formation of engineered migratory bodies.
  • Figure 4a-b The effect of knockout of SWELL1-encoding gene Lrrc8a in cells on the formation of engineered migratory bodies.
  • Figure 5a-b Effect of different cation treatments on the formation of engineered migrators.
  • a Formation of engineered migrators under different cations; statistical results of diameters of engineered migrators in b.a.
  • Figure 6a-b Effect of overexpression of Tspan4 on the number of engineered migratory bodies.
  • a. The formation of engineered migrators caused by overexpression of Tspan4;
  • b. The statistical results of the number of engineered migrators in a.
  • FIG. 7a-b MGC803-T4-GFP cells produced migratory bodies on thermosensitive coated dishes. Photographs (a) and quantification of cell detachment rate (b) of migratory bodies produced by MGC803-T4-GFP cells under different conditions in different culture dishes.
  • Figure 8a-d Engineered migratory bodies induced in different cell lines.
  • a. Engineered migratory bodies induced by different rodent cell lines; partial enlargement of b.a (scale bar 5 ⁇ m).
  • c. Engineered migratory bodies induced by different T4-GFP transfected human embryonic kidney cell lines/lines.
  • d. WGA-stained engineered migratory bodies induced by different human cell lines.
  • Figure 9a-b NRK (a) and MC38 (b) cells cultured in suspension at different cell concentrations were induced to produce engineered migratory bodies.
  • Figure 10 Schematic diagram of the separation and purification process of engineered migratory bodies induced by engineered NRK cells.
  • Figure 11a-c Morphological observation of engineered migratory bodies induced by engineered NRK cells.
  • Figure 12 Western blot of isolated and purified engineered migratory bodies.
  • Figure 13a-b Time-dependent permeability of engineered migratory bodies to Cy5 and dextran-TMR observed by confocal microscopy.
  • a Laser confocal microscope observation results of engineered migrators;
  • b Statistical results of dextran-TMR permeability ratios of engineered migrators placed at room temperature for 1.5h, 6h, 12h, 24h, 48h.
  • Figure 14a-c Stability of engineered migratory bodies.
  • a. The morphological results of engineered migratory bodies observed by laser confocal microscope on days 0, 1, 2, 3, 5, and 7;
  • b. Western blot analysis of engineered migratory bodies loaded with chicken egg white after storage at room temperature for different days Protein (OVA) and mCherry protein amounts;
  • OVA OVA-specific antibodies produced after immunizing mice with engineered migratory bodies stored at room temperature for different days.
  • Figure 15 The effect of cholesterol extraction reagent M ⁇ CD on the stability of engineered migrators observed by confocal laser microscopy.
  • Figure 16a-b Schematic diagram of the loading process of membrane proteins, soluble proteins and small molecules on engineered migratory body (a) and schematic diagram of loading soluble protein with OVA as an example (b).
  • Figure 17 Localization of various loaded membrane proteins on engineered migratory bodies observed by confocal microscopy.
  • Figure 18 Localization of Spike protein on engineered migratory bodies observed by confocal laser microscopy.
  • Figure 19 Map of vector pB-Hygro-GFP.
  • FIG. 20 Map of vector pmCherry-N1.
  • Figure 21 Map of the vector pB-Hygro-mCherry.
  • Figure 22 Localization of t-STX2-OVA fusion protein on engineered migratory bodies observed by confocal laser microscopy.
  • Figure 23 Co-localization of Tspan4-HaloTag-GFP and HaloTag Ligand-TMR on engineered migratory bodies observed by confocal laser microscopy.
  • Figure 24a-c S protein-specific immune responses induced in mice immunized with engineered migratory bodies loaded with the SARS-CoV-2 Spike protein (e migratory bodies).
  • a Schematic diagram of animal experiment design;
  • b Western blot of S1 protein in spike-e migratory, control-e migratory group and purified S1 protein as control.
  • c the concentration of spike (S) protein-specific IgG in mouse serum after accepting different methods of immunization;
  • Figure 25a-c Characterization of engineered NRK cells to produce migratory bodies.
  • NRK cells overexpressing Tspan4-GFP were fixed with 2.5% glutaraldehyde after hypotonic treatment and observed by scanning electron microscope. Scale bars: 20 ⁇ m for the left panel and 2 ⁇ m for the right panel.
  • Figure 26a-h Comparison of characteristics of transsomes with engineered transsomes (e transsomes).
  • c NRK cells overexpressed Tspan4-GFP and stained with tetramethylrhodamine-labeled WGA. The top picture shows cells in an isotonic solution, and the bottom picture shows cells after hypotonic treatment.
  • the picture is the Z-axis overlay effect picture after the laser confocal microscope layer scanning. Scale bar: 5 ⁇ m.
  • d Electron micrograph of the migration body (Ma et al, Cell Res.2015).
  • e TEM images of engineered migratory bodies produced by NRK cells overexpressing T4-GFP. Scale bar: 1 ⁇ m.
  • the comparison of mass spectrometric analysis between the purified e-migratory and the control migratory (f) and the 25 most enriched proteins in the engineered migratory (g) that were missing in the engineered migratory the numbers in the table represent Log2 (fold change in expression relative to the soma).
  • Figure 27a-f Comparison of production of vesicles/exosomes and engineered migratory bodies by MC-38 cells overexpressing Tspan4-GFP.
  • a Purification process of vesicles/exosomes;
  • b NTA detection of MC-38 vesicles/exosomes after purification;
  • c Transmission electron microscope (TEM) detection of MC-38 vesicles/exosomes;
  • d Western blot analysis of vesicles/exosomes, engineered migratory bodies and cell bodies from the same cell source.
  • e Comparison of the production and yield of vesicles/exosomes and engineered migratory bodies from the same cell source.
  • f In vivo distribution.
  • Tspan4-GFP MC-38 cells Small vesicles/exosomes or engineered migratory bodies produced by Tspan4-GFP MC-38 cells were labeled with DiD and injected intravenously with the same amount of fluorescent dye, and the tissues were taken at different times for fluorescence imaging.
  • Figure 28a-c Quantitative mass spectrometric analysis of engineered migratory bodies and vesicles/exosomes from the same cell source.
  • Left PCA analysis; middle: signal pathway analysis heat map; note: the three data points of the engineered migrator have high consistency and overlap in the figure.
  • Right List of the 10 most enriched proteins in engineered migratory bodies and vesicles/exosomes. Note: The three sets of data points of the engineered migratory body are highly consistent, and the 10 most enriched proteins are completely consistent.
  • relative displacement generally refers to transmitting a displacement relative to a certain reference point or plane. Refers to the displacement of the cell center relative to the environment (such as the attached surface), or when the cell center is at rest, its membrane surface remains relative to the surrounding microenvironment (such as the attached solid surface, or the liquid phase in the microenvironment) or relative to The relative displacement from the cell center (such as expansion, deformation from flat to raised, etc.).
  • migratory body generally refers to a structure produced by cells when relative displacement occurs.
  • a migratory body can be a new type of organelle.
  • vesicle structures can be generated at the tips or intersections of contractile filaments left behind in the cell tail during cell migration.
  • the cell body continuously transports intracellular substances to the migratory body, and then the contractile filament breaks, and the migratory body is released, which can be transported to distant tissues in the local area or through body fluids such as blood, and then be transported by the extracellular space or uptake by surrounding cells.
  • migratory bodies may be involved in the transmission of intracellular substances and signals between cells, thereby mediating intercellular communication.
  • membrane microdomain generally refers to a region having a biological membrane structure.
  • cell membrane microdomains based on sphingolipids and cholesterol (Ch) including lipid rafts, Tetraspanin-enriched microdomains (Tetraspanin-enriched microdomain, TEM), etc.
  • intraluminal vesicle generally refers to membranous vesicles (such as organelles or larger vesicles) that form or reside within a lumen or in the space of a lumen or tubular structure .
  • a lumen may also be used herein to describe the interior space of a cellular component or structure.
  • intraluminal vesicles can be produced by organelles.
  • a luminal vesicle can comprise a luminal vesicle within a transsome, or a luminal vesicle that is not currently in a transsome but is expected to be translocated into a transsome.
  • exogenous load generally refers to substances that are not naturally present in chromosomes or host cells.
  • the cell itself does not produce, or does not produce (eg, express) the substance in appreciable amounts.
  • cells that produce the substance differ from natural cells, eg, the substance differs in structure or function.
  • hypoosmolarity generally refers to an osmotic pressure lower than that of an isotonic solution of cells.
  • An isotonic solution is a solution having an osmotic pressure equivalent to that of blood plasma.
  • cytoskeleton generally refers to the protein fiber network framework system in cells, such as the system composed of microtubules, microfilaments and intermediate fibers.
  • Tetraspanin refers generally to the tetraspanin superfamily, which comprises four transmembrane domains. These proteins can form so-called tetraspanins-enriched microdomains (TEMs) (Rubinstein, E. (2011). The complexity of tetraspanins. Biochem Soc Trans 39, 501-505.).
  • the TEM size is about 100 nanometers, rich in a series of proteins and lipid raft lipids, such as cholesterol, etc.
  • TEMA tetraspanin-enriched macrodomains
  • the Tetraspanin family can contain 33 members, including Tspan1, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, Tspan10, Tspan11, Tspan12, Tspan13, Tspan14, Tspan15, Tspan16, Tspan17, Tspan18, Tspan19, T span20 (UPK1B), Tspan21(UPK1A), Tspan22(PRPH2), Tspan23(ROM1), Tspan24(CD151), Tspan25(CD53), Tspan26(CD37), Tspan27(CD82), Tspan28(CD81), Tspan29(CD9), Tspan30 (CD63), Tspan31, Tspan32 and Tspan33. Data from biochemical studies and knockout mice suggest that these Tetraspanin family members play important roles in membrane biology.
  • an "isolated or purified” mobile can be prepared by at least one purification step that removes or substantially removes these contaminant components.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a method for preparing a migratory body, and the method may include causing a relative displacement of cells and then producing a migratory body derived from the cells.
  • the relative displacement of the present application may refer to the displacement of the cell center relative to the environment (such as the attached surface), or in the case of the cell center being stationary, its membrane surface is still relative to the surrounding microenvironment (such as the attached solid surface, or microenvironment). liquid phase in the environment) or relative displacement with respect to the center of the cell (e.g. expansion, deformation from flat to bulge, etc.).
  • the generation and/or extent of said relative displacement can be intervened by physical and/or chemical and/or biological means.
  • it may further include isolating the migratory bodies produced by the cells.
  • it may include performing hypotonic treatment on the cells, destroying the cytoskeleton of the cells, inhibiting the cell volume regulation function of the cells, detaching the cells from their adhesion surfaces, and/or Or causing a relative displacement between the cell membrane of said cell and its adhesion surface, and may comprise increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cell.
  • it may include destroying the cytoskeleton of the cell, inhibiting the cell volume regulation function of the cell, detaching the cell from its adhesion surface, and/or making the cell membrane of the cell adhere to it. Relative displacement occurs between the attached surfaces and may comprise increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cells.
  • the cells may comprise that the cells are subjected to hypotonic treatment, inhibiting the cell volume regulation function of the cells, detaching the cells from their adhesion surfaces, and/or making the cell membrane of the cells Relative displacement between the adhesion surfaces occurs and may comprise increasing the amount and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cells.
  • the cells may comprise that the cells are subjected to hypotonic treatment, destroying the cytoskeleton of the cells, detaching the cells from their adhesion surfaces, and/or allowing the cell membranes of the cells to adhere to them Relative displacement occurs between surfaces and may comprise increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cell.
  • it may include performing hypotonic treatment on the cells, destroying the cytoskeleton of the cells, inhibiting the cell volume regulation function of the cells, and/or allowing the cell membrane of the cells to adhere to it Relative displacement occurs between surfaces and may comprise increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cell.
  • the cells may undergo hypotonic treatment, destroy the cytoskeleton of the cells, inhibit the cell volume regulation function of the cells, and/or detach the cells from their adherent surfaces , and may comprise increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cells.
  • it may include inhibiting the cell volume regulation function of the cell, detaching the cell from its adhesion surface, and/or causing a relative displacement between the cell membrane of the cell and its adhesion surface, And may include increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cells.
  • it may comprise that the cells are treated with hypotonic pressure, the cells are detached from their adhesion surfaces, and/or the relative displacement between the cell membrane of the cells and its adhesion surface occurs, and It may comprise increasing the quantity and/or function of tetraspanin protein, functional fragments and/or functional variants thereof in said cells.
  • the cells may include that the cells are treated with hypotonic pressure, destroy the cytoskeleton of the cells, and/or inhibit the cell volume regulation function of the cells, and may include increasing tetraspanin in the cells. Quantity and/or function of proteins, functional fragments and/or functional variants thereof.
  • the cells may include that the cells are treated with hypotonic pressure and/or the relative displacement between the cell membrane of the cells and its adhesion surface occurs, and may include increasing the tetraspanin protein in the cells, which Quantity and/or function of functional fragments and/or functional variants.
  • it may include destroying the cytoskeleton of the cell and/or causing a relative displacement between the cell membrane of the cell and its adhesion surface, and may include increasing the tetraspanin protein in the cell, and its function Quantity and/or function of sexual fragments and/or functional variants.
  • it may include inhibiting the cell volume regulation function of the cell and/or causing a relative displacement between the cell membrane of the cell and its adhesion surface, and may include increasing the tetraspanin protein in the cell, Quantity and/or function of functional fragments and/or functional variants thereof.
  • it may include performing hypotonic treatment on the cells.
  • hypotonic treatment may comprise placing the cells in a hypotonic buffer solution.
  • the hypotonic treatment may include placing the cells in a buffer solution, and reducing the osmotic pressure of the buffer solution into a hypotonic buffer.
  • the reduction may include linear reduction and/or stepwise reduction.
  • the osmotic pressure of the hypotonic buffer solution may be lower than about 305 mOsmol/L.
  • the osmolarity of the hypotonic buffer solution can be lower than about 305 mOsmol/L, lower than about 300 mOsmol/L, lower than about 270 mOsmol/L, lower than about 250 mOsmol/L, lower than about 200 mOsmol/L, lower than about 150mOsmol/L, less than about 100mOsmol/L, less than about 90mOsmol/L, less than about 80mOsmol/L, less than about 70mOsmol/L, less than about 60mOsmol/L, less than about 50mOsmol/L, less than about 40mOsmol /L, less than about 30mOsmol/L, less than about 20mOsmol/L, less than about 15mOsmol/L, less than about 10mOsmol/L, less than about
  • the osmotic pressure of the hypotonic buffer solution may be about 10 mOsmol/L to about 274.5 mOsmol/L.
  • the osmotic pressure of the hypotonic buffer solution may be about 10 mOsmol/L to about 274.5 mOsmol/L.
  • the osmotic pressure of the hypotonic buffer solution may be from about 10 mOsmol/L to about 300 mOsmol/L, from about 20 mOsmol/L to about 300 mOsmol/L, from about 30 mOsmol/L to about 300 mOsmol/L, from about 50 mOsmol/L to about 300 mOsmol /L, about 70mOsmol/L to about 300mOsmol/L, about 100mOsmol/L to about 300mOsmol/L, about 150mOsmol/L to about 300mOsmol/L, about 200mOsmol/L to about 300mOsmol/L, about 250mOsmol/L to about 300mOsmol /L, about 10mOsmol/L to about 250mOsmol/L, about 20mOsmol/L to about 250mOsmol/L, about 30mOsmol/L to about 250mOsmol/L, about 50mOsmol/L
  • it may comprise destroying the cytoskeleton of the cell.
  • disrupting the cytoskeleton of the cell may comprise contacting the cell with an agent that disrupts the cytoskeleton.
  • the agent for disrupting the cytoskeleton may include a microfilament and/or microtubule depolymerizing agent.
  • the reagent for disrupting the cytoskeleton may include Latrunculin A, Latrunculin B, cytochalasin A, cytochalasin B, cytochalasin C, cytochalasin D and/or cytochalasin E .
  • it may comprise inhibiting the cell volume regulating function of the cells.
  • inhibiting the cell volume regulation function of the cells may include reducing the quantity and/or function of volume regulation proteins in the cells.
  • the quantity and/or function of the volume-regulating protein of the cell that the present application inhibits the cell volume-regulating function of the cell is decreased by about 5%, decreased by about 10%, decreased by about 20%, decreased by about 50% %, or drop by about 100%.
  • the volume-regulating protein may include volume-regulating ion channels and/or transporters.
  • the volume-regulated ion channel may include a volume-regulated anion channel VRAC and/or a volume-regulated cation channel VRCC.
  • VRAC volume-regulated anion channel
  • VRCC volume-regulated cation channel
  • the transporter may comprise a co-transporter.
  • the co-transporter may include KCC1, KCC3 and/or KCC4.
  • inhibiting the cell volume regulating function of the cells may comprise placing the cells in a buffer solution with weakened volume regulating ability.
  • the buffer may contain increased cations.
  • the cations may include K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , Ba 2+ , Mn 2+ , Fe 2+ , Ni 2+ , Zn 2+ , Al 3+ , Fe 3+ , CH 3 NH 3 + , C 2 H 5 NH 3 + , (CH 3 ) 2 NH 2 + , (C 2 H 5 ) 2 NH 2 + , (C 2 H 5 ) 3 N + , ammonia ion and/or choline ion.
  • the buffer may contain increased anions.
  • the anions may include Br - , Cl - , I - , F - , OH - , HCO 3 - , H 2 PO 4 - , NO 2 - , NO 3 - , CN - , HPO 4 2- , CO 3 2- , SO 4 2- and/or PO 4 3- .
  • the method may comprise detaching the cell from its adhesion surface.
  • the method may comprise causing a relative displacement between the cell membrane of the cell and its adhesion surface.
  • it may also include increasing the quantity and/or function of the tetraspanin protein, its functional fragments and/or functional variants in the cells.
  • it may include making the cells overexpress the tetraspanin protein, its functional fragments and/or functional variants.
  • the tetraspanin protein can be selected from: Tspan1, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, Tspan10, Tspan11, Tspan12, Tspan13, Tspan14, Tspan15, Tspan16, Tspan17 , Tspan18, Tspan19, Tspan20(UPK1B), Tspan21(UPK1A), Tspan22(PRPH2), Tspan23(ROM1), Tspan24(CD151), Tspan25(CD53), Tspan26(CD37), Tspan27(CD82), Tspan28(CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32 and Tspan33.
  • the method may further include reducing the size of the migration body.
  • the reducing the size of the migration body may include extruding the migration body using a filter or a squeezer.
  • the pore size of the filter or squeezer can be from about 30 nm to about 10000 nm.
  • the pore size of the filter or squeezer can be from about 30 nm to about 100 nm, from about 30 nm to about 1000 nm, from about 30 nm to about 10000 nm, from about 50 nm to about 100 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 10000 nm, From about 100 nm to about 1000 nm, from about 100 nm to about 10000 nm, or from about 1000 nm to about 10000 nm.
  • the size of the migrator may be from about 50 nm to about 8000 nm.
  • the size of the migrator can be from about 50 nm to about 100 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 10000 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 10000 nm, or from about 1000 nm to about 10000 nm.
  • the migratory bodies can be produced from the contractile filaments of the cells.
  • sodium/potassium ATPase and/or its functional fragments may be enriched on the membrane of the migratory body.
  • integrins and/or functional fragments thereof may be enriched on the membrane of the migratory body.
  • tetraspanin protein, its functional variant and/or its functional fragment may be enriched on the membrane of the migratory body.
  • the membrane of the migration body may be enriched with cholesterol.
  • enrichment can refer to comparison with other parts on the cell membrane of the corresponding cell, or comparison with the situation on the corresponding cell membrane before said transfectant is produced.
  • enrichment means that the density of said molecules or substances on the transsomes of the present application is higher than the density of corresponding molecules or substances on the cell membranes from which these transsomes are produced.
  • membrane microdomains refer to the microdomains of cell membranes based on sphingolipids and cholesterol (Ch), including lipid rafts, tetraspanin-rich microdomains ( Tetraspanin-enriched microdomain, TEM) and so on.
  • the migration body can be produced in vitro or ex vivo.
  • the content of the migratory body may be at least partially reduced or absent compared to a native migratory body produced by the same cell.
  • the natural migratory body can refer to the migratory body produced by the spontaneous migration of cells without changing the external culture conditions (such as hypotonicity, temperature, etc.); Migrators produced after induction of relative displacement.
  • the at least partially reduced content may comprise intraluminal vesicles.
  • the engineered migratory bodies provided herein have fewer intraluminal vesicles.
  • the intraluminal vesicle may refer to a vesicle with a phospholipid bilayer structure in the outer membrane structure.
  • the preparation methods of the present application may be an in vitro or ex vivo method.
  • the preparation methods of the present application may be performed in vitro.
  • the preparation methods of the present application may not be performed on living subjects.
  • the cells may be cultured cells in vitro.
  • the cells may be cultured in suspension or adherently.
  • the cells may comprise primary cells.
  • the primary cells may include tissue cells derived from organisms, and the organisms may include humans, monkeys, mice, rats, rabbits, chickens and/or insects.
  • the primary cells may include hepatocytes, spleen cells, kidney cells, tissue macrophages, brain glial cells, osteoclasts, bone marrow cells, leukocytes, fibroblasts and/or fat cells.
  • primary cells of the present application may include Kupffer cells.
  • a type of phagocyte located on the inner surface of liver sinusoids can remove substances such as foreign antigens, antigen-antibody complexes and cell debris in the blood.
  • the white blood cells may include B cells, T cells, NK cells, dendritic cells, neutrophils and/or macrophages.
  • the cells may include tumor cells.
  • the tumor cells may include tumor cell lines, primary or limited passage tumor cells derived from patients, tumor stromal cells, and/or tumor organoids.
  • the cells may include CHO cells, CHO-K1 cells, HEK293 cells, HEK293T cells, HEK293FT cells, HEK293F cells, Vero cells, NRK cells, L929 cells, MC38 cells, 4T1 cells, DC2 .4 cells, MGC803 cells, Jurkat cells, NK-92MI cells, BJ cells and/or HepG2 cells.
  • the cells may include leukocytes, stem cells and/or fibroblasts.
  • the stem cells may comprise mesenchymal stem cells.
  • the present application provides migratory bodies prepared in the methods of the present application.
  • the present application provides a novel extracellular vesicle that can be produced by engineering—an engineered migratory body and/or a migratory body that forces cells to produce efficiently through engineering methods, and/or contains any of the above migratory bodies
  • the composition is characterized in that, the size of the engineered migration body is 50-8000 nanometers (nm), the membrane has the enrichment of proteins such as integrin (integrin) and Tetraspanin (Tspan) family members, and is composed of membrane microstructure
  • the natural migration body produced by this kind of cell is similar; it can also be specifically adjusted according to the application requirements to obtain a vesicle-like structure with a structure and bio
  • the application provides migratory bodies/engineered migratory bodies as a drug-loaded delivery carrier, which can be used in different diseases (such as tumors, inflammation/autoimmune diseases, cardiovascular diseases, neurodegenerative diseases and other nervous system diseases, etc.) therapeutic and vaccine applications.
  • diseases such as tumors, inflammation/autoimmune diseases, cardiovascular diseases, neurodegenerative diseases and other nervous system diseases, etc.
  • the migratory body is a new type of organelle, which is a single-layer membrane vesicle structure with a diameter of 0.5-3 ⁇ m produced at the tip or intersection of the contractile filament left by the cell tail during cell migration.
  • the cell body continuously transports intracellular materials to the migratory body.
  • the contractile filament breaks, and the migratory body is released, which can be transported to distant tissues locally or through body fluids such as blood, and then taken up by the extracellular space or surrounding cells.
  • migrasomes as the membrane-coated carrier of signal molecules, determine the temporal and spatial distribution of signal molecules, thereby exerting the function of regulating organ development (Jiang D et. at., Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation, Nat Cell Biol, 2019, 21(8):966-977).
  • Migrasomes can mediate the transfer of proteins and mRNA between cells, and transfer proteins and mRNAs to recipient cells through migrasomes, thereby changing the life activities of recipient cells (Zhu M et.al., Lateral transfer of mRNA and protein by migrasomes modifies the recipient cells, Cell Res, 2020, doi; 10.1038/s41422-020-00415-3). Migrasomes can regulate the quality of mitochondria, and maintain the homeostasis of intracellular mitochondria by cleaning out damaged mitochondria (Jiang H et.al., Mitocytosis, a migrasome-mediated mitochondrial quality control process, Cell Press, doi: 10.1016 /j.cell.2021.04.027).
  • Migratome has a regulatory effect on cancer cells in the tumor microenvironment.
  • migratome in pancreatic cancer cells, can induce an inhibitory immune microenvironment to promote tumor growth (Zhang Ronghua, Pancreatic cancer cell migratory body has an effect on cancer cells in the tumor microenvironment and related Mutual regulation of immune cell phenotype and function, 2020).
  • the detached migratory body is a type of extracellular vesicle that differs from known extracellular vesicles in many ways.
  • the difference with exosomes 1) The structure of the two is different: the migratory body is attached to the contractile filament before being released and presents a structure containing small vesicles within a large vesicle; while exosomes do not exist this Structure; 2) The size of the two is different: the diameter of the exosome is about 50-150nm, while the diameter of the migratory body is about 0.5-3 ⁇ m; 3)
  • the protein composition of the two is obviously different: between the migratory body and the exosome Only 27% of the protein composition is the same among them, such as NDST1 (bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1), PIGK (phosphatidylinositol glycan-anchored biosynthetic class K), CP
  • engineered migrators with structures similar to natural migrators can be induced by various methods.
  • Engineered migratory bodies are distinct from "hypoosmolarity-induced vesicles" known in the art (e.g., Cohen S, Ushiro H, Stoscheck C, Chinkers MA native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.J
  • the vesicle shown in Figure 1 in Biol Chem 257:1523-1531 they have at least the following differences: 1) The vesicle production site is different: the engineered migrator is produced on the contractile filament around the cell; and Cohen et al.
  • vesicles are produced on the upper surface of cells; 2) vesicle sizes are different: the size of the engineered migrator is micron-sized, and the diameter rarely exceeds 5 ⁇ m; while the vesicles induced by Cohen et al. using low osmotic pressure can be as large as 20 ⁇ m; 3 ) is different from the hypotonic induction method used by Cohen et al.
  • hypotonicity is not a necessary condition for producing engineered migratory bodies, and engineered migratory bodies can be induced by other methods.
  • Engineered extracellular vesicles are currently widely used in important biomedical fields such as drug delivery and vaccine preparation.
  • engineered exosomes Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, Lee JJ, Kalluri R. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017 Jun 22; 546 (7659):498-503.doi:10.1038/nature223411
  • its main advantages mainly include: natural biofilm source, low toxicity; low immunogenicity, not easy to be cleared by the immune system, so the circulation time in the body is longer; stable properties , capable of long-term storage at -80°C.
  • the engineered migratory body discovered in this application is of biological origin, and has the advantages of low toxicity and low immunogenicity of extracellular vesicles of biological origin such as exosomes; it is a brand new extracellular vesicle, and the molecules it can transport , biological distribution in the body and the like have unique characteristics; meanwhile, the preparation method is simple and the yield is high.
  • the engineered migratory body discovered in this application has extremely high application value in biomedical fields such as drug delivery and vaccine preparation.
  • a method for preparing an engineered migratory body which includes artificially inducing cells to produce an engineered migratory body, and isolating and/or purifying the engineered migratory body; and/or forcing the cell membrane to communicate with the culture environment (including attached solid surface and the surrounding liquid environment), including but not limited to natural cell migration or the relative displacement of the cell membrane and the environment at the sub-cellular scale (sub-cellular scale).
  • the present application provides an in vitro or ex vivo prepared migration body, wherein the size of the migration body can be from about 50 nm to about 8000 nm.
  • the migratory body of the present application it can be produced from the contractile filaments of cells in vitro.
  • the membrane of the migration body can be enriched with sodium/potassium ATPase and/or its functional fragments.
  • integrins and/or functional fragments thereof may be enriched on the membrane of the migration body.
  • the membrane of the migratory body may be enriched with tetraspanin protein, its functional variant and/or its functional fragment.
  • cholesterol may be enriched on the membrane of the migration body.
  • the migration body may be enriched with membrane microdomains.
  • the content of the migratory body may be at least partially reduced or absent.
  • the at least partially reduced content may comprise intraluminal vesicles.
  • exogenous load may refer to a substance that is not produced by the cell itself, does not produce (eg, express) in a considerable amount, or that the substance is produced in the cell itself, but as an exogenous load, the substance has no structural or functional effect. This aspect is different from the above-mentioned substance produced in the cell itself.
  • the migration body may include the migration body described in the application.
  • the present application provides a delivery system that may comprise a migratory body and one or more exogenous payloads.
  • the exogenous load can be directly or indirectly bound, connected or embedded in the membrane and/or inside of the migrating body.
  • the migration body may include the migration body described in the application.
  • the migratory body can be derived from cells.
  • the exogenous load may contain one or more targeting substances and/or therapeutically active substances.
  • the exogenous load may comprise proteins, lipids, polynucleotides, small molecule compounds, complexes, polysaccharides, polymers, nanoparticles, microparticles and/or organelles.
  • the exogenous load may include membrane protein, soluble protein and/or polypeptide.
  • the exogenous load may include DNA and/or RNA.
  • the exogenous load may include antibodies or antigen-binding antibody fragments thereof, integrins or fragments thereof, immunogenic proteins, cytokines, chemokines, receptor proteins or fragments thereof, Enzymes, tumor suppressor gene products, siRNA, microRNA, antisense oligonucleotides ASO, mRNA, DNA, gene editing tools and/or cytotoxic agents.
  • gene editing tools can include nucleases such as Cas proteins, CRISPR-Cas systems, Cre recombinases, zinc finger endonucleases, transcription activator-like effector nucleases, and gene epigenetics. editing tools.
  • the exogenous load may include PAMP, DAMP, CD47, CD24, IL-12, IL-15, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX and/or their functions active fragment.
  • exogenous load can be directly or indirectly connected to the mobilizer through gene editing, exogenous expression, liquid-to-solid conversion, membrane fusion, charge adsorption, physical adsorption and/or chemical connection combined.
  • exogenous expression can include, for example, plasmid expression, such as establishing a cell line that transiently or stably overexpresses a gene.
  • the exogenous load can be bound or embedded into the migrating body through direct or indirect connection with the membrane component of the migrating body.
  • the membrane component of the migratory body may include membrane proteins, cholesterol, phospholipids, sugar chains on glycoproteins and/or polysaccharides.
  • the indirect connection may include connection through a click chemical reaction.
  • the indirect connection may include providing the exogenous load connected to the first member of the binding pair, and bringing it into contact with the migration body, the membrane of the migration body contains A second member of said binding pair, wherein said first member is capable of binding to said second member.
  • the first member and the second member of the binding pair can be selected from antigens and their antibodies; receptors and their ligands; biotin and avidin; HaloTag and its ligands; CP05 and CD63.
  • the exogenous cargo can be expressed as a membrane protein on the inner or outer surface of the membrane of the migratory body.
  • the exogenous cargo can be expressed on the inner or outer surface of the membrane of the migratory body as a fusion protein fused with a membrane protein or a part thereof.
  • the exogenous load can be expressed on the inner or outer surface of the migratory membrane as a fusion protein fused with a membrane protein or a part thereof through gene editing and/or exogenous expression .
  • the present application provides a method for preparing a delivery system, the method may include providing a migratory body, and allowing the migratory body to carry an exogenous load.
  • said migratory body carrying exogenous load may include reversibly carrying exogenous load.
  • the migratory body carrying the exogenous load may include irreversibly carrying the exogenous load.
  • the mobilization body may be an isolated or purified mobilization body.
  • the making the migrating body carry the exogenous load may include directly or indirectly connecting or embedding the exogenous load into the membrane of the migrating body and/or the interior of the migrating body.
  • it may also include isolating or purifying the migratory body from cells.
  • it may include providing a complex of the exogenous load and the first member of the binding pair; causing the cell to produce a migratory body, the migratory body comprising the second member of the binding pair; and making the A migratory body is contacted with the complex to form the delivery system.
  • the migration body may include the migration body described in the application.
  • the migratory body can be derived from cells.
  • the exogenous load may contain one or more targeting substances and/or therapeutically active substances.
  • the exogenous load may comprise proteins, lipids, polynucleotides, small molecular compounds, complexes, polysaccharides, polymers, nanoparticles, microparticles and/or organelles.
  • the exogenous load may include membrane protein, soluble protein and/or polypeptide.
  • the exogenous load may include DNA and/or RNA.
  • the exogenous load may include antibodies or antigen-binding antibody fragments thereof, integrins or fragments thereof, immunogenic proteins, cytokines, chemokines, receptor proteins or fragments thereof, enzymes , tumor suppressor gene products, siRNA, microRNA, antisense oligonucleotides ASO, mRNA, DNA, gene editing tools and/or cytotoxic agents.
  • the exogenous load may include PAMP, DAMP, CD47, CD24, IL-12, IL-15, coagulation factor VII, coagulation factor VIII, coagulation factor IX and/or their functional activities fragment.
  • the exogenous load can be directly or indirectly combined with the migration body through gene editing, exogenous expression, liquid-to-solid conversion, membrane fusion, charge adsorption, physical adsorption and/or chemical connection .
  • the exogenous load can be connected or embedded in the migrating body by directly or indirectly combining with the membrane component of the migrating body.
  • the membrane component of the migratory body may include membrane proteins, cholesterol, phospholipids, sugar chains on glycoproteins and/or polysaccharides.
  • the indirect binding may comprise linking through a click chemical reaction.
  • the indirect binding may comprise providing the exogenous load linked to the first member of the binding pair, and bringing it into contact with the migration body, the membrane of the migration body comprising the A second member of said binding pair, wherein said first member is capable of binding to said second member.
  • the first member and the second member of the binding pair can be selected from antigens and their antibodies; receptors and their ligands; biotin and avidin; HaloTag and its ligands; and CP05 and CD63.
  • the exogenous load can be expressed as a membrane protein on the inner or outer surface of the membrane of the migratory body.
  • the exogenous load can be expressed on the inner or outer surface of the membrane of the migratory body as a fusion protein fused with a membrane protein or a part thereof.
  • an mRNA binding protein can comprise a protein forming a complex for binding said mRNA, eg, the RNA binding protein comprises an RNA recognition motif.
  • RNA binding proteins contain native or inactivated nuclease activity.
  • the RNA binding protein or its functional domain may comprise Cys2-His2, Gag-knuckle, Treble-clet, zinc band, Zn2/Cys6 motifs.
  • Exemplary mRNA binding proteins may include, but are not limited to, RNA binding proteins (RNA binding protein, RBP).
  • the present application provides a method for preparing a delivery system, the method may include: allowing cells to express an exogenous load on the cell membrane; load.
  • the exogenous expression can be achieved by gene editing or exogenous expression, such as plasmid expression.
  • the exogenous load can be protein
  • the protein may be a membrane protein.
  • the protein may be a soluble protein, and it may be fused with a membrane protein or a part thereof to form a fusion protein.
  • the present application provides a composition, which may comprise the migratory body described in the present application or the delivery system described in the present application.
  • a method for preparing an engineered migration body comprising
  • step (a) carry out at least 1 kind of processing in (2)-(5), carry out at least 2 kinds of processing in (1)-(5), at least 3 kinds of processing , at least 4 treatments or all 5 treatments.
  • step (a) includes performing hypotonic treatment on cells.
  • hypotonicity treatment is carried out by placing the cells in a hypotonic buffer solution, the osmotic pressure of the hypotonic buffer solution being 30.5-274.5mOsmol/L , such as 30.5-150mOsmol/L.
  • step (a) comprises destroying the cytoskeleton of the cell.
  • the reagent for destroying the cytoskeleton includes a microfilament depolymerization agent, for example selected from Latrunculin A, Latrunculin B, cytochalasin A, cytochalasin B, cytochalasin C, cytochalasin Cytochalasin D and Cytochalasin E.
  • a microfilament depolymerization agent for example selected from Latrunculin A, Latrunculin B, cytochalasin A, cytochalasin B, cytochalasin C, cytochalasin Cytochalasin D and Cytochalasin E.
  • step (a) comprises inhibiting the cell volume regulation function of the cells.
  • VRAC volume-regulated anion channels
  • VRCC volume-regulated cation channels
  • TRPV4 volume-regulated cation channels
  • TRPM3 co-transporters , such as KCC1, KCC3 and KCC4.
  • step (a) comprises making the cells overexpress one or more proteins selected from Tetraspanin family members.
  • Tetraspanin family members include Tspan1, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, Tspan10, Tspan11, Tspan12, Tspan13, Tspan14, Tspan15, Tspan16, Tspan17, Tspan18, Tspan19, Tspan20(UPK1B), Tspan21(UPK1A), Tspan22(PRPH2), Tspan23(ROM1), Tspan24(CD151), Tspan25(CD53), Tspan26(CD37), Tspan27(CD82), Tspan28(CD81), Tspan29 (CD9), Tspan30 (CD63), Tspan31, Tspan32 and Tspan33.
  • step (a) comprises making the cells overexpress Tspan4.
  • the size of the reduced engineered migrator is nanoscale, eg, 50-200 nm.
  • a delivery system comprising an isolated or purified transporter and a cargo attached directly or indirectly to the membrane of the transporter.
  • migratory body is selected from naturally occurring migratory bodies and artificially induced engineered migratory bodies.
  • the load is selected from proteins such as membrane proteins and soluble proteins, peptides, nucleic acids such as DNA and RNA, and small molecule compounds.
  • the load is selected from therapeutic proteins, immunogenic proteins, cytokines, enzymes, siRNA, microRNA, antisense oligonucleotides (ASO), mRNA, CRISPR systems, cytotoxic agents, therapeutic small molecules and targeting molecules.
  • the load is selected from therapeutic proteins, immunogenic proteins, cytokines, enzymes, siRNA, microRNA, antisense oligonucleotides (ASO), mRNA, CRISPR systems, cytotoxic agents, therapeutic small molecules and targeting molecules.
  • the targeting molecule is selected from an antibody or an antigen-binding fragment thereof; an integrin; find me/eat me signal (find-me/eat me signal), such as PAMP and DAMP; and Don't-eat-me signals, such as CD47 and CD24.
  • the membrane of the migratory body comprises a second member of the binding pair
  • the payload is attached to the membrane of the migratory body through the binding of the first member and the second member .
  • a method of producing a delivery system comprising an isolated or purified migratory and a cargo selected from the group consisting of proteins, peptides, nucleic acids (such as DNA and RNA) and small molecule compounds, wherein the method comprises:
  • transsome isolating or purifying the transsome from the cell, the transsome being a naturally occurring transsome or an artificially induced engineered transsome;
  • the delivery system is created by attaching the cargo directly or indirectly to the membrane of the migrator.
  • a method of producing a delivery system comprising an engineered mobilizer and a payload selected from the group consisting of proteins, peptides, nucleic acids (such as DNA and RNA) and small molecule compounds, wherein the payload is associated with the first binding pair One member is connected, and the membrane of described migration body comprises the second member of described binding pair, wherein said method comprises:
  • the complex of the payload and the first member of the binding pair is contacted with the engineered migrator, whereby the delivery system is produced by association of the first member with the second member.
  • a method of producing a delivery system comprising an engineered migratory body and a payload, the payload being mRNA, and the membrane of the migratory body comprising an mRNA binding protein, the mRNA bound by protein at its 3'-UTR A site is combined with the mRNA binding protein, wherein the method comprises:
  • a delivery system comprising an engineered transsome and mRNA is produced from the cell, wherein the mRNA is attached to the membrane of the engineered transsome by binding to the mRNA binding protein.
  • a method of producing a delivery system comprising an engineered mobilizer and a cargo that is a protein and expressed on the surface of an engineered mobilizer membrane comprising:
  • a delivery system comprising the engineered transsome and a protein expressed on the surface of the engineered transsome membrane is produced from the cells.
  • the migration body delivery system of the present application has higher biocompatibility, more types of delivered drugs, larger capacity and easier membrane modification to increase or change targeting.
  • the migratory body delivery system retains high biocompatibility and easy-to-modify characteristics, and is larger than traditional small vesicles (such as exosomes), which can be more effective Delivery of drugs, especially large molecular weight proteins and nucleic acids.
  • the transfer body delivery system is highly reconfigurable, and the size of the transfer body and the substances loaded on the membrane can be adjusted according to the drugs to be delivered and the cells to be targeted.
  • the migratory body also has the advantage of low toxicity, and it is easier to realize the transformation and modification of membrane components.
  • the preparation of engineered migratory bodies does not require ultra-high-speed centrifugation, nor does it need to extract blood samples every time, which greatly reduces the preparation time and cost.
  • the migration body delivery system of the present application has the characteristics of low toxicity, high loading capacity, easy modification, fast production, and low cost, and the antigenicity can be adjusted, and has a unique antigen presentation effect , can be used as a vaccine delivery platform.
  • Migratory bodies Although migratory bodies have a vesicular structure, they are different from extracellular vesicles and exosomes. Migratory bodies may not be strictly extracellular vesicles. Although detached transsomes are extracellular vesicles, many functions of transsomes are performed before they detach from the cell body. This is why migratory bodies can be considered organelles rather than a type of extracellular vesicle. Generation of extracellular vesicles (detached transsomes) is only one of the many functions of transsomes.
  • the detached migratory body is a type of extracellular vesicle, but it can differ from exosomes in many ways.
  • the structure of the two is different: the migratory body is attached to the contractile filament before being released and presents a structure containing small vesicles within a large vesicle; this structure does not exist in exosomes; 2) the size of the two is different : The diameter of exosomes is about 50-150nm, while the diameter of migratory bodies is about 0.5-3 ⁇ m; 3)
  • the protein composition of the two is significantly different: only 27% of the protein composition is the same between migratory bodies and exosomes NDST1 (bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1), PIGK (phosphatidylinositol glycan-anchored biosynthetic class K), CPQ (carboxypeptidase Q) and EOGT (
  • exosomes first act as multivesicular bodies (MVB) vesicles are produced, and exosomes are released when MVB fuses with the plasma membrane; whereas migratory bodies are assembled from large domains on the plasma membrane (Huang Y, Zucker B, Zhang S, Elias S, Zhu Y, Chen H,Ding T,Li Y,Sun Y,Lou J,Kozlov MM*,Yu L*.Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains.Nat Cell Biol.2019 Aug;21(8):991- 1002).
  • MVB multivesicular bodies
  • engineered migratory body refers to a migratory body produced by engineering and/or artificially inducing a cell. It should be noted that an “engineered migratory” may not imply that it is synthetic, it may still be of cell origin.
  • the engineered migratory bodies of the present application may be different from the "hypoosmolarity-induced vesicles" known in the art (e.g., Cohen S, Ushiro H, Stoscheck C, Chinkers MA native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.
  • the vesicles shown in Figure 1 in J Biol Chem 257:1523-1531 they have at least the following differences: 1) The vesicle production site is different: the engineered migration body is produced on the contractile filament around the cell ; while the vesicles of Cohen et al.
  • the size of the vesicles is different: the size of the engineered migratory body is micron-scale, and the diameter rarely exceeds 5 ⁇ m; while the vesicles induced by Cohen et al. using low osmotic pressure can be As large as 20 ⁇ m; 3) Different from the method of Cohen et al. using hypotonic induction, hypotonicity is not a necessary condition for producing engineered migratory bodies in this application. The relative displacement of the surface induces migratory bodies.
  • the migratory bodies herein may include naturally occurring migratory bodies and artificially induced engineered migratory bodies.
  • the isolated or purified motile may be free of detectable contaminant components, or have levels or amounts of contaminant components at or below acceptable levels or amounts.
  • engineered migratory bodies can be isolated and/or purified by centrifugation.
  • engineered migratory bodies can be isolated and/or purified by centrifugation at 300-17000 xg.
  • engineered mobilizers can be isolated and/or purified by filtration.
  • inducing the cells to produce the engineered migratory body may include treating the cells with low osmotic pressure.
  • the hypotonic treatment can be performed by placing the cells in a hypotonic buffer solution having an osmotic pressure of 10%-90% of that of an isotonic buffer solution, such as 15%- 85%, 20%-80%, 25%-75%, 30%-70%, 35%-65%, 40%-60%, 50%-55%, or any value or subrange therebetween, such as 10% , 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% %.
  • the hypotonicity treatment can be performed by placing the cells in a buffer solution and reducing the osmotic pressure of the buffer solution.
  • decreasing the osmolarity of the buffer solution comprises a linear decrease or a stepwise decrease.
  • the salt concentration of the buffer solution can be gradually decreased at predetermined time intervals.
  • the salt concentration of the buffer solution can be gradually reduced for at least 3 times (for example, 3-5 times), each time reducing the salt concentration by 1/6-1/2.
  • inducing a cell to produce an engineered migratory body may comprise disrupting the cell's cytoskeleton.
  • the cytoskeleton of a cell can be disrupted by contacting the cell with an agent that disrupts the cytoskeleton.
  • agents that disrupt the cytoskeleton include, for example, microfilament and or microtubule depolymerizing agents selected from the group consisting of Latrunculin A, Latrunculin B, Cytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D and cytochalasin E.
  • the agent that disrupts the cytoskeleton is Latrunculin A.
  • the concentration of the cytoskeleton disrupting agent can be any suitable concentration.
  • the concentration of microfilament and/or microtubule depolymerizing agent can be at least 0.01 ⁇ M, at least 0.1 ⁇ M, at least 0.2 ⁇ M, at least 0.5 ⁇ M, at least 1 ⁇ M, at least 2 ⁇ M, at least 3 ⁇ M, at least 4 ⁇ M, at least 5 ⁇ M, at least 6 ⁇ M , at least 7 ⁇ M, at least 8 ⁇ M, at least 9 ⁇ M, at least 10 ⁇ M.
  • Cells can respond to changes in osmotic pressure through regulated volume changes. Therefore, inhibition of cell volume regulation functions can promote the formation of engineered migratory bodies.
  • inducing a cell to produce an engineered migratory body can comprise inhibiting a cell volume regulating function of said cell.
  • the cell volume regulating function of the cell can be inhibited by inhibiting the expression or activity of the cell's protein that regulates cell volume.
  • proteins that regulate cell volume may be selected from ion channels and transporters.
  • proteins that regulate cell volume may be selected from volume-regulated anion channels (VRACs), such as SWELL 1; volume-regulated cation channels (VRCCs), such as TRPV4 and TRPM3; and cotransporters, such as KCC1, KCC3 and KCC4.
  • VRACs volume-regulated anion channels
  • VRCCs volume-regulated cation channels
  • cotransporters such as KCC1, KCC3 and KCC4.
  • the expression or activity of the protein can be inhibited by any method or reagent known in the art, such as destroying the gene sequence encoding the protein, RNAi, protein activity inhibitors and the like.
  • the cell volume regulating function of the cells can be inhibited by placing the cells in a buffer solution comprising a cation or anion having a reduced ability to regulate changes in cell volume.
  • a buffer solution comprising a cation or anion having a reduced ability to regulate changes in cell volume.
  • the term "decreased ability to regulate cell volume changes" is relative to cations (such as sodium ions) or anions that impart normal cell volume regulation ability to cells.
  • inducing cells to produce engineered migratory bodies may include making the cells overexpress one or more proteins selected from members of the Tetraspanin family or key enzymes and structural proteins of the migratory body production pathway, or using migratory bodies to produce agonistic agent stimulation.
  • Tetraspanin family members can include Tspan1, Tspan2, Tspan3, Tspan4, Tspan5, Tspan6, Tspan7, Tspan8, Tspan9, Tspan10, Tspan11, Tspan12, Tspan13, Tspan14, Tspan15, Tspan16, Tspan17, Tspan18, Tspan19, Tspan20 ( UPK1B), Tspan21(UPK1A), Tspan22(PRPH2), Tspan23(ROM1), Tspan24(CD151), Tspan25(CD53), Tspan26(CD37), Tspan27(CD82), Tspan28(CD81), Tspan29(CD9), Tspan30(CD63), Tspan31, Tspan32 and Tspan33.
  • cells can be induced to produce engineered migratory bodies by overexpressing one or more Tspan proteins.
  • cells can be detached quickly by adjusting the cell attachment properties to reduce the contact area between the cells and the culture surface, or cause a relative displacement between the cell membrane and the surface, thereby inducing migratory bodies.
  • the cells can be subjected to hypotonic treatment at 4, 8, 16, 24, 37 degrees Celsius, or not, and the cell detachment process can be performed at 4, 8, 16, 24 degrees Celsius. And can separate cell inclusions and migratory bodies.
  • the preparation method of the engineered migration body of the present application further includes reducing the size of the engineered migration body.
  • the engineered migration body can be reduced in size by any means known in the art, such as by squeezing the engineered migration body using a filter or squeezer of a specific pore size to reduce the size of the engineered migration body.
  • the size of the engineered migrator can be reduced by processing the engineered migrator with an extruder containing a filter membrane of a specific pore size.
  • the pore size of the filter or squeezer can be 30nm-10000nm, such as 50nm-8000nm, 50nm-1000nm, 50nm-10000nm, 100nm-1000nm, 100nm-10000nm, 1000nm-10000nm, 10-400nm, 20 - 300nm, 30-200nm, 40-100nm, 50-80nm, or any value or subrange therebetween.
  • the size of the reduced engineered migrator can be nanoscale, such as 1-1000 nm, 10-900 nm, 20-300 nm, 30-200 nm, 40-100 nm, 50-80 nm, or any value therebetween or subrange.
  • the cells used to produce the engineered migratory body can be any cells, such as cells cultured in vitro or cells in vivo; cells cultured in suspension or adherent in a suspended or adherent state, with or without modification Strains/lines, normal cells, primary cells or cells of disease origin including cancer cells.
  • the cells used to produce engineered migratory bodies can be derived from any cell line suitable for in vitro proliferation, modification and expression of exogenous molecules and production of engineered migratory bodies.
  • the cells may be animal cells, particularly mammalian cells, including murine and human cells.
  • suitable cells include, but are not limited to, normal rat kidney cells (NRK cells), mouse fibroblasts such as NIH3T3 cells, mouse breast cancer 4T1 cells, mouse colon cancer MC38 cells, commonly used human embryonic kidney (HEK) Cell lines/lines such as HEK293 or HEK293FT cells, human gastric cancer MGC-803 cells, human T lymphoma Jurkat cells, human skin fibroblast BJ cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSC), or any other suitable cells.
  • NRK cells normal rat kidney cells
  • mouse fibroblasts such as NIH3T3 cells
  • mouse breast cancer 4T1 cells mouse colon cancer MC38 cells
  • HEK human embryonic kidney
  • HEK human embryonic kidney
  • MGC-803 human T lymphoma Jurkat cells
  • human skin fibroblast BJ cells human skin fibroblast BJ cells
  • MSC mesenchymal stem cells
  • the term "load” as used herein may refer to any substance capable of being loaded onto a migratory body so as to be effectively delivered.
  • the payload can be delivered to the target cell through the interaction of surface molecules of the migratory body with the target cell.
  • One or more of the loadings of the present application can be loaded onto the membrane of the engineered migration body during or after formation of the engineered migration body, or directly onto the membrane of the naturally occurring migration body.
  • payloads include, but are not limited to, therapeutic agents such as synthetic bioactive compounds, natural bioactive compounds, antibacterial compounds, antiviral compounds, proteins or peptides (e.g., enzymes or antibodies), nucleotides (e.g., containing detectable moieties, or toxins).
  • nucleotides or nucleotides that disrupt transcription nucleic acids
  • nucleic acids for example, DNA or mRNA molecules encoding polypeptides such as enzymes, immunogenic proteins, cytokines, tumor suppressor genes, antibodies, etc., or RNA molecules with regulatory functions such as microRNA, dsDNA, antisense oligonucleotides (ASO), lncRNA, and siRNA
  • genome editing systems lipids, carbohydrates, small molecules (e.g., small-molecule drugs and toxins), targeting molecules, polysaccharides, complexes, Organelles, nano and micro particles or any combination thereof.
  • the payload can be a microRNA or siRNA, eg, a microRNA or siRNA that specifically binds to a transcript encoding a mutant or non-mutated oncogene. Combination of microRNA or siRNA can enable mRNA decoding and protein synthesis.
  • genes include, but are not limited to, ABLI, BLC1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2 , MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, YES, VEGF, FGF, G-CSF, CXCR4, etc.
  • the payload can be a genome editing system.
  • Genome editing systems include, but are not limited to, meganuclease systems, zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, and clustered regularly interspaced short palindromic repeat (CRISPR) systems.
  • the payload can be a CRISPR system.
  • the CRISPR system can be a CRISPR-Cas9 system.
  • the CRISPR-Cas9 system comprises a nucleotide sequence encoding a Cas9 protein, a nucleotide sequence encoding a CRISPR RNA that hybridizes to a target sequence (crRNA), and a nucleotide sequence encoding a transactivating CRISPR RNA (tracrRNA). crRNA and tracrRNA can be fused into guide RNA.
  • the CRISPR-Cas9 system can further comprise a nuclear localization signal (NLS). Migratants loaded with the CRISPR-Cas9 system can be used to alter gene expression and function in disease management, regenerative medicine, and tissue engineering.
  • the payload can be a therapeutic protein or fragment thereof, such as an antibody or fragment thereof.
  • the payload can be a protein (including antibodies or antibody fragments, immunogenic proteins, cytokines, enzymes, tumor suppressor gene products, etc.) or fragments thereof, such as chicken ovalbumin (OVA), SARS-CoV-2 Spike protein or its S1 fragment.
  • OVA chicken ovalbumin
  • SARS-CoV-2 Spike protein or its S1 fragment.
  • the payload can be a targeting molecule.
  • targeting molecule refers to a molecule capable of specifically binding another molecule (target molecule).
  • targeting molecules can be used to specifically localize migratory bodies presenting the targeting molecule on the surface to an entity, such as a tissue or cell expressing the target molecule, thereby increasing the targeting of the delivery system.
  • the delivery systems of the present application may comprise at least two different targeting molecules to further increase targeting specificity or otherwise improve targeting to target cells or tissues.
  • targeting molecules can specifically bind surface proteins overexpressed on cancer cells, such as antibodies or antigen-binding fragments thereof.
  • targeting molecules may be selected from antibodies or antigen-binding fragments thereof; integrins; cytokines, chemokines and/or cytokine, chemokine receptors; polysaccharides; find me/eat me signals (find -me/eat me signal), such as PAMP and DAMP; and don't-eat-me signal (don't-eat-me signal), such as CD47 and CD24.
  • the antibody or antigen-binding fragment thereof can deliver the migratory body loaded with the antibody or antigen-binding fragment thereof to target cells expressing the corresponding antigen by binding to its corresponding antigen.
  • integrins or other targeting molecules can be paired with tissue-specific extracellular matrix, thereby realizing the targeting of migratory bodies loaded with integrins or other targeting molecules to specific organs.
  • find-me/eat me signal refers to a signal exposed or released by an apoptotic cell to initiate phagocytic uptake which in turn activates tolerance pathways to prevent immune responses against self-antigens.
  • the find me/eat me signal is loaded on the migratory body, and the find me/eat me signal can be recognized by macrophages, so that the migratory body is phagocytized by specific cells.
  • Exemplary find-me/eat-me signals include, for example, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).
  • PAMP Pathogen-associated molecular pattern
  • PAMPs can be expressed on the surface of pathogens or free from immune cells, and can also appear in the cytosol of immune cells, as well as various pathogen-carrying intracellular compartments in the sol, such as endosomes and phagolysosomes.
  • PAMP mainly includes two categories.
  • the first type is bacterial cell wall components mainly composed of sugars and lipids, such as lipopolysaccharide, peptidoglycan, lipoteichoic acid, mannose, lipid, lipoarabinomannan, lipoprotein, and flagellin, etc. .
  • LPS lipopolysaccharide
  • proteoglycan proteoglycan
  • glycolipid glycolipid
  • mycobacteria mycobacteria and manna produced by yeast sugar.
  • the first category is viral products and bacterial nuclear components, such as unmethylated oligonucleotides CpGDNA, single-stranded RNA, and double-stranded RNA.
  • DAMP Dermatal-associated molecular pattern
  • RIG-1-like Pattern recognition receptors such as receptors or NOD-like receptors induce autoimmunity or immune tolerance, and play an important role in the occurrence and development of arthritis, atherosclerosis, tumors, systemic lupus erythematosus and other diseases.
  • DAMPs are present in the nucleus, cytoplasm (e.g., high mobility group box (HMGB) 1, S100 proteins), extracellular matrix (e.g., hyaluronic acid), and plasma (e.g., complement C3a, C4a, C5a) or as exosomes (such as heat shock proteins).
  • cytoplasm e.g., high mobility group box (HMGB) 1, S100 proteins
  • extracellular matrix e.g., hyaluronic acid
  • plasma e.g., complement C3a, C4a, C5a
  • exosomes such as heat shock proteins.
  • Nonprotein forms of DAMPs include adenosine triphosphate, uric acid, heparan sulfate, RNA, and DNA. These proteins and non-proteins are confined inside the cell under healthy conditions and are released outside the cell when the cell is damaged.
  • the term "don't-eat-me signal” refers to the ligand expressed on the surface of tumor cells for binding to the surface of immune cells, thereby inhibiting the killing effect of immune cells on tumor cells signal of.
  • exemplary don't eat me signals include, for example, CD47 and CD24.
  • the don't eat me signal is loaded on the migratory body by means of gene editing, membrane fusion, etc., so that the migratory body escapes the clearance of the immune system, thereby prolonging the circulation time in the blood and achieving better tissue infiltration.
  • the migratory body can have a single-layer membrane structure, and its membrane origin is cell membrane and intracellular vesicle. Compared with the cell membrane, the membrane of the migratory body is specifically enriched in some proteins (such as Tetraspanin) and lipids (such as cholesterol, sphingomyelin).
  • proteins such as Tetraspanin
  • lipids such as cholesterol, sphingomyelin
  • the loads of the present application may be directly or indirectly attached or embedded into the membrane of the migration body and/or the interior of the migration body by any means known in the art.
  • the cargo can be attached or embedded into the membrane of the mobile and/or the interior of the mobile via liquid-to-solid transition, membrane fusion, charge adsorption, physical adsorption, or chemical linkage.
  • the load can be connected or embedded into the membrane of the migration body and/or the interior of the migration body in a manner selected from the group consisting of: (1) with the membrane of the migration body and/or internal components of the migration body such as membrane Linked with sugar chains or polysaccharides on proteins, cholesterol, phospholipids, and glycoproteins; (2) with proteins or peptides, sugar chains or polysaccharides on glycoproteins, phospholipids, and cholesterol that are connected or embedded in the membrane of the migratory body and/or inside the migratory body etc., preferably the protein or peptide, sugar chains or polysaccharides on glycoproteins, phospholipids, cholesterol, etc.
  • the membrane of the migratory body and/or the interior of the migratory body contains the second member of the binding pair, and the cargo is linked or embedded in the membrane of the migratory body by the binding of the first member and the second member and/or migrate internally.
  • the cargo is attached or embedded in the membrane of the migratory body and/or interior of the migratory body by association with the membrane of the migratory body and/or interior components of the migratory body, such as membrane proteins or lipids.
  • the cargo is attached to or embedded in the membrane of the migratory body and/or the interior of the migratory body by linking to a membrane protein of the migratory body, such as Tetraspanin.
  • the cargo is attached to or embedded in the membrane of the migratory body and/or the interior of the migratory body by linking to a Tetraspanin protein.
  • the cargo is attached or embedded in the membrane of the migratory body and/or the interior of the migratory body by linking to the migratory membrane lipids (such as cholesterol and sphingomyelin), proteins or peptides, sugar chains or polysaccharides on glycoproteins .
  • lipids such as cholesterol and sphingomyelin
  • proteins or peptides such as cholesterol and sphingomyelin
  • sugar chains or polysaccharides on glycoproteins such as glycoproteins.
  • binding pairs include antigen-antibody, receptor-ligand, biotin-avidin, HaloTag and its ligand, and the like.
  • the binding pair is HaloTag and its ligand, or CP05 and CD63.
  • the cargo is mRNA
  • the membrane of the migratory body and/or the interior of the migratory body comprises an mRNA binding protein that is linked or embedded into the migratory body through binding of its protein binding site to the mRNA binding protein.
  • the mRNA binding proteins and their protein binding sites can be those known in the art.
  • the mRNA binding protein is L7Ae and the protein binding site is the C/D Box.
  • the mRNA binding protein is MS2BP and the protein binding site is MS2 stem-loop (MS2SL).
  • the cargo can be expressed as a membrane protein on the inner or outer surface of the migratory membrane. In other embodiments, the cargo can be expressed on the inner or outer surface of the migratory membrane as a fusion protein to a membrane protein or a portion thereof.
  • Any membrane protein known in the art can be used as the membrane anchor protein fused to the soluble protein. Examples of membrane proteins fused as soluble proteins include, but are not limited to, cell receptors, ion channels, transporters, etc., such as Tspan-4, CD81, CD9, CD63, PDGFR, Lamp2b, syntaxin 2 (STX2), etc.
  • the membrane protein fused as a soluble protein may be STX2.
  • the membrane protein fused as a soluble protein may be a truncated STX2 (t-STX2).
  • STX2 truncated STX2
  • the N-terminus of STX2 can be modified to remove its intracellular function to obtain t-STX2.
  • the soluble protein can then be linked to the extracellular C-terminus of t-STX2 to form a soluble protein-t-STX2 fusion protein, allowing the soluble protein to be expressed on the migratory membrane as an artificial plasma membrane localized fusion protein.
  • the present application relates to a method of producing a delivery system comprising an isolated or purified mobilizer and a cargo selected from the group consisting of proteins, peptides, nucleic acids (eg DNA and RNA), lipids, small molecules compounds, polysaccharides, complexes, nano/microparticles, organelles, or simultaneously loaded with more than one of the loads, etc., wherein the method comprises: isolating or purifying the migratory body from the cell, the migratory body being a naturally occurring migratory body or artificially induced engineered migrator; and directly or indirectly linking or embedding the payload into the membrane of the migratory and/or the interior of the migratory, thereby producing the delivery system.
  • a cargo selected from the group consisting of proteins, peptides, nucleic acids (eg DNA and RNA), lipids, small molecules compounds, polysaccharides, complexes, nano/microparticles, organelles, or simultaneously loaded with more than one of the
  • the cargo is connected or embedded into the membrane of the mobile body and/or the interior of the mobile body through liquid-to-solid conversion, membrane fusion, charge adsorption, physical adsorption or chemical linkage.
  • the load is connected to or embedded in the membrane of the migration body and/or inside the migration body in a manner selected from the following: (1) combining the load with the transfer body
  • the membrane of the body and/or the internal components of the migration body such as membrane proteins, cholesterol, phospholipids, sugar chains or polysaccharides on glycoproteins; and (2) linking the load to the migration body membrane and/or migration body
  • Proteins or peptides, sugar chains or polysaccharides on glycoproteins, phospholipids, cholesterol, etc. are connected, preferably, the protein or peptides, sugar chains or polysaccharides on glycoproteins, phospholipids, cholesterol, etc. are connected to the membrane of the migration body and/or by click chemistry or migrate inside the body.
  • naturally occurring migratory bodies can be isolated or purified from cells by methods known in the art.
  • the artificially induced engineered migratory is produced by the preparation method of the engineered migratory disclosed herein.
  • the load can be one or more of the loads described elsewhere herein.
  • the present application is directed to a method of producing a delivery system comprising an engineered mobilizer and a cargo selected from the group consisting of proteins, peptides, nucleic acids (e.g., DNA and RNA), lipids, small molecule compounds, Polysaccharides, complexes, nano/microparticles, organelles, or more than one such load simultaneously loaded, etc., wherein the load is connected to the first member of the binding pair, and the membrane of the migration body and/or the interior of the migration body contains the The second member of the binding pair, wherein the method comprises: causing the cell to express the second member of the binding pair on the cell membrane; engineering the cell to contain the second member of the binding pair on the membrane and contacting the complex of the payload and the first member of the binding pair with the engineered migrator, whereby the delivery system is produced by binding of the first member and the second member.
  • a cargo selected from the group consisting of proteins, peptides, nucleic acids (e.g., DNA and RNA),
  • the method comprises introducing into the cell a nucleotide sequence comprising a coding sequence of the second member of the binding pair, so that the cell expresses the second member of the binding pair on the cell membrane. member. In some embodiments, the method comprises introducing into the cell a nucleotide sequence comprising a coding sequence for a second member of the binding pair, and culturing the cell under conditions that allow the cell to express the second member of the binding pair, such that the cell expresses the second member of the binding pair on the cell membrane.
  • the method comprises: a) introducing into the cell a nucleotide sequence comprising a coding sequence for a second member of the binding pair; b) under conditions that allow the cell to express the second member of the binding pair c) producing an engineered migratory body comprising the second member of the binding pair on the membrane from the cell; d) linking the payload to the first member of the binding pair to form a complex; and e) contacting the engineered migratory body with the complex to produce the delivery system.
  • step d) above is performed before, after or simultaneously with step a).
  • the second member of the binding pair is a membrane protein or a soluble protein. In some embodiments, the second member of the binding pair is a membrane protein and is expressed on the inner or outer surface of the engineered migratory membrane. In some embodiments, the second member of the binding pair is a soluble protein and is expressed on the inner or outer surface of the engineered migratory membrane as a fusion protein to a membrane protein or a portion thereof.
  • the binding pair includes antigen-antibody, receptor-ligand, biotin-avidin, HaloTag and its ligand, and the like. In some embodiments, the binding pair is HaloTag and its ligand, or CP05 and CD63.
  • the engineered migratory is produced by the methods of making the engineered migratory disclosed herein.
  • the load can be one or more of the loads described elsewhere herein.
  • the present application relates to a method of producing a delivery system comprising an engineered migratory and a payload, the payload being mRNA, and the membrane of the migratory and/or the interior of the migratory comprising an mRNA binding protein,
  • the mRNA binds to the mRNA binding protein through its protein binding site, wherein the method comprises: allowing the cell to express the mRNA; before, after or simultaneously with the above steps, allowing the cell to express the mRNA on the cell membrane said mRNA binding protein; and producing from said cell a delivery system comprising an engineered migratory body and mRNA, wherein said mRNA is attached to the engineered migratory body's membrane and/or migratory body by binding to said mRNA binding protein internal.
  • the method comprises introducing into a cell a coding sequence of an mRNA comprising a protein binding site, so that the cell expresses the mRNA.
  • the method comprises introducing into a cell a nucleotide sequence comprising a coding sequence of an mRNA binding protein, such that the cell expresses the mRNA binding protein on a cell membrane.
  • the method comprises introducing into a cell a coding sequence of an mRNA comprising a protein binding site and a nucleotide sequence comprising a coding sequence of an mRNA binding protein, such that the cell expresses the mRNA and the mRNA-binding protein.
  • the mRNA binding protein is a membrane protein or a soluble protein. In some embodiments, the mRNA binding protein is a membrane protein and is expressed on the inner or outer surface of the membrane of the engineered migratory body. In some embodiments, the mRNA binding protein is a soluble protein and is expressed on the inner or outer surface of the engineered migratory membrane as a fusion protein to a membrane protein or a portion thereof.
  • the mRNA binding proteins and their protein binding sites can be those known in the art.
  • the mRNA binding protein is L7Ae and the protein binding site is the C/D Box.
  • the mRNA binding protein is MS2BP and the protein binding site is MS2 stem-loop (MS2SL).
  • the engineered migratory is produced by the methods of making the engineered migratory disclosed herein.
  • the present application is directed to a method of producing a delivery system comprising an engineered mobilizer and a cargo that is a protein and expressed on an inner or outer surface of a membrane of the engineered migratory body, wherein the method comprises: causing the cell to express the protein on the cell membrane; and producing a delivery system comprising the engineered migratory body and the protein expressed on the inner or outer surface of the engineered migratory body membrane from the cell.
  • the method comprises introducing into a cell a nucleotide sequence comprising a protein coding sequence such that the cell expresses the protein on a cell membrane. In some embodiments, the method comprises introducing into a cell a nucleotide sequence comprising a protein coding sequence, and culturing the cell under conditions that allow the cell to express the protein, such that the cell expresses the protein on the cell membrane. protein.
  • the method comprises: introducing into a cell a nucleotide sequence comprising a coding sequence for a protein of interest; cultivating the cell under conditions that allow the cell to express the protein of interest; and producing from the cell an engineered Delivery systems for migratory bodies and proteins expressed on the inner or outer surface of the membrane of the engineered migratory bodies.
  • the protein is a membrane protein. In other embodiments, the protein is a soluble protein and is expressed on the surface of the migratory membrane as a fusion protein with a membrane protein or a portion thereof.
  • membrane proteins that are fused to soluble proteins are described above, including but not limited to cell receptors, ion channels, transporters, etc., such as Tspan-4, CD81, CD9, CD63, PDGFR, Lamp2b, syntaxin 2 ( STX2) etc.
  • engineered migratory bodies are produced by methods of preparing engineered migratory bodies disclosed herein.
  • the present application also provides a pharmaceutical composition or a diagnostic composition, which comprises the engineered migration body of the present application, and one or more pharmaceutically acceptable excipients, diluents or carrier combinations. Therefore, the present application also provides the use of the engineered migration body of the present application in the preparation of a pharmaceutical composition.
  • the present application also provides a method for preparing a pharmaceutical or diagnostic composition, which includes adding and mixing the engineered migratory of the present application together with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the engineered migratory body can be used as the sole active ingredient in a pharmaceutical or diagnostic composition, or it can be accompanied by other active ingredients such as steroids or other drug molecules.
  • the compositions can be administered to a patient individually or can be administered in combination (eg simultaneously, sequentially or separately) with other agents, drugs or hormones.
  • the pharmaceutical composition may comprise a therapeutically effective amount of the engineered migratory body of the present application.
  • therapeutically effective amount refers to the amount of therapeutic agent required to treat, ameliorate or prevent a targeted disease or condition or exhibit a detectable therapeutic or preventive effect.
  • the therapeutically effective amount can be estimated initially in cell culture assays or in animal models, typically rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine suitable doses and routes of administration in humans.
  • the precise therapeutically effective amount for a human subject will depend on the severity of the disease condition, the subject's general health, the subject's age, weight and sex, diet, time and frequency of administration, drug combination, response sensitivity and sensitivity to Tolerance/response to therapy depends.
  • compositions of the present application can be administered by various routes, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transdermal, subcutaneous, intraperitoneal, intranasal , enteral, topical, sublingual, intravaginal or rectal routes.
  • the pharmaceutical compositions of the present application can also be administered using needle-free injectors.
  • therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • a pharmaceutical composition containing a therapeutic dose of the engineered migratory body of the present application does not show significant toxicological effects in vivo.
  • ddH2O ddH2O
  • KCl KCl
  • KH2PO4 sangon
  • Na2HPO4-7H2O sangon
  • BSA VWR
  • Latrunculin A cayman
  • human fibronectin invitrogen or sigma
  • PBS Gibco
  • RPMI 1640 Gibco
  • FBS BI
  • WGA-AF488/AF594/AF647 invitrogen
  • BCA kit invitrogen
  • a NRK cell line stably expressing Tspan4-GFP was established, and the formation of the engineered migratory body was observed through the signal of Tspan4, one of the markers of the migratory body.
  • NRK cells overexpressing Tspan4-GFP were treated with 25% DPBS (corresponding to an osmotic pressure of 76.3 mOsmol/L), and the results are shown in Figure 1a.
  • Tspan4-GFP signal began to enrich on contractile filaments, and then formed micron-sized vesicles; after reaching its peak intensity, Tspan4-GFP signal began to diffuse from vesicles, accompanied by Shrinkage of vesicles; 460 seconds after hypoosmolarity treatment, most of the vesicle structures induced by hypoosmolarity disappeared.
  • the production process of these micron-sized vesicles and their attachment to contractile filaments resembles natural migratory bodies.
  • WGA wheat germ agglutinin
  • NRK cells overexpressing Tspan4-GFP were subjected to isotonic treatment (100% DPBS, 305mOsmol/L) and hypotonic treatment (76.3mOsmol/L) of 25% DPBS, respectively, and then treated with tetramethylrhodamine-labeled WGA.
  • the cells were stained and observed under a laser confocal microscope, and the results are shown in Figure 26c.
  • the results showed that the induced vesicle structures of NRK cells overexpressing Tspan4 were specifically stained by WGA, indicating that the produced vesicle structures were engineered migratory bodies.
  • NRK cells expressing Tspan4-GFP were subjected to isotonic pressure (100% DPBS, 305mOsmol/L) or different intensities of low osmotic pressure (50%, 25%, and 17 %DPBS, respectively corresponding to the osmotic pressure of 152.5, 76.3 and 50.8 mOsmol/L), the results are shown in Figure 1b, and the statistical results of the engineered migration body diameter are shown in Figure 1c. It can be found that the level of osmotic pressure is negatively correlated with the size of the engineered migration body, the lower the osmotic pressure, the larger the size of the induced engineered migration body.
  • the inventors established a protocol for stepwise reduction of osmotic pressure, and found that the gradual application of hypotonic pressure significantly increased the duration of engineered migratory bodies, and in cells undergoing 5-step hypotonic treatment, induced engineered migration The body remained after 20 minutes of hypotonic treatment.
  • Embodiment 2 produces engineered migration body by latrunculin A treatment
  • NRK cells expressing Tspan4-GFP were incubated with isotonic buffer solution DPBS containing different concentrations of actin depolymerization agent latrunculin A (0, 0.25, 0.5 and 1 ⁇ M) for 10 min, and then added water at intervals Three consecutive hypotonic stimulations were performed on the cells by reducing the salt concentration by 1/6 for 2 minutes.
  • the results after stepwise hypoosmotic treatment are shown in Figure 2a, and the statistical results of the number of engineered migratory bodies produced per cell are shown in Figure 2b.
  • Latrunculin A can significantly promote the increase of the number of engineered migratory bodies, and this promotion effect is dose-dependent. This suggests that the formation of engineered migratory bodies can be promoted by disrupting the cytoskeleton.
  • SWELL 1 a key component of volume-regulated anion channels that maintains constant cell volume by responding to changes in osmotic pressure, was knocked down or knocked out.
  • the gene Lrrc8a encoding SWELL 1 was knocked down in NRK cells expressing Tspan4-GFP, and the knockdown efficiency of Lrrc8a in cells was verified by qPCR. The results showed that the gene Lrrc8a encoding SWELL 1 was successfully knocked down to about 15% of wild-type cells (Fig. 3a).
  • the Lrrc8a gene knocked-down cells (Lrrc8a-KD) and non-knockdown control cells (NC) were respectively placed in DPBS, and the cells were subjected to three consecutive conditions by gradually adding water to reduce the salt concentration by 1/3 at intervals of 2 min. Stimulation by low osmotic pressure, and then observed with a laser confocal microscope, the results are shown in Figure 3b; the statistical results of the size of the engineered migrator are shown in Figure 3c.
  • Lrrc8a encoding SWELL 1 in NRK cells expressing Tspan4-GFP was knocked out, and the knockout of Lrrc8a in the cells was verified by Western blotting. No expression of SWELL 1 was observed in the knockout cells (Lrrc8a-KO). It indicated that the coding gene Lrrc8a of SWELL 1 was successfully knocked out (Fig. 4a).
  • Lrrc8a-KO cells (KO14# cell line and KO18# cell line) and non-knockout control cells (WT) were placed in DPBS, and the cells were treated by gradually adding water and reducing the salt concentration by 1/6 at intervals of 1 min. Five consecutive hypoosmotic pressure stimulations were then observed with a laser confocal microscope, and the results are shown in Figure 4b.
  • Cations are known to regulate cell volume. If different cations differ in their ability to regulate cell volume during osmotic pressure changes, the regulated cell volume changes can be attenuated by cation displacement, thereby facilitating the formation of engineered migratory bodies.
  • Sodium chloride in DPBS was replaced with potassium chloride, cesium chloride or choline chloride of equimolar concentration to prepare isotonic buffers containing different cations, and the cells were incubated with these isotonic buffers, and the corresponding The cells were subjected to five consecutive hypotonic stimulations by adding water gradually to the buffer solution to reduce the salt concentration by 1/6 at intervals of 2 min.
  • the results after gradually low osmotic pressure treatment are shown in Figure 5a, and the statistical results of the engineered migration body size are shown in Figure 5b.
  • Tspan4 is a key protein that promotes the formation of migratory bodies.
  • NRK cells that only overexpress mCherry-Kras or NRK cells that overexpress both Tspan4-GFP and mCherry-Kras were step-by-step Hypoosmolar stimulation. After NRK cells were incubated in KDPBS containing 2 ⁇ M latrunculin A for 10 min, three consecutive hypotonic stimulations were performed by reducing the salt concentration by 1/6 every 2 min, and then the engineered migratory bodies were observed by differential interference contrast microscopy (DIC) Formation. The results after stepwise hypotonic treatment are shown in Figure 6a, and the statistical results of the number of engineered migratory bodies produced per cell are shown in Figure 6b.
  • DIC differential interference contrast microscopy
  • Example 5 Rapid detachment of cells to produce migratory bodies by means of a temperature-sensitive coating on a petri dish
  • MGC803-T4-GFP cells were cultured on temperature-sensitive culture dishes. After destroying the cytoskeleton, they were subjected to hypotonic treatment at room temperature or 37 degrees, or without hypotonic treatment, and the culture dishes were left at room temperature for 45 minutes. More than 80% of the cells detached from the bottom of the dish and formed migratory bodies, and part of the migratory bodies detached from the bottom of the dish along with the cells (see Figure 7). Collect the supernatant and collect residual cells and migratory bodies by pipetting.
  • the above-mentioned hypotonic-treated cell line was further stained with tetramethylrhodamine-labeled WGA, a specific probe of the migratory body, and then observed under a confocal laser microscope.
  • the results are shown in Figure 8b.
  • Cell lines of human origin human embryonic kidney cells (HEK293T, HEK293FT; Fig. 8c), human gastric cancer cells (MGC803), human skin fibroblasts (BJ) and human peripheral blood leukemia T cells (Jurkat); Fig. 8d
  • engineered migrators could be produced by the methods of Examples 1-5.
  • the results showed that the induced vesicle structures of the eight cell lines were stained by WGA, a specific probe for migratory bodies, indicating the generation of engineered migratory bodies.
  • h-KDPBS hypotonic KDPBS
  • its salt concentration is close to the salt concentration in the solution system when hypotonic induction is completed;
  • h-KDPBS is 40% KDPBS;
  • h- KDPBS is 60% KDPBS), and the cells are washed twice;
  • the liquid in the centrifuge tube 2 was centrifuged at 4°C and 17000xg for 5min, the supernatant was discarded, and the precipitate was washed once with PBS, the supernatant was discarded, the precipitate was cracked with 30 ⁇ l 2% SDS, and the sample was boiled in a metal bath at 95°C.
  • the BCA method is used to determine the protein concentration, and the total protein amount in the centrifuge tube 1 can be converted, and the corresponding resuspension volume can be converted according to the injection dose.
  • step III the engineered migrator obtained in step III was observed with a negative-stain transmission electron microscope and a cryo-electron microscope, and the results are shown in Figures 11b and 11c, respectively.
  • step III-9 Take 1 ⁇ l of liquid in step III-9 or 10 and dilute to 100 ⁇ l. If the vesicles show two-color fluorescence, they can be directly diluted with PBS; if the vesicles only have single-color fluorescence, they can be stained with 1:500 WGA dye to improve the effect of grouping.
  • step III-1 Take a small amount of liquid from the supernatant of step III-1, centrifuge at 100 ⁇ g and then lyse the pellet with 2% SDS, and use it as a cell body sample; the sample obtained in step IV-1 is used as an engineered migratory sample.
  • the samples of the cell body (cell) and the engineered migratory body (eMig) were loaded with the same amount of protein, and western-blot was performed to detect the classic markers of various organelles: nucleus (histone H3), mitochondria (Tim23), Endoplasmic reticulum (calnexin), lysosome (Lamp2), cytoplasm (GAPDH), cell membrane (Na+-K+-ATPase), cell membrane focal adhesion (integrin ⁇ 5), Tspan4-GFP (GFP), the results are shown in the figure 12 shown.
  • the engineered migratory body obtained by separation and purification was added dropwise in a confocal small dish, and Cy5 (a fluorescent dye that cannot pass through the complete membrane) and dextran-TMR (Dex-TMR) were added to the buffer to indicate the vesicles the permeability.
  • Cy5 a fluorescent dye that cannot pass through the complete membrane
  • dextran-TMR Dex-TMR
  • the statistical results of the permeation ratio of the engineered migrator to dextran-TMR after being placed at room temperature for 1.5h, 6h, 12h, 24h, and 48h are shown in
  • Cholesterol extraction reagent M ⁇ CD (10 mM) was added to the droplets, and then confocal laser microscopy was performed. The results are shown in FIG. 15 . It was found that most of the engineered migrators were severely deformed and damaged within 10 minutes, which indicated that cholesterol played a crucial role in the stability of the engineered migrators.
  • the delivery of membrane proteins can be achieved by directly transferring the plasmid encoding the target gene and Tspan4 into the production cells (Fig. 16a).
  • the overexpression of Tspan4 combined with other steps in the preparation of engineered migratory bodies can greatly increase the production efficiency of engineered migratory bodies, and the overexpressed membrane proteins will be enriched in a large amount on the engineered migratory bodies produced, thereby realizing the membrane protein in the engineered migratory bodies. Loading on migratory bodies.
  • Membrane proteins that can be loaded include various cell receptors (such as various GPCRs, PD-1, VEGFR, etc.), extracellular enzymes (such as CD36, CD73), ion channels, transporters, and various antigens (such as S protein), etc. .
  • the membrane proteins DAG1, Tgfbr1, PDCD1 and Kras transfect the plasmid containing Tspan4-GFP and the target gene sequence into the production cell NRK cells, in order to facilitate the observation of the subcellular localization and expression of the target protein, the target gene fragments were fused through the linker to the mCherry tag.
  • the amino acid sequence and carrier information of each fusion protein are shown below.
  • Tspan4-linker-GFP carrier: pB-Hygro-GFP (vector map shown in Figure 19), insertion site BsrGI+BamHI): (SEQ ID NO: 1)
  • Tspan4 The amino acid sequence of Tspan4 is underlined; the amino acid sequence of GFP is shown in bold italics; the amino acid sequence between Tspan4 and GFP is the linker sequence (PG).
  • DAG1-linker-mCherry (vector: pmCherry-N1 (vector map shown in Figure 20), insertion site: EcoRI+KpnI): (SEQ ID NO: 2)
  • the amino acid sequence of DAG1 is underlined; the amino acid sequence of mCherry is shown in bold italics; the amino acid sequence between DAG1 and mCherry is the linker sequence (GDPVAT).
  • PDCD1-linker-mCherry vector: pB-Hygro-mCherry (vector map shown in Figure 21), insertion site: BsrGI+MluI): (SEQ ID NO: 3)
  • the amino acid sequence of PDCD1 is underlined; the amino acid sequence of mCherry is shown in bold italics; the amino acid sequence between PDCD1 and mCherry is a linker sequence (TVPRARDPPVAT).
  • Tgfbr1-linker-mCherry (vector: pmCherry-N1 (vector map shown in Figure 20), insertion site: EcoRI+KpnI): (SEQ ID NO: 4)
  • Tgfbr1 The amino acid sequence of Tgfbr1 is underlined; the amino acid sequence of mCherry is shown in bold italics; the amino acid sequence between Tgfbr1 and mCherry is a linker sequence (TVPRARDPPVAT).
  • mCherry-linker-Kras vector: pB-Hygro-mCherry (vector map shown in Figure 21), insertion site: BsrGI+MluI): (SEQ ID NO: 5)
  • amino acid sequence of mCherry is underlined; the amino acid sequence of Kras is shown in bold italics; the amino acid sequence between mCherry and Kras is a linker sequence (SGLRSRG).
  • the cells were screened for drug killing against the resistance carried on the plasmid, and cell lines stably expressing various mCherry-tagged membrane proteins were established. Then, the hypotonic treatment conditions for NRK cells in Example 5 were used to induce the cell line to produce engineered migratory bodies that had transcribed the corresponding membrane proteins, and then observed with a laser confocal microscope. The results showed that the four membrane proteins of interest were all located correctly on the engineered migratory body ( FIG. 17 ), indicating that membrane proteins could be integrated into the engineered migratory body.
  • the S protein For the S protein, a plasmid containing Tspan4-GFP and the target gene sequence was transfected into the production cell MC38 cells. In order to facilitate the observation of the subcellular localization and expression of the target protein, the mCherry tag was fused after the target gene fragment. After transfection, the cells were screened for drug killing against the resistance carried on the plasmid, and a cell line stably expressing the mCherry-tagged S protein was established. Then, the hypotonic treatment conditions for MC38 cells in Example 5 were used to induce the cell line to produce engineered migratory bodies loaded with S protein, and then observed with a laser confocal microscope. The results showed that the S protein was correctly positioned on the engineered migratory body ( FIG. 18 ), indicating that the S protein could be effectively integrated into the engineered migratory body.
  • the engineered migratory is leaky, it may not be used for direct delivery of cytosolic soluble proteins.
  • the soluble protein can be anchored on the membrane by making the production cell express the fusion protein of target protein-membrane protein, so as to prevent the leakage of soluble protein (Fig. 16a and Fig. 16b).
  • the N-terminus of the membrane protein syntaxin-2 STX2
  • STX2 the N-terminus of the membrane protein syntaxin-2
  • t-STX2 truncated STX2
  • the soluble protein was then linked to the extracellular C-terminus of t-STX2 to form a soluble protein-t-STX2 fusion protein ( FIG.
  • t-STX2 itself allows the soluble protein of interest to end up hanging outside the cell membrane, thus facilitating, for example, the protein's recognition by the immune system as an intact antigen.
  • membrane proteins are also commonly used as membrane anchor proteins, such as Tetraspanin-4, CD81, CD9, CD63, PDGFR, Lamp2b, etc.
  • Plasmids containing Tspan4-GFP and t-STX2-OVA-mCherry coding sequences were transfected into producer cells MC38 cells.
  • the amino acid sequence and carrier information of the fusion protein of t-STX2 and OVA are shown below.
  • amino acid sequence of t-STX2 is in bold; the amino acid sequence of OVA is underlined; the amino acid sequence of mCherry is in bold italics.
  • the cells were screened for drug killing against the resistance carried on the plasmid, and a cell line stably expressing the mCherry-tagged t-STX2-OVA fusion protein was established. Then, the hypotonic treatment conditions for MC38 cells in Example 5 were used to induce the cell line to produce engineered migratory bodies loaded with t-STX2-OVA fusion protein, and then observed by confocal laser microscopy. The results are shown in FIG. 22 .
  • target molecules such as small molecule drugs, small nucleic acid drugs, and peptides in engineered migratory bodies
  • target molecules such as small molecule drugs, small nucleic acid drugs, and peptides in engineered migratory bodies
  • target molecules such as small molecule drugs, small nucleic acid drugs, and peptides in engineered migratory bodies
  • antigen-antibody, receptor-ligand, and biotin-avidin binding systems Realized ( Figure 16a).
  • HaloTag receptor-ligand interaction of HaloTag with its artificially synthesized ligand
  • ligand can be used (Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish , R., Ohana, R.F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., & Wood, K.V. (2008).
  • HaloTag a novel protein labeling technology for cell imaging and protein analysis. ACS chemical biology, 3(6), 373–382.) to achieve the purpose of molecular migration in engineering on the loading.
  • a plasmid encoding the fusion protein Tspan4-HaloTag of the membrane protein Tspan4 and the receptor protein HaloTag was constructed.
  • the plasmid is transfected into the engineered migratory production cell to realize the transformation of the production cell.
  • the modified production cells are induced to produce engineered migratory bodies, and the engineered migratory bodies after separation and purification contain the receptor protein HaloTag on the membrane.
  • the modification of the target molecule is achieved by coupling the target molecule with the ligand of HaloTag to form a target molecule-ligand conjugate. Then, the target molecule-ligand conjugate is co-incubated with the engineered migratory containing HaloTag on the membrane in vitro, and the target molecule is fixed to the membrane of the engineered migratory and/or migrates through the interaction of HaloTag and its ligand. body interior.
  • the covalent binding of HaloTag to its ligand is specific, efficient and irreversible.
  • membrane proteins such as those described elsewhere herein, may also serve as membrane anchor proteins for the loading of molecules of interest on engineered migratory bodies.
  • other antigen-antibody, receptor-ligand, biotin-avidin and other binding systems can also be used to load target molecules on engineered mobilizers, such as the CP05 and CD63 binding system (X.
  • the receptor CD63 was first loaded onto the membrane of the engineered migrator, and then the target molecule-CP05 conjugate was loaded onto the engineered migratory membrane containing CD63 by using the binding of CD63 and its ligand CP05. on the migration membrane.
  • the use of receptors with a large number of natural ligands in the body can be avoided as much as possible.
  • a plasmid containing the Tspan4-HaloTag-GFP coding sequence was transfected into the production cell NRK cells, and a plasmid containing the Tspan4-GFP coding sequence was used as a control.
  • the amino acid sequence and carrier information of the fusion protein of Tspan4 and HaloTag are shown below.
  • Tspan4-linker-GFP-linker-Halo carrier: pB-Hygro-GFP (vector map shown in Figure 19), insertion site: BsrGI+MluI): (SEQ ID NO: 7)
  • Tspan4 is in bold; the amino acid sequence of GFP is underlined; the amino acid sequence of Halo is in bold italics.
  • Example 5 After transfection, the cells were screened for drug killing against the resistance carried on the plasmid, and a cell line with stable expression was established. Then, the hypotonic treatment conditions for NRK cells in Example 5 were used to induce the cell line to produce engineered migratory bodies. Add fluorescent HaloTag ligand-TMR dye to the engineered migrator, incubate at room temperature for 15 minutes, wash twice with dye-free buffer, drop into a confocal small dish and let it stand for 5 hours at room temperature for laser confocal microscope observation , the result is shown in Figure 23.
  • small nucleic acid drugs can be loaded onto the engineered migratory membrane by various other modification methods.
  • chemically modified siRNA or antisense oligonucleotides can be conjugated to cholesterol
  • small nucleic acid drugs can be loaded onto the membrane of engineered migratory bodies through the compatibility of cholesterol with cell membranes (S.S.Yerneni, S.
  • siRNA conjugates can also be immobilized on the membrane by means of click chemistry (T.Tian, H.X.Zhang, C.P.He, S.Fan, Y.L.Zhu, C.Qi, N.P.Huang, Z.D.Xiao, Z.H.Lu, B.A.Tannous, J. Gao, Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy, Biomaterials 150 (2016) 137–149).
  • This method needs to couple the siRNA to the peptide first, and then couple the peptide to the membrane by click chemistry.
  • mRNA-binding proteins L7Ae (Kojima, R., Bojar, D., Rizzi, G., Hamri, GC, El-Baba, MD, Saxena, P., S., Tan, KR, & Fussenegger, M. (2018).
  • the fusion protein plasmid and the mRNA plasmid are jointly transferred into the production cell, and the production cell is induced to produce an engineered migratory body, and the isolated and purified migratory body contains the target mRNA molecule on its membrane.
  • Example 11 The engineered migratory body loaded with SARS-CoV-2 spike protein induces an immune response in mice
  • the experimental flow is shown in Figure 24a.
  • the experimental animals were 8-week-old female mice of the C57BL/6 strain, 5 in each group.
  • the specific groups are:
  • NC-eMig engineered migration body expressing only Tspan4-GFP of 20 ⁇ g of total protein was injected into the tail vein as a negative control;
  • the S1 protein content in the S-eMig group was semi-quantitatively analyzed by western blotting ( FIG. 24 b ).
  • the first 6 lanes from the left are 1, 2, 5, 10, 20 and 50 ng of S1 recombinant protein, and the 7th to 10th lanes are respectively NC-eMig or S-eMig (eMig-1 and eMig- 2 represents samples from two independent experiments). Comparing the brightness of the bands in each lane, it can be calculated that the S1 protein in one dose (20 ⁇ g) of S-eMig is about 100 ng, which is far less than the amount of S1 recombinant protein (50 ⁇ g) used in the aluminum adjuvant group.
  • mice received one immunization on day 0, and peripheral blood was collected after being sacrificed on day 14, and the concentration of S protein-specific IgG antibody in the serum was detected by ELISA, and the results are shown in Figure 24c. It was found that neither the S1 protein group (group II) nor the NC-eMig negative control group (group III) could promote antibody production; the S1 protein + aluminum adjuvant group (group I) and the S-eMig group (group IV) ) can effectively promote antibody production, and the immune effects of the two are roughly similar.
  • the engineered migratory body can cause similar antibody response without adjuvant and the amount of protein is small, and it is a more efficient delivery vehicle, which can effectively promote immune response.
  • control migratory body from the same cell source (see Ma et al, Cell Res. 2015 for the production and purification process) and the engineered migratory body (see Figure 9 for the production and purification process) were produced and purified, and compared in many aspects.
  • NRK cells overexpressing Tspan4-GFP were fixed with 2.5% glutaraldehyde after hypotonic treatment and observed by scanning electron microscope (Fig. 25a).
  • a spinning disk confocal microscope to take 4D shots of the process of NRK cells overexpressing Tspan4-GFP producing engineered migratory bodies, it can be observed that during the hypotonic treatment, the cell body expands, the bottom surface shrinks, and a large number of filamentous structures are produced; Engineered migratory bodies were grown on the filamentous structures (Fig. 25b).
  • the naturally occurring migratory body is due to the displacement of the center point of the cell, the contractile filament attached to the migratory body is only located at the end of the cell migration, and the number of migratory body is small (Figure 26a); the engineered migratory body induces the cell membrane in different ways Relative displacement, the attached contractile filaments can be distributed in all directions of the cell, and the number of migratory bodies on each contractile filament and single cell can be significantly increased compared with the control migratory bodies (Fig. 26b). After the NRK cells overexpressing Tspan4-GFP were stained with tetramethylrhodamine-labeled WGA, the difference between the control migratory body (Fig.
  • Example 13 The engineered migratory body is distinguished from other hypotonic vesicles
  • the size of the engineered migrator is in the order of microns, and few have a diameter exceeding 5 ⁇ m.
  • the vesicles made by Cohen et al. using low osmotic pressure can be as large as 20 ⁇ m.
  • Example 14 Distinction of engineered migratory bodies from extracellular vesicles/exosomes
  • Migratory bodies and exosomes are both extracellular vesicles, but the mechanism of production and the size of the vesicles (exosomes are generally 50-150nm) and other characteristics are different.
  • Small extracellular vesicles (sEVs)/exocrine Exosomes (see Figure 27a for the production and purification process) and engineered migratory bodies (see Figure 9 for the production and purification process) were produced, purified, and compared in different aspects.
  • NTA detection of purified exosomes Figure 27b
  • TEM transmission electron microscopy
  • the MC-38 cells overexpressing Tspan4-GFP were plated in the cell culture dish after fibronectin treatment;
  • the applicant found that the unit yield of engineered migratory bodies is much higher than that of extracellular vesicles without ultracentrifugation , ultra-fast density gradient centrifugation and other methods for purification, the production speed is greatly increased.
  • the yield of engineered migratory bodies was about 35 times that of extracellular vesicles/exosomes (Fig. 27e).

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Abstract

提供了一种工程化迁移体及其制备方法和用途，具体涉及一种制备迁移体的方法，所述方法包括使细胞产生相对位移继而产生源自所述细胞的迁移体。还提供了一种所述方法制备的迁移体，体外或离体制备的迁移体以及包含所述迁移体的递送系统。

Description

工程化迁移体及其制备方法和用途 技术领域
涉及生物医学领域，具体涉及一种工程化迁移体、制备工程化迁移体的方法、包含工程化迁移体的递送系统及其制备方法。
背景技术
本领域现有的递送系统存在诸多问题，例如。生物来源的纳米颗粒及微颗粒递送系统拥有较高的生物兼容性及相对较好的靶向性，但生产均较为困难；工程化的外泌体存在明显的局限性，如产量低、纯化过程依赖超高速离心机、内容物主要为后期装载的核酸等；工程化改造的红细胞无法在体外实现持续的培养、增殖，因而每次制备都需要血液样品；红细胞也同样存在装载物限制性强的问题，装载物主要为核酸；膜修饰的纳米颗粒难以被人体正常代谢清除，可能在体内积累，具有潜在的毒性；病毒和类病毒颗粒可递送的药物基本局限于核酸和小分子量的蛋白，以及病毒和类病毒颗粒的生产及纯化过程较为困难、成本较高，修饰较为困难等等。因此，本领域需要能够克服现有的药物递送系统所存在的一种或多种缺陷的新型递送系统。
本申请发现可以通过多种方法诱导产生结构类似于天然迁移体的工程化迁移体，工程化迁移体不同于本领域已知的“低渗透压诱导的囊泡”。本申请同时发现的工程化迁移体为生物来源，具有生物来源的低毒性低免疫原性的优点；工程化迁移体是一种全新的细胞外囊泡，在可运送的分子、体内的生物分布等方面具有独特的优点；同时工程化迁移体制备方法简单，产量高。因此，本申请提供的工程化迁移体在药物递送和疫苗制备等生物医药领域具有极高的应用价值。
发明内容
本申请提供一种工程化迁移体、制备工程化迁移体的方法、包含工程化迁移体的递送系统及其制备方法。所述工程化迁移体包括但不限于以下优势：低毒性、低免疫原性、可运送多种外源荷载分子、适合的体内生物分布、制备方法简单、和/或产量高。本申请提供的工程化迁移体可以用于药物递送和疫苗制备等，具有优异的效果。
一方面，本申请提供了一种制备迁移体的方法，所述方法包括使细胞产生相对位移继而 产生源自所述细胞的迁移体。
在一种实施方式的方法中，其还包括分离所述细胞所产生的迁移体。
在一种实施方式的方法中，其包括对所述细胞进行低渗透压处理。
在一种实施方式的方法中，其中所述低渗透压处理包括使所述细胞置于低渗缓冲溶液中。
在一种实施方式的方法中，其中所述低渗透压处理包括使所述细胞置于缓冲溶液中，并将所述缓冲溶液的渗透压降低成为低渗缓冲液。
在一种实施方式的方法中，其中所述降低包括线性降低和/或阶梯式降低。
在一种实施方式的方法中，其中所述低渗缓冲溶液的渗透压低于约305mOsmol/L。
在一种实施方式的方法中，其中所述低渗缓冲溶液的渗透压为约10mOsmol/L至约274.5mOsmol/L。
在一种实施方式的方法中，其包括破坏所述细胞的细胞骨架。
在一种实施方式的方法中，其中破坏所述细胞的细胞骨架包括使所述细胞与破坏细胞骨架的试剂接触。
在一种实施方式的方法中，其中所述破坏细胞骨架的试剂包括微丝和/或微管解聚剂。
在一种实施方式的方法中，其中所述破坏细胞骨架的试剂包括Latrunculin A，Latrunculin B，细胞松弛素A，细胞松弛素B，细胞松弛素C，细胞松弛素D和/或细胞松弛素E。
在一种实施方式的方法中，其包括抑制所述细胞的细胞体积调节功能。
在一种实施方式的方法中，其中抑制所述细胞的细胞体积调节功能包括使所述细胞中体积调节蛋白的数量和/或功能下降。
在一种实施方式的方法中，其中所述体积调节蛋白包括体积调节离子通道和/或转运蛋白。
在一种实施方式的方法中，其中所述体积调节离子通道包括体积调节阴离子通道VRAC和/或体积调节阳离子通道VRCC。
在一种实施方式的方法中，其中所述体积调节阴离子通道VRAC包括SWELL1或其功能活性片段。
在一种实施方式的方法中，其中所述体积调节阳离子通道VRCC包括TRPV4，TRPM3和/或其功能活性片段。
在一种实施方式的方法中，其中所述转运蛋白包括协同转运蛋白。
在一种实施方式的方法中，其中所述协同转运蛋白包括KCC1、KCC3和/或KCC4。
在一种实施方式的方法中，其中抑制所述细胞的细胞体积调节功能包括将所述细胞置于 具有减弱的体积调节能力的缓冲溶液中。
在一种实施方式的方法中，其中所述缓冲液含有增加的阳离子。
在一种实施方式的方法中，其中所述阳离子包括K +，Na +，Cs +，Li +，Ca 2+，Mg 2+，Ba 2+，Mn 2+，Fe 2+，Ni 2+，Zn 2+，Al 3+，Fe 3+,CH 3NH 3 +，C 2H 5NH 3 +，(CH 3) 2NH 2 +，(C 2H 5) 2NH 2 +，(C 2H 5) 3N +，氨离子和/或胆碱离子。
在一种实施方式的方法中，其中所述缓冲液含有增加的阴离子。
在一种实施方式的方法中，其中所述阴离子包括Br -，Cl -，I -，F -，OH -，HCO 3 -，H 2PO 4 -，NO 2 -，NO 3 -，CN -，HPO 4 2-，CO 3 2-，SO 4 2-和/或PO 4 3-。
在一种实施方式的方法中，其中所述方法包括使所述细胞脱离其粘附表面。
在一种实施方式的方法中，其中所述方法包括使所述细胞的细胞膜与其粘附表面之间发生相对位移。
在一种实施方式的方法中，其还包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。
在一种实施方式的方法中，其包括使所述细胞过表达所述tetraspanin蛋白，其功能性片段和/或功能性变体。
在一种实施方式的方法中，其中所述tetraspanin蛋白选自：Tspan1、Tspan2、Tspan3、Tspan4、Tspan5、Tspan6、Tspan7、Tspan8、Tspan9、Tspan10、Tspan11、Tspan12、Tspan13、Tspan14、Tspan15、Tspan16、Tspan17、Tspan18、Tspan19、Tspan20(UPK1B)、Tspan21(UPK1A)、Tspan22(PRPH2)、Tspan23(ROM1)、Tspan24(CD151)、Tspan25(CD53)、Tspan26(CD37)、Tspan27(CD82)、Tspan28(CD81)、Tspan29(CD9)、Tspan30(CD63)、Tspan31、Tspan32和Tspan33。
在一种实施方式的方法中，所述方法还包括减小所述迁移体的尺寸。
在一种实施方式的方法中，其中所述减小所述迁移体的尺寸包括使用过滤器或挤压器挤压所述迁移体。
在一种实施方式的方法中，其中所述过滤器或挤压器的孔径为约30nm至约10000nm。
在一种实施方式的方法中，其中所述迁移体的尺寸为约50nm至约8000nm。
在一种实施方式的方法中，其中所述迁移体产生于所述细胞的收缩丝。
在一种实施方式的方法中，其中所述迁移体的膜上富集有钠/钾ATP酶和/或其功能性片段。
在一种实施方式的方法中，其中所述迁移体的膜上富集有整合素和/或其功能性片段。
在一种实施方式的方法中，其中所述迁移体的膜上富集有tetraspanin蛋白，其功能性变体和/或其功能性片段。
在一种实施方式的方法中，其中所述迁移体的膜上富集有胆固醇。
在一种实施方式的方法中，其中所述迁移体富集有膜微结构域。
在一种实施方式的方法中，其中所述迁移体在体外产生或离体产生。
在一种实施方式的方法中，其中与相应细胞产生的天然迁移体相比，所述迁移体的内容物至少部分减少或缺失。
在一种实施方式的方法中，其中所述至少部分减少的内容物包含腔内囊泡。
在一种实施方式的方法中，其为体外或离体方法。
在一种实施方式的方法中，其中所述细胞为经体外培养的细胞。
在一种实施方式的方法中，其中所述细胞为经悬浮培养或经贴壁培养的细胞。
在一种实施方式的方法中，其中所述细胞包括原代细胞。
在一种实施方式的方法中，其中所述原代细胞包括源自生物体的组织细胞，所述生物体包括人，猴，小鼠，大鼠，兔，鸡和/或昆虫。
在一种实施方式的方法中，其中所述原代细胞包括肝细胞，脾细胞，肾细胞，组织巨噬细胞，脑胶质细胞，破骨细胞，骨髓细胞，白细胞，成纤维细胞和/或脂肪细胞。
在一种实施方式的方法中，其中所述白细胞包括B细胞，T细胞，NK细胞，树突状细胞，中性粒细胞和/或巨噬细胞。
在一种实施方式的方法中，其中所述细胞包括肿瘤细胞。
在一种实施方式的方法中，其中所述肿瘤细胞包括肿瘤细胞系，源自患者的原代或经有限传代的肿瘤细胞，肿瘤基质细胞，和/或肿瘤类器官。
在一种实施方式的方法中，其中所述细胞包括CHO细胞、CHO-K1细胞、HEK293细胞、HEK293T细胞、HEK293FT细胞、HEK293F细胞、Vero细胞、NRK细胞、L929细胞、MC38细胞、4T1细胞、DC2.4细胞、MGC803细胞、Jurkat细胞、NK-92MI细胞、BJ细胞和/或HepG2细胞。
在一种实施方式的方法中，其中所述细胞包括白细胞、干细胞和/或成纤维细胞。
在一种实施方式的方法中，其中所述干细胞包含间充质干细胞。
在一种实施方式的方法中制备的迁移体。
另一方面，本申请提供了一种体外或离体制备的迁移体，其中所述迁移体的尺寸为约 50nm至约8000nm。
在一种实施方式的迁移体中，其在体外产生于细胞的收缩丝。
在一种实施方式的迁移体中，其中所述迁移体的膜上富集有钠/钾ATP酶和/或其功能性片段。
在一种实施方式的迁移体中，其中所述迁移体的膜上富集有整合素和/或其功能性片段。
在一种实施方式的迁移体中，其中所述迁移体的膜上富集有tetraspanin蛋白，其功能性变体和/或其功能性片段。
在一种实施方式的迁移体中，其中所述迁移体的膜上富集有胆固醇。
在一种实施方式的迁移体中，其中所述迁移体富集有膜微结构域。
在一种实施方式的迁移体中，其中与相应细胞产生的天然迁移体相比，所述迁移体的内容物至少部分减少或缺失。
在一种实施方式的迁移体中，其中所述至少部分减少的内容物包含腔内囊泡。
另一方面，本申请提供了一种迁移体用于递送外源荷载的用途。
在一种实施方式的用途中，其中所述迁移体包含本申请所述的迁移体。
另一方面，本申请提供了一种递送系统，其包含迁移体和一种或多种外源荷载。
在一种实施方式的递送系统中，其中所述外源荷载直接或间接结合、连接或嵌入至所述迁移体的膜和/或内部。
在一种实施方式的递送系统中，其中所述迁移体包括本申请所述的迁移体。
在一种实施方式的递送系统中，其中所述迁移体源自细胞。
在一种实施方式的递送系统中，其中所述外源荷载包含一种或多种靶向物质和/或治疗活性物质。
在一种实施方式的递送系统中，其中所述外源荷载包含蛋白质，脂质，多核苷酸，小分子化合物，复合物、多糖、聚合物、纳米颗粒、微米颗粒和/或细胞器。
在一种实施方式的递送系统中，其中所述外源荷载包括膜蛋白，可溶性蛋白和/或多肽。
在一种实施方式的递送系统中，其中所述外源荷载包括DNA和/或RNA。
在一种实施方式的递送系统中，其中所述外源荷载包括抗体或其抗原结合抗体片段、整合素或其片段、免疫原性蛋白、细胞因子、趋化因子、受体蛋白或其片段、酶、抑癌基因产物、siRNA、microRNA、反义寡核苷酸ASO、mRNA、DNA、基因编辑工具和/或细胞毒性剂。
在一种实施方式的递送系统中，其中所述外源荷载包括PAMP、DAMP、CD47、CD24、 IL-12、IL-15、凝血因子VII、凝血因子VIII、凝血因子IX和/或它们的功能活性片段。
在一种实施方式的递送系统中，其中所述外源荷载通过基因编辑、外源表达、液态固态转换、膜融合、电荷吸附、物理吸附和/或化学连接而与所述迁移体直接或间接结合。
在一种实施方式的递送系统中，其中所述外源荷载通过与所述迁移体的膜组分直接或间接连接而结合或嵌入至所述迁移体。
在一种实施方式的递送系统中，其中所述迁移体的膜组分包含膜蛋白、胆固醇、磷脂、糖蛋白上的糖链和/或多糖。
在一种实施方式的递送系统中，其中所述间接连接包括通过点击化学反应连接。
在一种实施方式的递送系统中，其中所述间接连接包括提供与结合对的第一成员连接的所述外源荷载，并使其与所述迁移体接触，所述迁移体的膜上包含所述结合对的第二成员，其中所述第一成员能够与所述第二成员结合。
在一种实施方式的递送系统中，其中所述结合对的第一成员和第二成员选自抗原及其抗体；受体及其配体；生物素和亲和素；HaloTag及其配体；以及CP05和CD63。
在一种实施方式的递送系统中，其中所述外源荷载作为膜蛋白表达在所述迁移体膜的内表面或外表面。
在一种实施方式的递送系统中，其中所述外源荷载作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
在一种实施方式的递送系统中，其中所述外源荷载通过基因编辑和/或外源表达而作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
另一方面，本申请提供了一种制备递送系统的方法，所述方法包括提供迁移体，和使所述迁移体携带外源荷载。
在一种实施方式的方法中，其中所述迁移体为经分离或纯化的迁移体。
在一种实施方式的方法中，其中所述使迁移体携带外源荷载包括使所述外源荷载直接或间接连接或嵌入至所述迁移体的膜和/或所述迁移体的内部。
在一种实施方式的方法中，其还包括由细胞分离或纯化所述迁移体。
在一种实施方式的方法中，其包括提供所述外源荷载与结合对的第一成员的复合物；使细胞产生迁移体，所述迁移体包含结合对的第二成员；以及使所述迁移体与所述复合物接触以形成所述递送系统。
在一种实施方式的方法中，其中所述迁移体包括本申请所述的迁移体。
在一种实施方式的方法中，其中所述迁移体源自细胞。
在一种实施方式的方法中，其中所述外源荷载包含一种或多种靶向物质和/或治疗活性物质。
在一种实施方式的方法中，其中所述外源荷载包含蛋白质，脂质，多核苷酸，小分子化合物，复合物、多糖、聚合物、纳米颗粒、微米颗粒和/或细胞器。
在一种实施方式的方法中，其中所述外源荷载包括膜蛋白，可溶性蛋白和/或多肽。
在一种实施方式的方法中，其中所述外源荷载包括DNA和/或RNA。
在一种实施方式的方法中，其中所述外源荷载包括抗体或其抗原结合抗体片段、整合素或其片段、免疫原性蛋白、细胞因子、趋化因子、受体蛋白或其片段、酶、抑癌基因产物、siRNA、microRNA、反义寡核苷酸ASO、mRNA、DNA、基因编辑工具和/或细胞毒性剂。
在一种实施方式的方法中，其中所述外源荷载包括PAMP、DAMP、CD47、CD24、IL-12、IL-15、凝血因子VII、凝血因子VIII、凝血因子IX和/或它们的功能活性片段。
在一种实施方式的方法中，其中所述外源荷载通过基因编辑、外源表达、液态固态转换、膜融合、电荷吸附、物理吸附和/或化学连接而与所述迁移体直接或间接结合。
在一种实施方式的方法中，其中所述外源荷载通过与所述迁移体的膜组分直接或间接结合而连接或嵌入至所述迁移体。
在一种实施方式的方法中，其中所述迁移体的膜组分包含膜蛋白、胆固醇、磷脂、糖蛋白上的糖链和/或多糖。
在一种实施方式的方法中，其中所述间接结合包括通过点击化学反应连接。
在一种实施方式的方法中，其中所述间接结合包括提供与结合对的第一成员连接的所述外源荷载，并使其与所述迁移体接触，所述迁移体的膜上包含所述结合对的第二成员，其中所述第一成员能够与所述第二成员结合。
在一种实施方式的方法中，其中所述结合对的第一成员和第二成员选自抗原及其抗体；受体及其配体；生物素和亲和素；HaloTag及其配体；以及CP05和CD63。
在一种实施方式的方法中，其中所述外源荷载作为膜蛋白表达在所述迁移体膜的内表面或外表面。
在一种实施方式的方法中，其中所述外源荷载作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
另一方面，本申请提供了一种制备递送系统的方法，所述方法包括：使细胞表达mRNA； 使所述细胞产生迁移体，所述迁移体包含mRNA结合蛋白，且所述mRNA通过所述mRNA结合蛋白连接至所述迁移体。
另一方面，本申请提供了一种制备递送系统的方法，所述方法包括：使细胞在细胞膜上表达外源荷载；和使所述细胞产生迁移体，所述迁移体包含所述外源荷载。
在一种实施方式的方法中，其中所述外源荷载为蛋白质。
在一种实施方式的方法中，其中所述蛋白质为膜蛋白。
在一种实施方式的方法中，其中所述蛋白质为可溶性蛋白，并且其与膜蛋白或其部分融合而形成融合蛋白。
另一方面，本申请提供了一种组合物，其包含本申请所述的迁移体或本申请所述的递送系统。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1a-c：低渗透压刺激对工程化迁移体形成的影响。a.76.3mOsmol/L渗透压刺激下，不同时间点工程化迁移体的形成(标尺5μm)；b.不同渗透压下工程化迁移体的形成；c.b中的工程化迁移体的直径的统计结果。
图2a-b：不同浓度latrunculin A处理对工程化迁移体形成的影响。a.不同latrunculin A浓度下工程化迁移体的形成；b.a中的工程化迁移体数量的统计结果。
图3a-c：细胞中SWELL1编码基因Lrrc8a的敲低对工程化迁移体形成的影响。a.通过qPCR确定的细胞中Lrrc8a的敲低效率；b.通过激光共聚焦显微镜观察到的Lrrc8a被敲低的细胞在逐步低渗透压刺激下产生的工程化迁移体；c.b中的工程化迁移体的直径的统计结果。
图4a-b：细胞中SWELL1编码基因Lrrc8a的敲除对工程化迁移体形成的影响。a.通过 蛋白质免疫印迹确定的细胞中Lrrc8a的敲除；b.通过激光共聚焦显微镜观察到的Lrrc8a被敲除的细胞在逐步低渗透压刺激下产生的工程化迁移体。
图5a-b：不同阳离子处理对工程化迁移体形成的影响。a.不同阳离子下工程化迁移体的形成；b.a中的工程化迁移体的直径的统计结果。
图6a-b：过表达Tspan4对工程化迁移体数量的影响。a.过表达Tspan4导致的工程化迁移体的形成；b.a中的工程化迁移体数量的统计结果。
图7a-b：MGC803-T4-GFP细胞在温敏涂层培养皿产生迁移体。MGC803-T4-GFP细胞在不同培养皿不同条件下产生迁移体的照片(a)和细胞脱离率定量(b)。
图8a-d：在不同细胞系中诱导产生的工程化迁移体。a.不同啮齿类动物细胞系经诱导产生的工程化迁移体；b.a的局部放大(标尺5μm)。c.不同T4-GFP转染的人胚肾细胞株/系经诱导产生的工程化迁移体。d.WGA染色的不同人细胞系经诱导产生的工程化迁移体。
图9a-b：不同细胞浓度的悬浮培养的NRK(a)和MC38(b)细胞经不同诱导产生工程化迁移体。
图10：工程化NRK细胞诱导产生的工程化迁移体的分离纯化流程示意图。
图11a-c：工程化NRK细胞诱导产生的工程化迁移体的形态观察。a.工程化迁移体的激光共聚焦显微镜图像；b.工程化迁移体的负染透射电镜照片；c.工程化迁移体的冷冻电镜照片。
图12：分离纯化的工程化迁移体的蛋白质印迹。
图13a-b：通过激光共聚焦显微镜观察的工程化迁移体对Cy5和右旋糖苷-TMR随时间变化的通透性。a.工程化迁移体的激光共聚焦显微镜观察结果；b.工程化迁移体室温放置1.5h、6h、12h、24h、48h后对右旋糖苷-TMR通透比例的统计结果。
图14a-c：工程化迁移体的稳定性。a.通过激光共聚焦显微镜观察的工程化迁移体在第0、1、2、3、5和7天的形态结果；b.免疫印迹分析室温存放不同天数后的工程化迁移体所装载鸡卵白蛋白(OVA)和mCherry蛋白量；c.以室温存放不同天数后的工程化迁移体免疫小鼠后产生的OVA-特异的抗体。
图15：通过激光共聚焦显微镜观察的胆固醇抽提试剂MβCD对工程化迁移体稳定性的影响。
图16a-b：工程化迁移体上膜蛋白、可溶性蛋白及小分子的装载流程示意图(a)和以OVA为例装载可溶蛋白的示意图(b)。
图17：通过激光共聚焦显微镜观察到的各种装载的膜蛋白在工程化迁移体上的定位。
图18：通过激光共聚焦显微镜观察到的刺突蛋白在工程化迁移体上的定位。
图19：载体pB-Hygro-GFP的图谱。
图20：载体pmCherry-N1的图谱。
图21：载体pB-Hygro-mCherry的图谱。
图22：通过激光共聚焦显微镜观察到的t-STX2-OVA融合蛋白在工程化迁移体上的定位。
图23：通过激光共聚焦显微镜观察到的Tspan4-HaloTag-GFP和HaloTag配体-TMR在工程化迁移体上的共定位。
图24a-c：经装载SARS-CoV-2刺突蛋白的工程化迁移体(e迁移体)免疫的小鼠体内诱导的S蛋白特异性免疫应答。a.动物实验设计示意图；b.刺突-e迁移体中S1蛋白的蛋白免疫印迹，对照-e迁移体组和纯化的S1蛋白作为对照。c.接受不同方法免疫后小鼠血清中的刺突(S)蛋白特异性IgG的浓度；
图25a-c：工程化NRK细胞生产迁移体的表征。a)过表达Tspan4-GFP的NRK细胞在低渗处理后，用2.5％戊二醛固定并用扫描电镜观察。标尺：左图为20μm，右图为2μm。b)运用转盘共聚焦显微镜对过表达Tspan4-GFP的NRK细胞产生工程化迁移体的过程进行4D拍摄，可以观察到在低渗处理的过程中，细胞胞体膨胀、底面收缩并产生大量丝状结构；在丝状结构上又生长出工程化迁移体。c)A-431细胞生产低渗囊泡的电镜照片和最终产出囊泡的显微照片(参考文献:Cohen S,Ushiro H,Stoscheck C,Chinkers MA native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.J Biol Chem 257:1523-1531.)。
图26a-h：迁移体与工程化迁移体(e迁移体)的特征比较。左：a：表达TSPAN4-mCherry的NRK细胞培养后未经处理以共聚焦显微镜观察。标尺：5μm。b：激光共聚焦显微镜下观察到的过表达Tspan4-GFP的NRK细胞经低渗等工艺产生的工程化迁移体，标尺：5μm。c:过表达Tspan4-GFP，并用四甲基罗丹明标记的WGA染色后的NRK细胞。上图为处于等渗溶液中的细胞，下图为低渗处理后的细胞。图片为激光共聚焦显微镜层扫拍摄后Z轴叠加效果图。标尺：5μm。d:迁移体电镜照片(Ma et al,Cell Res.2015)。e:过表达T4-GFP的NRK细胞产生的工程化迁移体的透射电镜图像。标尺：1μm。3次生产纯化的e迁移体和对照迁移体进行质谱分析后所做对比(f)和工程化迁移体中缺失，迁移体对照中最为富集的25个蛋白(g)：表中的数字代表Log2(相对胞体的表达倍数变化)。(h)相比迁移体(上排)，工程化迁移体(下排)表现ERM家族和其他多种蛋白的富集。左面3列(分别为Tspan-4- GFP,Ezrin-mCherry成像和两者的图像Z-stack合并)；右二为Fxyd5-mCherry成像；右一为Atp1β1-mCherry成像。(参考文献：Ma L,Li Y,Peng J,Wu D,Zhao X,Cui Y,Chen L,Yan X,Du Y,Yu L.Discovery of the migrasome,an organelle mediating release of cytoplasmic contents during cell migration.Cell Res.2015Jan；25(1):24-38.doi:10.1038/cr.2014.135；Zhao X,Lei Y,Zheng J,Peng J,Li Y,Yu L,Chen Y.Identification of markers for migrasome detection.Cell Discov.2019 May 21；5:27.doi:10.1038/s41421-019-0093-y.Erratum in:Cell Discov.2022Apr 6；8(1):32.PMID:31123599；PMCID:PMC6527679.)
图27a-f：过表达Tspan4-GFP的MC-38细胞生产小囊泡/外泌体和工程化迁移体的对比。a.小囊泡/外泌体纯化流程；b.纯化后MC-38小囊泡/外泌体的NTA检测；c.MC-38小囊泡/外泌体的透射电镜(TEM)检测；d.同一细胞来源的小囊泡/外泌体、工程化迁移体及细胞胞体的蛋白印迹分析。e.同一细胞来源的小囊泡/外泌体及工程化迁移体的生产和产量的比较。f.体内分布。Tspan4-GFP的MC-38细胞生产的小囊泡/外泌体或工程化迁移体以DiD标记后以等荧光染料量静脉注射后不同时间取组织进行荧光成像。
图28a-c：同一细胞来源的工程化迁移体和小囊泡/外泌体的定量质谱分析。左：PCA分析；中：信号通路分析热图；注释：工程化迁移体的三个数据点一致性高，图中重合。右：所制备的工程化迁移体和小囊泡/外泌体最为富集的10个蛋白清单。注释：工程化迁移体的三组数据点一致性高，最富集的10个蛋白完全一致。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“相对位移”通常是指相对于某一参考点或参考面发送位移。是指细胞中心相对环境(例如贴附表面)的位移，或在细胞中心静止的情况下，其膜表面仍然相对周围微环境(如贴附的固体表面，或微环境中的液相)或相对于细胞中心(如膨胀、从扁平到隆起等形变)的相对位移。
在本申请中，术语“迁移体”通常是指一种由细胞在发生相对位移时产生的结构。例如，迁移体可以是一种新型细胞器。例如，可以在细胞迁移过程中，在细胞尾部留下的收缩丝的尖端或交叉部位产生的囊泡结构。例如，在细胞迁移过程中，胞体持续地向迁移体运输胞内 物质，随后收缩丝断裂，迁移体被释放，可在产生的局部或通过血液等体液运输到远端组织，继而被细胞外空间或周围的细胞所摄取。例如，迁移体可能参与胞内物质及信号在细胞之间的传递，从而介导细胞间通讯。
在本申请中，术语“膜微结构域”通常是指具有生物膜结构的区域。例如，细胞膜的以鞘脂(sphingolipids)和胆固醇(Ch)为基础的微结构域(microdomain)，包括脂筏(lipid raft)，Tetraspanin富集的微结构域(Tetraspanin-enriched microdomain,TEM)等。
在本申请中，术语“腔内囊泡”(intraluminal vesicle)通常是指形成或存在于管腔内或管腔或管状结构的空间内的膜性囊泡(例如细胞器或更大的囊泡)。管腔(lumen)在本文中也可以用于描述细胞成分或结构的内部空间。例如，管腔内囊泡可由细胞器产生。例如，腔内囊泡可以包含迁移体中的腔内囊泡、或者包含目前未处于迁移体但预计将转移到迁移体内的腔内囊泡。
在本申请中，术语“外源荷载”通常是指并未天然存在于染色体或宿主细胞中的物质。例如，细胞本身不产生，或者不以相当的量产生(例如表达)该物质。例如，产生该物质的细胞与天然细胞有所区别，例如该物质在结构或功能方面有所不同。
在本申请中，术语“低渗透压”通常是指比细胞的等渗溶液的渗透压更低的渗透压。等渗溶液是指渗透压相当于血浆渗透压的溶液。
在本申请中，术语“细胞骨架”通常是指是指细胞中的蛋白纤维网架体系，例如由微管、微丝和中间纤维组成的体系。
在本申请中，术语“Tetraspanin”通常是四跨膜蛋白超家族，其包含四个跨膜结构域。这些蛋白可以形成所谓的四跨膜蛋白富集的微结构域(TEM)(Rubinstein,E.(2011).The complexity of tetraspanins.Biochem Soc Trans 39,501-505.)。TEM尺寸为约100纳米，富含一系列蛋白和脂筏类脂质，如胆固醇等。在迁移体形成过程中，很多小的TEM会聚集形成微米级别的宏结构域，称为四跨膜蛋白富集的宏结构域(TEMA)，TEMA的形成可以与迁移体在收缩丝上的生长有关。例如，Tetraspanin家族可以包含33个成员，包括Tspan1、Tspan2、Tspan3、Tspan4、Tspan5、Tspan6、Tspan7、Tspan8、Tspan9、Tspan10、Tspan11、Tspan12、Tspan13、Tspan14、Tspan15、Tspan16、Tspan17、Tspan18、Tspan19、Tspan20(UPK1B)、Tspan21(UPK1A)、Tspan22(PRPH2)、Tspan23(ROM1)、Tspan24(CD151)、Tspan25(CD53)、Tspan26(CD37)、Tspan27(CD82)、Tspan28(CD81)、Tspan29(CD9)、Tspan30(CD63)、Tspan31、Tspan32和Tspan33。来自生化研究和基因敲除小鼠的数据表明这些Tetraspanin家族成员在膜生物学 中发挥着重要作用。
在本申请中，术语“分离”和“纯化”可互换使用，并且通常是指从其产生环境的组分中鉴别、分离和/或回收，使得该“分离或纯化的”迁移体不含或基本上不含来自其产生环境的、可能干扰其治疗或诊断用途的其它污染物组分。污染物组分可包括非生物物质(包括化学物质)或生物物质，例如细胞器、核酸、蛋白质(例如可溶性蛋白)、脂质或代谢物。因此，可以通过至少一个移除或基本上移除这些污染物组分的纯化步骤制备“分离或纯化的”迁移体。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
一方面，本申请提供了一种制备迁移体的方法，所述方法可以包括使细胞产生相对位移继而产生源自所述细胞的迁移体。例如，本申请的相对位移可以是指细胞中心相对环境(例如贴附表面)的位移，或在细胞中心静止的情况下，其膜表面仍然相对周围微环境(如贴附的固体表面，或微环境中的液相)或相对于细胞中心(如膨胀、从扁平到隆起等形变)的相对位移。例如，所述相对位移的产生和/或程度可以通过物理和/或化学和/或生物的手段进行干预。
例如，本申请的方法中，其还可以包括分离所述细胞所产生的迁移体。
例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、破坏所述细胞的细胞骨架、抑制所述细胞的细胞体积调节功能、使所述细胞脱离其粘附表面、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。
例如，本申请的方法中，其可以包括破坏所述细胞的细胞骨架、抑制所述细胞的细胞体积调节功能、使所述细胞脱离其粘附表面、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、抑制所述细胞的细胞体积调节功能、使所述细胞脱离其粘附表面、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或 功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、破坏所述细胞的细胞骨架、使所述细胞脱离其粘附表面、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、破坏所述细胞的细胞骨架、抑制所述细胞的细胞体积调节功能、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、破坏所述细胞的细胞骨架、抑制所述细胞的细胞体积调节功能、和/或使所述细胞脱离其粘附表面，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。
例如，本申请的方法中，其可以包括抑制所述细胞的细胞体积调节功能、使所述细胞脱离其粘附表面、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、使所述细胞脱离其粘附表面、和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理、破坏所述细胞的细胞骨架、和/或抑制所述细胞的细胞体积调节功能，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。
例如，本申请的方法中，其可以包括所述细胞进行低渗透压处理和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括破坏所述细胞的细胞骨架和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括抑制所述细胞的细胞体积调节功能和/或使所述细胞的细胞膜与其粘附表面之间发生相对位移，以及可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。
例如，本申请的方法中，其可以包括对所述细胞进行低渗透压处理。
例如，本申请的方法中，其中所述低渗透压处理可以包括使所述细胞置于低渗缓冲溶液 中。
例如，本申请的方法中，其中所述低渗透压处理可以包括使所述细胞置于缓冲溶液中，并将所述缓冲溶液的渗透压降低成为低渗缓冲液。
例如，本申请的方法中，其中所述降低可以包括线性降低和/或阶梯式降低。
例如，本申请的方法中，其中所述低渗缓冲溶液的渗透压可以低于约305mOsmol/L。例如，所述低渗缓冲溶液的渗透压可以低于约305mOsmol/L、低于约300mOsmol/L、低于约270mOsmol/L、低于约250mOsmol/L、低于约200mOsmol/L、低于约150mOsmol/L、低于约100mOsmol/L、低于约90mOsmol/L、低于约80mOsmol/L、低于约70mOsmol/L、低于约60mOsmol/L、低于约50mOsmol/L、低于约40mOsmol/L、低于约30mOsmol/L、低于约20mOsmol/L、低于约15mOsmol/L、低于约10mOsmol/L、低于约5mOsmol/L或低于约2mOsmol/L。
例如，本申请的方法中，其中所述低渗缓冲溶液的渗透压可以为约10mOsmol/L至约274.5mOsmol/L。例如，本申请的方法中，其中所述低渗缓冲溶液的渗透压可以为约10mOsmol/L至约274.5mOsmol/L。例如，所述低渗缓冲溶液的渗透压可以为约10mOsmol/L至约300mOsmol/L、约20mOsmol/L至约300mOsmol/L、约30mOsmol/L至约300mOsmol/L、约50mOsmol/L至约300mOsmol/L、约70mOsmol/L至约300mOsmol/L、约100mOsmol/L至约300mOsmol/L、约150mOsmol/L至约300mOsmol/L、约200mOsmol/L至约300mOsmol/L、约250mOsmol/L至约300mOsmol/L、约10mOsmol/L至约250mOsmol/L、约20mOsmol/L至约250mOsmol/L、约30mOsmol/L至约250mOsmol/L、约50mOsmol/L至约250mOsmol/L、约70mOsmol/L至约250mOsmol/L、约100mOsmol/L至约250mOsmol/L、约150mOsmol/L至约250mOsmol/L、约200mOsmol/L至约250mOsmol/L、约10mOsmol/L至约200mOsmol/L、约20mOsmol/L至约200mOsmol/L、约30mOsmol/L至约200mOsmol/L、约50mOsmol/L至约200mOsmol/L、约70mOsmol/L至约200mOsmol/L、约100mOsmol/L至约200mOsmol/L、约150mOsmol/L至约200mOsmol/L、约10mOsmol/L至约150mOsmol/L、约20mOsmol/L至约150mOsmol/L、约30mOsmol/L至约150mOsmol/L、约50mOsmol/L至约150mOsmol/L、约70mOsmol/L至约150mOsmol/L、约100mOsmol/L至约150mOsmol/L、约10mOsmol/L至约100mOsmol/L、约20mOsmol/L至约100mOsmol/L、约30mOsmol/L至约100mOsmol/L、约50mOsmol/L至约100mOsmol/L、约70mOsmol/L至约100mOsmol/L、约10mOsmol/L至约30mOsmol/L、约20mOsmol/L至约30mOsmol/L、约30mOsmol/L至约90mOsmol/L、 或约30mOsmol/L至约70mOsmol/L。其中所述低渗透压处理包括使所述细胞置于缓冲溶液中，并将所述缓冲溶液的渗透压降低成为低渗缓冲液。
例如，本申请的方法中，其可以包括破坏所述细胞的细胞骨架。
例如，本申请的方法中，其中破坏所述细胞的细胞骨架可以包括使所述细胞与破坏细胞骨架的试剂接触。例如，本申请的方法中，其中所述破坏细胞骨架的试剂可以包括微丝和/或微管解聚剂。例如，本申请的方法中，其中所述破坏细胞骨架的试剂可以包括Latrunculin A，Latrunculin B，细胞松弛素A，细胞松弛素B，细胞松弛素C，细胞松弛素D和/或细胞松弛素E。
例如，本申请的方法中，其可以包括抑制所述细胞的细胞体积调节功能。
例如，本申请的方法中，其中抑制所述细胞的细胞体积调节功能可以包括使所述细胞中体积调节蛋白的数量和/或功能下降。例如相对于未经修饰的细胞，本申请抑制所述细胞的细胞体积调节功能的细胞的体积调节蛋白的数量和/或功能下降约5％、下降约10％、下降约20％、下降约50％、或下降约100％。
例如，本申请的方法中，其中所述体积调节蛋白可以包括体积调节离子通道和/或转运蛋白。例如，本申请的方法中，其中所述体积调节离子通道可以包括体积调节阴离子通道VRAC和/或体积调节阳离子通道VRCC。例如，本申请的方法中，其中所述体积调节阴离子通道VRAC可以包括SWELL1或其功能活性片段。例如，本申请的方法中，其中所述体积调节阳离子通道VRCC可以包括TRPV4，TRPM3和/或其功能活性片段。
例如，本申请的方法中，其中所述转运蛋白可以包括协同转运蛋白。例如，本申请的方法中，其中所述协同转运蛋白可以包括KCC1、KCC3和/或KCC4。
例如，本申请的方法中，其中抑制所述细胞的细胞体积调节功能可以包括将所述细胞置于具有减弱的体积调节能力的缓冲溶液中。例如，本申请的方法中，其中所述缓冲液可以含有增加的阳离子。例如，本申请的方法中，其中所述阳离子可以包括K +，Na +，Cs +，Li +，Ca 2+，Mg 2+，Ba 2+，Mn 2+，Fe 2+，Ni 2+，Zn 2+，Al 3+，Fe 3+,CH 3NH 3 +，C 2H 5NH 3 +，(CH 3) 2NH 2 +，(C 2H 5) 2NH 2 +，(C 2H 5) 3N +，氨离子和/或胆碱离子。
例如，本申请的方法中，其中所述缓冲液可以含有增加的阴离子。例如，本申请的方法中，其中所述阴离子可以包括Br -，Cl -，I -，F -，OH -，HCO 3 -，H 2PO 4 -，NO 2 -，NO 3 -，CN -，HPO 4 2-，CO 3 2-，SO 4 2-和/或PO 4 3-。
例如，本申请的方法中，其中所述方法可以包括使所述细胞脱离其粘附表面。
例如，本申请的方法中，其中所述方法可以包括使所述细胞的细胞膜与其粘附表面之间发生相对位移。
例如，本申请的方法中，其还可以包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。例如，本申请的方法中，其可以包括使所述细胞过表达所述tetraspanin蛋白，其功能性片段和/或功能性变体。例如，本申请的方法中，其中所述tetraspanin蛋白可以选自：Tspan1、Tspan2、Tspan3、Tspan4、Tspan5、Tspan6、Tspan7、Tspan8、Tspan9、Tspan10、Tspan11、Tspan12、Tspan13、Tspan14、Tspan15、Tspan16、Tspan17、Tspan18、Tspan19、Tspan20(UPK1B)、Tspan21(UPK1A)、Tspan22(PRPH2)、Tspan23(ROM1)、Tspan24(CD151)、Tspan25(CD53)、Tspan26(CD37)、Tspan27(CD82)、Tspan28(CD81)、Tspan29(CD9)、Tspan30(CD63)、Tspan31、Tspan32和Tspan33。
例如，本申请的方法中，所述方法还可以包括减小所述迁移体的尺寸。
例如，本申请的方法中，其中所述减小所述迁移体的尺寸可以包括使用过滤器或挤压器挤压所述迁移体。
例如，本申请的方法中，其中所述过滤器或挤压器的孔径可以为约30nm至约10000nm。例如，所述过滤器或挤压器的孔径可以为约30nm至约100nm、约30nm至约1000nm、约30nm至约10000nm、约50nm至约100nm、约50nm至约1000nm、约50nm至约10000nm、约100nm至约1000nm、约100nm至约10000nm、或约1000nm至约10000nm。
例如，本申请的方法中，其中所述迁移体的尺寸可以为约50nm至约8000nm。例如，所述迁移体的尺寸可以为约50nm至约100nm、约50nm至约1000nm、约50nm至约10000nm、约100nm至约1000nm、约100nm至约10000nm、或约1000nm至约10000nm。
例如，本申请的方法中，其中所述迁移体可以产生于所述细胞的收缩丝。
例如，本申请的方法中，其中所述迁移体的膜上可以富集有钠/钾ATP酶和/或其功能性片段。例如，本申请的方法中，其中所述迁移体的膜上可以富集有整合素和/或其功能性片段。例如，本申请的方法中，其中所述迁移体的膜上可以富集有tetraspanin蛋白，其功能性变体和/或其功能性片段。例如，本申请的方法中，其中所述迁移体的膜上可以富集有胆固醇。例如，富集可以是指与相应细胞的细胞膜上的其他部分相比，或者跟未产生所述迁移体之前，相应细胞膜上的情况相比。例如，富集是指本申请的迁移体上所述分子或物质的密度高于产生这些迁移体的细胞膜上相应分子或物质的密度。
例如，本申请的方法中，其中所述迁移体可以富集有膜微结构域。例如，膜微结构域是 指细胞膜的以鞘脂(sphingolipids)和胆固醇(Ch)为基础的微结构域(microdomain)，包括脂筏(lipid raft)，富含四跨膜蛋白的微结构域(Tetraspanin-enriched microdomain,TEM)等。
例如，本申请的方法中，其中所述迁移体可以在体外产生或离体产生。
例如，本申请的方法中，其中与相同细胞产生的天然迁移体相比，所述迁移体的内容物可以至少部分减少或缺失。例如，以NRK细胞产生的工程化迁移体为例，与天然迁移体对比，有4000个或以上迁移体具有蛋白缺失，天然迁移体具有约1350个天然迁移体中没有的蛋白(即多出来的蛋白)，例如AMP3,Myh4,Gorasp2,Asz1,Lats2,Scn2b,Pacsin1,Alb。例如，“天然迁移体”可以是指由细胞未经改变外界培养条件(例如低渗、温度等)，自发性迁移产生出的迁移体；而“工程化迁移体”可以是指细胞经过外界刺激诱导发生相对位移后产生的迁移体。
例如，本申请的方法中，其中所述至少部分减少的内容物可以包含腔内囊泡。例如，本申请提供的工程化迁移体的内部较少有腔内囊泡。所述腔内囊泡可以是指由在外层膜结构中的具有磷脂双分子层结构的囊泡。
例如，本申请的方法中，其可以为体外或离体方法。例如，本申请的准备方法可以是在体外进行的。例如，本申请的准备方法可以是不在有生命的对象中进行的。
例如，本申请的方法中，其中所述细胞可以为经体外培养的细胞。
例如，本申请的方法中，其中所述细胞可以为经悬浮培养或经贴壁培养的细胞。
例如，本申请的方法中，其中所述细胞可以包括原代细胞。
例如，本申请的方法中，其中所述原代细胞可以包括源自生物体的组织细胞，所述生物体可以包括人，猴，小鼠，大鼠，兔，鸡和/或昆虫。
例如，本申请的方法中，其中所述原代细胞可以包括肝细胞，脾细胞，肾细胞，组织巨噬细胞，脑胶质细胞，破骨细胞，骨髓细胞，白细胞，成纤维细胞和/或脂肪细胞。例如，本申请的原代细胞可以包括Kupffer细胞。例如，一种位于肝窦内表面的吞噬细胞，能够清除血液中的外来抗原、抗原－抗体复合物和细胞碎片等物质。
例如，本申请的方法中，其中所述白细胞可以包括B细胞，T细胞，NK细胞，树突状细胞，中性粒细胞和/或巨噬细胞。
例如，本申请的方法中，其中所述细胞可以包括肿瘤细胞。
例如，本申请的方法中，其中所述肿瘤细胞可以包括肿瘤细胞系，源自患者的原代或经有限传代的肿瘤细胞，肿瘤基质细胞，和/或肿瘤类器官。
例如，本申请的方法中，其中所述细胞可以包括CHO细胞、CHO-K1细胞、HEK293细胞、HEK293T细胞、HEK293FT细胞、HEK293F细胞、Vero细胞、NRK细胞、L929细胞、MC38细胞、4T1细胞、DC2.4细胞、MGC803细胞、Jurkat细胞、NK-92MI细胞、BJ细胞和/或HepG2细胞。
例如，本申请的方法中，其中所述细胞可以包括白细胞、干细胞和/或成纤维细胞。
例如，本申请的方法中，其中所述干细胞可以包含间充质干细胞。
例如，本申请提供了本申请的方法中制备的迁移体。例如，本申请提供了一种新型的可工程化生产的细胞外囊泡--工程化迁移体和/或一种通过工程方法迫使细胞高效生产的迁移体，和/或包含以上任一迁移体的组合物，其特征在于，所述工程化迁移体大小在50-8000纳米(nm)，膜上有整合素(integrin)和Tetraspanin(Tspan)家族成员等蛋白的富集，并由膜微结构域的存在，在一些情况下生长于细胞周围的丝状结构上，其可自发释放或通过人工方法与细胞分离，得到完整的囊泡状结构、和/或其生物化学成分、形态、结构与该种细胞产生的天然迁移体相似；也可根据应用所需进行特异性调节，获得结构、生物化学成分与该细胞天然迁移体有较大不同的囊泡状结构。例如，本申请提供了迁移体/工程化迁移体作为一种装载药物的递送载体，在不同疾病(例如肿瘤、炎症/自身免疫病、心血管疾病、神经退行性疾病和其他神经系统疾病等)的治疗和疫苗领域的应用。
迁移体是一种新型细胞器，其是在细胞迁移过程中，在细胞尾部留下的收缩丝的尖端或交叉部位产生的直径为0.5-3μm的单层膜囊泡结构。在细胞迁移过程中，胞体持续地向迁移体运输胞内物质。随后，收缩丝断裂，迁移体被释放，可在产生的局部或通过血液等体液运输到远端组织，继而被细胞外空间或周围的细胞所摄取。这表明迁移体可能参与胞内物质及信号在细胞之间的传递，从而介导细胞间通讯(Liang Ma et.al.,Discovery of the migrasome,an organelle mediating release of cytoplasmic contents druing cell migration,Cell Res(2015)25:24-38)。
研究表明迁移体在胚胎发育、机体免疫应答、肿瘤、血管新生、组织再生等细胞迁移活跃的过程中发挥重要的信号传递作用。通过迁移体在斑马鱼胚胎发育中的研究发现，迁移体作为信号分子的膜包被载体，决定信号分子的时空分布，从而发挥调控器官发育的功能(Jiang D et.at.,Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation,Nat Cell Biol,2019,21(8):966-977)。迁移体可以介导细胞间的蛋白和mRNA传递，通过迁移体将蛋白和mRNA转移到受体细胞，从而改变受体细胞的生命活动(Zhu M et.al.,Lateral  transfer of mRNA and protein by migrasomes modifies the recipient cells,Cell Res,2020,doi；10.1038/s41422-020-00415-3)。迁移体可以调控线粒体的质量，通过将受损的线粒体清理出去，从而维持细胞内线粒体的稳态(Jiang H et.al.,Mitocytosis,a migrasome-mediated mitochondrial quality control process,Cell Press,doi:10.1016/j.cell.2021.04.027)。迁移体对肿瘤微环境中的癌细胞具有调控作用，如在胰腺癌细胞中迁移体可诱导抑制性免疫微环境从而促进肿瘤生长(张荣华，胰腺癌细胞迁移体对肿瘤微环境中癌细胞与相关免疫细胞表型及功能的相互调控作用研究，2020)。
脱离的迁移体是一种胞外囊泡，其与已知的细胞外囊泡具有诸多差异。例如，与外泌体之间的差异：1)两者结构不同：迁移体在被释放之前附着在收缩丝上并呈现大囊泡内包含小囊泡的结构；而外泌体不存在这种结构；2)两者的大小不同：外泌体的直径为约50-150nm，而迁移体的直径为约0.5-3μm；3)两者的蛋白质组成明显不同：在迁移体和外泌体之间只有27％的蛋白质组成是相同的，例如NDST1(双功能硫酸乙酰肝素N-脱乙酰酶/N-磺基转移酶1)、PIGK(磷脂酰肌醇聚糖锚定生物合成类K)、CPQ(羧肽酶Q)和EOGT(EGF结构域特异性O-连接的N-乙酰葡糖胺转移酶)富集在迁移体上，而不存在于外泌体中(Zhao X,Lei Y,Zheng J,Peng J,Li Y,Yu L,Chen Y.Identification of markers for migrasome detection.Cell Discov.2019May 21；5:27)；4)两者受不同的遗传途径调控，生物发生过程完全不同：外泌体首先作为多泡体(MVB)的囊泡产生，当MVB与质膜融合时释放外泌体；而迁移体是由质膜上大结构域组装形成的(Huang Y,Zucker B,Zhang S,Elias S,Zhu Y,Chen H,Ding T,Li Y,Sun Y,Lou J,Kozlov MM*,Yu L*.Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains.Nat Cell Biol.2019Aug；21(8):991-1002)。
本申请发现可以通过多种方法诱导产生结构类似于天然迁移体的工程化迁移体。工程化迁移体不同于本领域已知的“低渗透压诱导的囊泡”(例如，Cohen S,Ushiro H,Stoscheck C,Chinkers MA native 170 000epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.J Biol Chem 257:1523-1531中的图1所示的囊泡)，它们存在至少以下区别：1)囊泡产生位点不同：工程化迁移体产生于细胞周围的收缩丝上；而Cohen等人的囊泡产生于细胞上表面；2)囊泡尺寸不同：工程化迁移体的尺寸为微米级，直径很少超过5μm；而Cohen等人利用低渗透压诱导的囊泡可大至20μm；3)于Cohen等人利用低渗诱导的方法不同，本申请中低渗不是产生工程化迁移体的必要条件，可通过其它方法诱导出工程化迁移体。
工程化生产的细胞外囊泡目前广泛应用于药物递送和疫苗制备等重要生物医药领域。如工程化的外泌体(Kamerkar S,LeBleu VS,Sugimoto H,Yang S,Ruivo CF,Melo SA,Lee JJ,Kalluri R.Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.Nature.2017 Jun 22；546(7659):498-503.doi:10.1038/nature22341)，其主要优点主要包括：天然生物膜来源，毒性低；低免疫原性，不易被免疫系统清除，因而在体内循环时间更长；性质稳定，能够在-80℃长期储存。但其也存在明显的局限性，例如，产量低；纯化过程依赖超高速离心机；内容物主要为后期装载的核酸等。
本申请发现的工程化迁移体为生物来源，具有外泌体等生物来源的细胞外囊泡的低毒性低免疫原性的优点；其是一种全新的细胞外囊泡，其可运送的分子、在体内的生物分布等具有其独特的特点；同时其制备方法简单，产量高。综上，本申请发现的工程化迁移体在药物递送和疫苗制备等生物医药领域具有极高的应用价值。
一种制备工程化迁移体的方法，其包括对细胞进行人工诱导以产生工程化迁移体，及分离和/或纯化所述工程化迁移体；和/或迫使细胞膜与培养环境(包括贴附的固体表面和周围的液体环境)发生迁移，包括但不限于天然的细胞迁移方式或在细胞以下尺度(sub-cellular scale)的细胞膜与环境的相对位移。
另一方面，本申请提供了一种体外或离体制备的迁移体，其中所述迁移体的尺寸可以为约50nm至约8000nm。
例如，本申请的迁移体中，其在体外可以产生于细胞的收缩丝。
例如，本申请的迁移体中，其中所述迁移体的膜上可以富集有钠/钾ATP酶和/或其功能性片段。
例如，本申请的迁移体中，其中所述迁移体的膜上可以富集有整合素和/或其功能性片段。
例如，本申请的迁移体中，其中所述迁移体的膜上可以富集有tetraspanin蛋白，其功能性变体和/或其功能性片段。
例如，本申请的迁移体中，其中所述迁移体的膜上可以富集有胆固醇。
例如，本申请的迁移体中，其中所述迁移体可以富集有膜微结构域。
例如，本申请的迁移体中，其中与相应细胞产生的天然迁移体相比，所述迁移体的内容物可以至少部分减少或缺失。
例如，本申请的迁移体中，其中所述至少部分减少的内容物可以包含腔内囊泡。
另一方面，本申请提供了一种迁移体可以用于递送外源荷载的用途。例如，“外源荷载” 可以是指细胞本身不产生，不以相当的量产生(例如表达)的物质，或者该物质即使在细胞本身产生的，但作为外源荷载的该物质在结构或功能方面与上述在细胞本身产生的该物质有所区别。
例如，本申请的用途中，其中所述迁移体可以包含本申请所述的迁移体。
另一方面，本申请提供了一种递送系统，其可以包含迁移体和一种或多种外源荷载。
例如，本申请的递送系统中，其中所述外源荷载可以直接或间接结合、连接或嵌入至所述迁移体的膜和/或内部。
例如，本申请的递送系统中，其中所述迁移体可以包括本申请所述的迁移体。
例如，本申请的递送系统中，其中所述迁移体可以源自细胞。
例如，本申请的递送系统中，其中所述外源荷载可以包含一种或多种靶向物质和/或治疗活性物质。
例如，本申请的递送系统中，其中所述外源荷载可以包含蛋白质，脂质，多核苷酸，小分子化合物，复合物、多糖、聚合物、纳米颗粒、微米颗粒和/或细胞器。
例如，本申请的递送系统中，其中所述外源荷载可以包括膜蛋白，可溶性蛋白和/或多肽。
例如，本申请的递送系统中，其中所述外源荷载可以包括DNA和/或RNA。
例如，本申请的递送系统中，其中所述外源荷载可以包括抗体或其抗原结合抗体片段、整合素或其片段、免疫原性蛋白、细胞因子、趋化因子、受体蛋白或其片段、酶、抑癌基因产物、siRNA、microRNA、反义寡核苷酸ASO、mRNA、DNA、基因编辑工具和/或细胞毒性剂。例如，基因编辑工具可以包含核酸酶，例如Cas蛋白、CRISPR-Cas系统、Cre重组酶、锌指核酸内切酶、类转录激活因子效应物核酸酶(transcription activator-like effector nuclease)以及基因表观编辑工具。
例如，本申请的递送系统中，其中所述外源荷载可以包括PAMP、DAMP、CD47、CD24、IL-12、IL-15、凝血因子VII、凝血因子VIII、凝血因子IX和/或它们的功能活性片段。
例如，本申请的递送系统中，其中所述外源荷载可以通过基因编辑、外源表达、液态固态转换、膜融合、电荷吸附、物理吸附和/或化学连接而与所述迁移体直接或间接结合。例如，外源表达可以包含如质粒表达，如建立瞬时或稳定过表达某基因的细胞系。
例如，本申请的递送系统中，其中所述外源荷载可以通过与所述迁移体的膜组分直接或间接连接而结合或嵌入至所述迁移体。
例如，本申请的递送系统中，其中所述迁移体的膜组分可以包含膜蛋白、胆固醇、磷脂、 糖蛋白上的糖链和/或多糖。
例如，本申请的递送系统中，其中所述间接连接可以包括通过点击化学反应连接。
例如，本申请的递送系统中，其中所述间接连接可以包括提供与结合对的第一成员连接的所述外源荷载，并使其与所述迁移体接触，所述迁移体的膜上包含所述结合对的第二成员，其中所述第一成员能够与所述第二成员结合。
例如，本申请的递送系统中，其中所述结合对的第一成员和第二成员可以选自抗原及其抗体；受体及其配体；生物素和亲和素；HaloTag及其配体；以及CP05和CD63。
例如，本申请的递送系统中，其中所述外源荷载可以作为膜蛋白表达在所述迁移体膜的内表面或外表面。
例如，本申请的递送系统中，其中所述外源荷载可以作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
例如，本申请的递送系统中，其中所述外源荷载可以通过基因编辑和/或外源表达而作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
另一方面，本申请提供了一种制备递送系统的方法，所述方法可以包括提供迁移体，和使所述迁移体携带外源荷载。例如，所述迁移体携带外源荷载可以包含可逆地携带外源荷载。例如，所述迁移体携带外源荷载可以包含不可逆地携带外源荷载。
例如，本申请的方法中，其中所述迁移体可以为经分离或纯化的迁移体。
例如，本申请的方法中，其中所述使迁移体携带外源荷载可以包括使所述外源荷载直接或间接连接或嵌入至所述迁移体的膜和/或所述迁移体的内部。
例如，本申请的方法中，其还可以包括由细胞分离或纯化所述迁移体。
例如，本申请的方法中，其可以包括提供所述外源荷载与结合对的第一成员的复合物；使细胞产生迁移体，所述迁移体包含结合对的第二成员；以及使所述迁移体与所述复合物接触以形成所述递送系统。
例如，本申请的方法中，其中所述迁移体可以包括本申请所述的迁移体。
例如，本申请的方法中，其中所述迁移体可以源自细胞。
例如，本申请的方法中，其中所述外源荷载可以包含一种或多种靶向物质和/或治疗活性物质。
例如，本申请的方法中，其中所述外源荷载可以包含蛋白质，脂质，多核苷酸，小分子化合物，复合物、多糖、聚合物、纳米颗粒、微米颗粒和/或细胞器。
例如，本申请的方法中，其中所述外源荷载可以包括膜蛋白，可溶性蛋白和/或多肽。
例如，本申请的方法中，其中所述外源荷载可以包括DNA和/或RNA。
例如，本申请的方法中，其中所述外源荷载可以包括抗体或其抗原结合抗体片段、整合素或其片段、免疫原性蛋白、细胞因子、趋化因子、受体蛋白或其片段、酶、抑癌基因产物、siRNA、microRNA、反义寡核苷酸ASO、mRNA、DNA、基因编辑工具和/或细胞毒性剂。
例如，本申请的方法中，其中所述外源荷载可以包括PAMP、DAMP、CD47、CD24、IL-12、IL-15、凝血因子VII、凝血因子VIII、凝血因子IX和/或它们的功能活性片段。
例如，本申请的方法中，其中所述外源荷载可以通过基因编辑、外源表达、液态固态转换、膜融合、电荷吸附、物理吸附和/或化学连接而与所述迁移体直接或间接结合。
例如，本申请的方法中，其中所述外源荷载可以通过与所述迁移体的膜组分直接或间接结合而连接或嵌入至所述迁移体。
例如，本申请的方法中，其中所述迁移体的膜组分可以包含膜蛋白、胆固醇、磷脂、糖蛋白上的糖链和/或多糖。
例如，本申请的方法中，其中所述间接结合可以包括通过点击化学反应连接。
例如，本申请的方法中，其中所述间接结合可以包括提供与结合对的第一成员连接的所述外源荷载，并使其与所述迁移体接触，所述迁移体的膜上包含所述结合对的第二成员，其中所述第一成员能够与所述第二成员结合。
例如，本申请的方法中，其中所述结合对的第一成员和第二成员可以选自抗原及其抗体；受体及其配体；生物素和亲和素；HaloTag及其配体；以及CP05和CD63。
例如，本申请的方法中，其中所述外源荷载可以作为膜蛋白表达在所述迁移体膜的内表面或外表面。
例如，本申请的方法中，其中所述外源荷载可以作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
另一方面，本申请提供了一种制备递送系统的方法，所述方法可以包括：使细胞表达mRNA；使所述细胞产生迁移体，所述迁移体包含mRNA结合蛋白，且所述mRNA通过所述mRNA结合蛋白连接至所述迁移体。例如，mRNA结合蛋白可以包含用于结合所述mRNA的蛋白形成复合物，例如，所述RNA结合蛋白包含RNA识别基序。RNA结合蛋白包含天然或失活的核酸酶活性。RNA结合蛋白或其功能结构域可以包含Cys2-His2、Gag-knuckle、Treble-clet、锌带、Zn2/Cys6类基序。示例性mRNA结合蛋白可以包含但不限于RNA结合蛋白(RNA  binding protein，RBP)。
另一方面，本申请提供了一种制备递送系统的方法，所述方法可以包括：使细胞在细胞膜上表达外源荷载；和使所述细胞产生迁移体，所述迁移体包含所述外源荷载。所述表达外源合在可以通过基因编辑或外源表达，例如质粒表达等实现。
例如，本申请的方法中，其中所述外源荷载可以为蛋白质。
例如，本申请的方法中，其中所述蛋白质可以为膜蛋白。
例如，本申请的方法中，其中所述蛋白质可以为可溶性蛋白，并且其可以与膜蛋白或其部分融合而形成融合蛋白。
另一方面，本申请提供了一种组合物，其可以包含本申请所述的迁移体或本申请所述的递送系统。
1.制备工程化迁移体的方法，其包括
(a)对细胞进行以下一项或多项处理以诱导所述细胞产生工程化迁移体：
(1)低渗透压处理；
(2)破坏所述细胞的细胞骨架；
(3)抑制所述细胞的细胞体积调节功能；和
(4)使所述细胞过表达选自Tetraspanin家族成员的一种或多种蛋白；以及
(5)诱导细胞快速从贴附表面脱离或诱导细胞膜和贴附表面的相对位移。
(b)分离和/或纯化所述工程化迁移体。
2.根据技术方案1的方法，其中在步骤(a)中，进行(2)-(5)中至少1种处理，进行(1)-(5)中的至少2种处理，至少3种处理，至少4种处理或者进行全部5种处理。
3.根据技术方案1或2的方法，其中步骤(a)包括对细胞进行低渗透压处理。
4.根据技术方案1-3中任一项的方法，其中通过将细胞置于低渗缓冲溶液中，从而进行低渗透压处理，所述低渗缓冲溶液的渗透压为30.5-274.5mOsmol/L，例如30.5-150mOsmol/L。
5.根据技术方案1-3中任一项的方法，其中通过将细胞置于缓冲溶液中，并将所述缓冲溶液的渗透压降低至30.5-274.5mOsmol/L，例如30.5-150mOsmol/L，从而进行低渗透压处理。
6.根据技术方案5的方法，其中降低所述缓冲溶液的渗透压包括线性降低或阶梯式逐步降低。
7.根据技术方案1-6中任一项的方法，其中步骤(a)包括破坏所述细胞的细胞骨架。
8.根据技术方案7的方法，其中通过使所述细胞与破坏细胞骨架的试剂接触，从而破坏所述细胞的细胞骨架。
9.根据技术方案8的方法，其中所述破坏细胞骨架的试剂包括微丝解聚剂，例如选自Latrunculin A、Latrunculin B、细胞松弛素A、细胞松弛素B、细胞松弛素C、细胞松弛素D和细胞松弛素E。
10.根据技术方案1-9中任一项的方法，其中步骤(a)包括抑制所述细胞的细胞体积调节功能。
11.根据技术方案10的方法，其中通过抑制所述细胞的调节细胞体积的蛋白的表达或活性，从而抑制所述细胞的细胞体积调节功能。
12.根据技术方案11的方法，其中所述调节细胞体积的蛋白选自离子通道和转运蛋白。
13.根据技术方案12的方法，其中所述调节细胞体积的蛋白选自体积调节的阴离子通道(VRAC)，例如SWELL 1；体积调节的阳离子通道(VRCC)，例如TRPV4和TRPM3；和协同转运蛋白，例如KCC1、KCC3和KCC4。
14.根据技术方案10的方法，其中通过将所述细胞置于包含具有减弱的调节细胞体积变化能力的阳离子或阴离子的缓冲溶液中，从而抑制所述细胞的细胞体积调节功能。
15.根据技术方案14的方法，其中所述阳离子选自K +，Na +，Cs +，Li +，Ca 2+，Mg 2+，Ba 2+，Mn 2+，Fe 2+，Ni 2+，Zn 2+，Al 3+，Fe 3+,CH 3NH 3 +，C 2H 5NH 3 +，(CH 3) 2NH 2 +，(C 2H 5) 2NH 2 +，(C 2H 5) 3N +，氨离子和/或胆碱离子中的一种或多种。
16.根据技术方案14的方法，其中所述阴离子选自Br -，Cl -，I -，F -，OH -，HCO 3 -，H 2PO 4 -，NO 2 -，NO 3 -，CN -，HPO 4 2-，CO 3 2-，SO 4 2-和/或PO 4 3-中的一种或多种。
17.根据技术方案1-16中任一项的方法，其中步骤(a)包括使所述细胞过表达选自Tetraspanin家族成员的一种或多种蛋白。
18.根据技术方案17的方法，其中所述Tetraspanin家族成员包括Tspan1、Tspan2、Tspan3、Tspan4、Tspan5、Tspan6、Tspan7、Tspan8、Tspan9、Tspan10、Tspan11、Tspan12、Tspan13、Tspan14、Tspan15、Tspan16、Tspan17、Tspan18、Tspan19、Tspan20(UPK1B)、Tspan21(UPK1A)、Tspan22(PRPH2)、Tspan23(ROM1)、Tspan24(CD151)、Tspan25(CD53)、Tspan26(CD37)、Tspan27(CD82)、Tspan28(CD81)、Tspan29(CD9)、Tspan30(CD63)、Tspan31、Tspan32和Tspan33。
19.根据技术方案17的方法，其中步骤(a)包括使所述细胞过表达Tspan4。
20.根据技术方案1-19中任一项的方法，其中所述方法进一步包括减小工程化迁移体的 尺寸。
21.根据技术方案20的方法，其中通过使用特定孔径的过滤器挤压工程化迁移体从而减小工程化迁移体的尺寸；
任选地，减小的工程化迁移体的尺寸为纳米级，例如50-200nm。
22.根据技术方案1-21中任一项的方法，其中所述细胞为正常细胞或癌细胞。
23.由技术方案1-22中任一项的方法制备的工程化迁移体。
24.递送系统，其包含分离或纯化的迁移体和荷载，所述荷载直接或间接连接至所述迁移体的膜。
25.根据技术方案24的递送系统，其中所述迁移体选自天然产生的迁移体和经人工诱导产生的工程化迁移体。
26.根据技术方案25的递送系统，其中所述工程化迁移体由技术方案1-22中任一项的方法产生。
27.根据技术方案24-26中任一项的递送系统，其中所述荷载选自蛋白质例如膜蛋白和可溶性蛋白、肽、核酸例如DNA和RNA、和小分子化合物。
28.根据技术方案24-26中任一项的递送系统，其中所述载荷选自治疗性蛋白、免疫原性蛋白、细胞因子、酶、siRNA、microRNA、反义寡核苷酸(ASO)、mRNA、CRISPR系统、细胞毒性剂、治疗性小分子和靶向分子。
29.根据技术方案28的递送系统，其中所述靶向分子选自抗体或其抗原结合片段；整合素；找到我/吃我信号(find-me/eat me signal)，例如PAMP和DAMP；和不要吃我信号(don’t-eat-me signal)，例如CD47和CD24。
30.根据技术方案24-29中任一项的递送系统，其中所述荷载通过物理吸附或化学连接而连接或嵌入至迁移体的膜。
31.根据技术方案22-29中任一项的递送系统，其中所述荷载通过选自以下的方式连接或嵌入至所述迁移体的膜：
(1)与迁移体的膜组分例如膜蛋白或胆固醇连接；
(2)与连接至迁移体膜的蛋白或肽连接，优选所述蛋白或肽通过点击化学连接至迁移体的膜；和
(3)与结合对的第一成员连接，其中所述迁移体的膜包含所述结合对的第二成员，所述荷载通过所述第一成员和第二成员的结合连接至迁移体的膜。
32.根据技术方案31的递送系统，其中所述结合对包括受体-配体结合对。
33.根据技术方案32的递送系统，其中所述结合对为HaloTag及其配体，或CP05和CD63。
34.根据技术方案31的递送系统，其中所述荷载为mRNA，并且所述迁移体的膜包含mRNA结合蛋白，所述mRNA通过其3’-UTR的蛋白结合位点与所述mRNA结合蛋白的结合连接至迁移体的膜。
35.根据技术方案24-29中任一项的递送系统，其中所述荷载作为膜蛋白表达在迁移体膜的表面。
36.根据技术方案24-29中任一项的递送系统，其中所述荷载作为与膜蛋白或其部分融合的融合蛋白表达在迁移体膜的表面。
37.产生递送系统的方法，所述递送系统包含分离或纯化的迁移体和荷载，所述荷载选自蛋白质、肽、核酸(例如DNA和RNA)和小分子化合物，其中所述方法包括：
从细胞分离或纯化迁移体，所述迁移体是天然产生的迁移体或经人工诱导产生的工程化迁移体；和
将所述荷载直接或间接连接至所述迁移体的膜，从而产生所述递送系统。
38.根据技术方案37的方法，其中将所述荷载通过物理吸附或化学连接而连接至迁移体的膜。
39.根据技术方案37的方法，其中将所述荷载通过选自以下的方式连接至所述迁移体的膜：
(1)将所述荷载与迁移体的膜组分例如膜蛋白或胆固醇连接；
(2)将所述荷载与连接至迁移体膜的蛋白或肽连接，优选所述蛋白或肽通过点击化学连接至迁移体的膜。
40.产生递送系统的方法，所述递送系统包含工程化迁移体和荷载，所述荷载选自蛋白质、肽、核酸(例如DNA和RNA)和小分子化合物，其中所述荷载与结合对的第一成员连接，并且所述迁移体的膜包含所述结合对的第二成员，其中所述方法包括：
使所述细胞在细胞膜上表达所述结合对的第二成员；
由所述细胞产生在膜上包含所述结合对的第二成员的工程化迁移体；和
将所述荷载与所述结合对的第一成员的复合物与所述工程化迁移体接触，从而通过所述第一成员与第二成员的结合产生所述递送系统。
41.根据技术方案40的方法，其中所述结合对包括受体-配体结合对。
42.根据技术方案41的方法，其中所述结合对为HaloTag及其配体，或CP05和CD63。
43.产生递送系统的方法，所述递送系统包含工程化迁移体和荷载，所述荷载为mRNA，并且所述迁移体的膜包含mRNA结合蛋白，所述mRNA通过其3’-UTR的蛋白结合位点与所述mRNA结合蛋白结合，其中所述方法包括：
使所述细胞表达所述mRNA，
在上述步骤之前、之后或同时，使所述细胞在细胞膜上表达所述mRNA结合蛋白；和
由所述细胞产生包含工程化迁移体和mRNA的递送系统，其中所述mRNA通过与所述mRNA结合蛋白的结合连接至所述工程化迁移体的膜。
44.产生递送系统的方法，所述递送系统包含工程化迁移体和荷载，所述荷载为蛋白质并且表达在工程化迁移体膜的表面，其中所述方法包括：
使所述细胞在细胞膜上表达所述蛋白质；和
由所述细胞产生包含工程化迁移体和表达在工程化迁移体膜的表面的蛋白质的递送系统。
45.根据技术方案44的方法，其中所述蛋白质为膜蛋白。
46.根据技术方案44的方法，其中所述蛋白质为可溶性蛋白，并且作为与膜蛋白或其部分融合的融合蛋白表达在迁移体膜的表面。
47.根据技术方案37-46中任一项的方法，其中所述工程化迁移体由技术方案1-22中任一项的方法产生。
与药物偶联递送系统及非生物来源的药物递送系统相比，本申请的迁移体递送系统具有更高的生物兼容性，递送药物种类更多、容量更大且更容易进行膜改造增加或改变靶向性。与工程化外泌体和工程化改造的红细胞相比，迁移体递送系统保留了高生物兼容性以及易改造特性，并且比传统小囊泡(如外泌体)体积更大，可以更加有效地递送药物，尤其是大分子量的蛋白以及核酸。同时，迁移体递送系统的可改造性很高，迁移体的尺寸、膜上装载的物质都可按需要递送的药物和需要靶向的细胞进行调整。迁移体还具有低毒性的优点，更容易实现膜组分的改造和修饰。另外，工程化迁移体的制备不需要超高速离心，也不需要每次提取血液样品，大幅降低了制备时间和成本。
因此，相比现有的药物递送系统，本申请的迁移体递送系统兼具毒性低、载量高、易改造、生产快、成本低的特性，且抗原性可调，具有独特的抗原呈递作用，可用作疫苗递送平台。
本申请人发现，通过对细胞进行以下一项或多项处理：低渗透压处理、破坏细胞的细胞骨架、抑制细胞的细胞体积调节功能和使细胞过表达Tetraspanin家族成员、调节细胞快速脱离表面或细胞膜的相对位移，可以诱导细胞产生工程化迁移体。
虽然迁移体具有囊泡结构，但其不同于细胞外囊泡和外泌体。迁移体可以不属于严格意义上的细胞外囊泡。虽然脱离细胞的迁移体是一种胞外囊泡，但是迁移体的许多功能是在他们脱离细胞体之前完成的。这就是为何将迁移体视为细胞器而可以不是一种细胞外囊泡的原因。产生细胞外囊泡(脱离的迁移体)只是迁移体的众多功能之一。
脱离的迁移体是一种胞外囊泡，但其与外泌体可以存在众多差异。例如，1)两者结构不同：迁移体在被释放之前附着在收缩丝上并呈现大囊泡内包含小囊泡的结构；而外泌体不存在这种结构；2)两者的大小不同：外泌体的直径为约50-150nm，而迁移体的直径为约0.5-3μm；3)两者的蛋白质组成明显不同：在迁移体和外泌体之间只有27％的蛋白质组成是相同的，例如NDST1(双功能硫酸乙酰肝素N-脱乙酰酶/N-磺基转移酶1)、PIGK(磷脂酰肌醇聚糖锚定生物合成类K)、CPQ(羧肽酶Q)和EOGT(EGF结构域特异性O-连接的N-乙酰葡糖胺转移酶)富集在迁移体上，而不存在于外泌体中(Zhao X,Lei Y,Zheng J,Peng J,Li Y,Yu L,Chen Y.Identification of markers for migrasome detection.Cell Discov.2019 May 21；5:27)；4)两者受不同的遗传途径调控，生物发生过程完全不同：外泌体首先作为多泡体(MVB)的囊泡产生，当MVB与质膜融合时释放外泌体；而迁移体是由质膜上大结构域组装形成的(Huang Y,Zucker B,Zhang S,Elias S,Zhu Y,Chen H,Ding T,Li Y,Sun Y,Lou J,Kozlov MM*,Yu L*.Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains.Nat Cell Biol.2019 Aug；21(8):991-1002)。
如本文所用的术语“工程化迁移体”是指对细胞进行工程化改造和/或进行人工诱导所产生的迁移体。应当注意，“工程化迁移体”可以不暗示其是合成的，其仍可以是细胞来源的。
本申请的工程化迁移体可以不同于本领域已知的“低渗透压诱导的囊泡”(例如，Cohen S,Ushiro H,Stoscheck C,Chinkers MA native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.J Biol Chem 257:1523-1531中的图1所示的囊泡)，它们存在至少以下区别：1)囊泡产生位点不同：工程化迁移体产生于细胞周围的收缩丝上；而Cohen等人的囊泡产生于细胞上表面；2)囊泡尺寸不同：工程化迁移体的尺寸为微米级，直径很少超过5μm；而Cohen等人利用低渗透压诱导的囊泡可大至20μm；3)于Cohen等人利用低渗诱导的方法不同，本申请中低渗不是产生工程化迁移体的必要条件，可通过调 节细胞贴附特性使细胞快速脱离，造成细胞膜于贴附表面的相对位移，诱导出迁移体。
除非另有明确说明，否则本文中的迁移体可以包括天然产生的迁移体和人工诱导产生的工程化迁移体。
在一些实施方案中，分离或纯化的迁移体可以没有可检测的污染物组分，或者污染物组分的水平或量等于或低于可接受的水平或量。
在一些实施方案中，可以通过离心来分离和/或纯化工程化迁移体。例如，可以通过在300-17000xg下离心来分离和/或纯化工程化迁移体。在一些实施方案中，可以通过过滤来分离和/或纯化工程化迁移体。
本申请人发现，对细胞进行低渗压处理能够诱导大量的微米级囊泡在细胞的收缩丝上快速形成，这种微米级囊泡即人工诱导产生的工程化迁移体。因此，在本申请工程化迁移体制备方法的一些实施方案中，诱导细胞产生工程化迁移体可以包括对细胞进行低渗透压处理。在一些实施方案中，可以通过将细胞置于低渗缓冲溶液中，从而进行低渗透压处理，所述低渗缓冲溶液的渗透压为等渗缓冲溶液的10％-90％，例如15％-85％、20％-80％、25％-75％、30％-70％、35％-65％、40％-60％、50％-55％或其间的任何数值或子范围，例如10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、30％、40％、50％、60％、70％、80％、90％。在另一些实施方案中，可以通过将细胞置于缓冲溶液中，并将缓冲溶液的渗透压降低，从而进行低渗透压处理。在一些实施方案中，降低缓冲溶液的渗透压包括线性降低或阶梯式逐步降低。例如，在一些实施方案中，可以以预定的时间间隔，逐步降低缓冲溶液的盐浓度。例如，可以逐步降低缓冲溶液的盐浓度至少3次(例如3-5次)，每次降低1/6-1/2的盐浓度。
本申请人还发现，通过破坏细胞骨架使细胞收缩，收缩会导致细胞边缘向中心回缩，同时细胞通过粘着斑粘附在培养板底部的各个点，从而使质膜在原位粘附，作为膜系绳生成的锚点。细胞的收缩会导致在收缩前被细胞占据的区域上形成大量的膜系绳，这些新形成的膜系绳导致工程化迁移体的形成显著增强。因此，在一些实施方案中，诱导细胞产生工程化迁移体可以包括破坏所述细胞的细胞骨架。在一些实施方案中，可以通过使细胞与破坏细胞骨架的试剂接触，从而破坏所述细胞的细胞骨架。在一些实施方案中，破坏细胞骨架的试剂包括例如微丝和或微管解聚剂，例如选自Latrunculin A、Latrunculin B、细胞松弛素A、细胞松弛素B、细胞松弛素C、细胞松弛素D和细胞松弛素E。在一些实施方案中，破坏细胞骨架的试剂为Latrunculin A。在一些实施方案中，破坏细胞骨架的试剂的浓度可以是任何合适的浓度。例如，微丝和/或微管解聚剂的浓度可以是至少0.01μM、至少0.1μM、至少0，2μM、 至少0.5μM、至少1μM、至少2μM、至少3μM、至少4μM、至少5μM、至少6μM、至少7μM、至少8μM、至少9μM、至少10μM。
细胞可以通过受调节的体积变化来对抗渗透压的变化。因此，抑制细胞体积调节功能可以促进工程化迁移体的形成。在一些实施方案中，诱导细胞产生工程化迁移体可以包括抑制所述细胞的细胞体积调节功能。在一些实施方案中，可以通过抑制细胞的调节细胞体积的蛋白的表达或活性，从而抑制所述细胞的细胞体积调节功能。例如，调节细胞体积的蛋白可以选自离子通道和转运蛋白。在一些实施方案中，调节细胞体积的蛋白可以选自体积调节的阴离子通道(VRAC)，例如SWELL 1；体积调节的阳离子通道(VRCC)，例如TRPV4和TRPM3；和协同转运蛋白，例如KCC1、KCC3和KCC4。可以通过本领域已知的任何方法或试剂来抑制蛋白质的表达或活性，例如破坏编码蛋白质的基因序列、RNAi、蛋白活性抑制剂等。
已知阳离子和阴离子可以调节细胞体积，但不同的离子调节细胞体积变化的能力是不同的。因此，调节细胞体积变化能力较弱的离子对抗渗透压变化的能力也较弱，从而能够促进迁移体的形成。因此，在一些实施方案中，可以通过将细胞置于包含具有减弱的调节细胞体积变化能力的阳离子或阴离子的缓冲溶液中，从而抑制所述细胞的细胞体积调节功能。如本文所用的术语“减弱的调节细胞体积变化能力”是相对于使细胞具有正常的细胞体积调节能力的阳离子(例如钠离子)或阴离子而言的。
在一些实施方案中，诱导细胞产生工程化迁移体可以包括使所述细胞过表达选自Tetraspanin家族成员或迁移体产生通路的关键酶和结构蛋白的一种或多种蛋白或以迁移体产生激动剂刺激。例如，Tetraspanin家族成员可以包括Tspan1、Tspan2、Tspan3、Tspan4、Tspan5、Tspan6、Tspan7、Tspan8、Tspan9、Tspan10、Tspan11、Tspan12、Tspan13、Tspan14、Tspan15、Tspan16、Tspan17、Tspan18、Tspan19、Tspan20(UPK1B)、Tspan21(UPK1A)、Tspan22(PRPH2)、Tspan23(ROM1)、Tspan24(CD151)、Tspan25(CD53)、Tspan26(CD37)、Tspan27(CD82)、Tspan28(CD81)、Tspan29(CD9)、Tspan30(CD63)、Tspan31、Tspan32和Tspan33。在一些实施方案中，可以通过使细胞过表达一种或多种Tspan蛋白，从而诱导细胞产生工程化迁移体。
在一些实施方案中，可以通过调节细胞贴附特性使细胞快速脱离，以减少细胞与培养表面的接触面积，或造成细胞膜与表面的相对位移，从而诱导出迁移体。所述细胞可以在4，8，16，24，37摄氏度进行低渗处理，或不经过低渗处理，该细胞脱离过程可以在4，8，16，24摄氏度进行。并且可以分离细胞包体和迁移体。
在一些实施方案中，本申请的工程化迁移体的制备方法还包括减小工程化迁移体的尺寸。 可以通过本领域已知的任何方式来减小工程化迁移体的尺寸，例如通过使用特定孔径的过滤器或挤压器挤压工程化迁移体从而减小工程化迁移体的尺寸。在一些实施方案中，可以通过使用含有特定孔径滤膜的挤出机对工程化迁移体进行处理，从而减小工程化迁移体的尺寸。在一些实施方案中，滤膜或挤压器的孔径可以为30nm-10000nm，例如50nm-8000nm、50nm-1000nm、50nm-10000nm、100nm-1000nm、100nm-10000nm、1000nm-10000nm、10-400nm、20-300nm、30-200nm、40-100nm、50-80nm或其间的任何数值或子范围。在一些实施方案中，减小的工程化迁移体的尺寸可以为纳米级，例如1-1000nm、10-900nm、20-300nm、30-200nm、40-100nm、50-80nm或其间的任何数值或子范围。
在本文中，用于产生工程化迁移体的细胞可以是任何细胞，例如体外培养的细胞或体内的细胞；经过或未经改造的在悬浮或贴壁的状态下的悬浮或贴壁培养的细胞株/系、正常细胞、原代细胞或包括癌细胞在内的疾病来源的细胞。。此外，用于产生工程化迁移体的细胞可源自适合于体外增殖、修饰并表达外源分子并产生工程化迁移体的任何细胞系。在一些实施方案中，所述细胞可以是动物细胞，特别是哺乳动物细胞，包括鼠和人的细胞。合适的细胞的实例包括但不限于正常大鼠肾细胞(NRK细胞)、小鼠成纤维细胞如NIH3T3细胞、小鼠乳腺癌4T1细胞、小鼠结肠癌MC38细胞、常用的人胚胎肾(HEK)细胞株/系如HEK293或HEK293FT细胞、人胃癌MGC-803细胞、人T淋巴细胞瘤Jurkat细胞、人皮肤成纤维BJ细胞、中国仓鼠卵巢(CHO)细胞、间充质干细胞(MSC)、或任何其他合适的细胞。
如本文所用的术语“载荷”可以是指能够装载在迁移体上从而被有效递送的任何物质。例如，荷载可以通过迁移体的表面分子与靶细胞相互作用而被递送至靶细胞。本申请的荷载中的一种或多种可以在工程化迁移体形成期间或形成之后装载到工程化迁移体的膜上，也可以直接装载到天然产生的迁移体的膜上。载荷的实例包括但不限于治疗剂，例如合成生物活性化合物、天然生物活性化合物、抗菌化合物、抗病毒化合物、蛋白质或肽(例如酶或抗体)、核苷酸(例如，包含可检测部分或毒素的核苷酸或破坏转录的核苷酸)、核酸(例如，编码多肽如酶、疫原性蛋白、细胞因子、抑癌基因、抗体等的DNA或mRNA分子，或具有调节功能的RNA分子如microRNA、dsDNA、反义寡核苷酸(ASO)、lncRNA和siRNA)、基因组编辑系统、脂质、碳水化合物、小分子(例如，小分子药物和毒素)、靶向分子、多糖、复合物、细胞器、纳米和微米颗粒或其任意组合。
在一些实施方案中，荷载可以是microRNA或siRNA，例如特异性地结合到编码突变型或非突变型致癌基因的转录物中的microRNA或siRNA。microRNA或siRNA的结合可以 mRNA译码和蛋白质合成。此类基因包括但不限于ABLI、BLC1、BCL6、CBFA1、CBL、CSFIR、ERBA、ERBB、EBRB2、ETS1、ETS1、ETV6、FGR、FOX、FYN、HCR、HRAS、JUN、KRAS、LCK、LYN、MDM2、MLL、MYB、MYC、MYCL1、MYCN、NRAS、PIM1、PML、RET、SRC、TAL1、TCL3、YES、VEGF、FGF、G-CSF、CXCR4等。
在一些实施方案中，荷载可以是基因组编辑系统。基因组编辑系统包括但不限于大范围核酸酶系统、锌指核酸酶(ZFN)系统、类转录激活因子效应物核酸酶(TALEN)系统和规律成簇的间隔短回文重复(CRISPR)系统。在一些实施方案中，荷载可以是CRISPR系统。在一些实施方案中，CRISPR系统可以是CRISPR-Cas9系统。CRISPR-Cas9系统包含编码Cas9蛋白质的核苷酸序列、编码与靶序列(crRNA)杂交的CRISPR RNA的核苷酸序列、和编码反式激活CRISPR RNA(tracrRNA)的核苷酸序列。crRNA和tracrRNA可以融合成向导RNA。CRISPR-Cas9系统可以进一步包含核定位信号(NLS)。装载有CRISPR-Cas9系统的迁移体可以用于改变疾病处理、再生医学和组织工程的基因表达和功能。
在一些实施方案中，荷载可以是治疗性蛋白或其片段，例如抗体或其片段。在一些实施方案中，荷载可以是蛋白(包括抗体或抗体片段、免疫原性蛋白、细胞因子、酶、抑癌基因产物等)或其片段，例如鸡卵白蛋白(OVA)、SARS-CoV-2的刺突蛋白或其S1片段。
在一些实施方案中，荷载可以是靶向分子。如本文所用的术语“靶向分子”是指能够特异性结合另一分子(靶分子)的分子。例如，靶向分子可用于将在表面上呈递靶向分子的迁移体特异性定位至某一实体，例如表达靶分子的组织或细胞，从而提高递送系统的靶向性。在一些实施方案中，本申请的递送系统可包含至少两种不同的靶向分子，从而进一步提高靶向特异性或以其他方式改善对靶细胞或靶组织的靶向性。例如，靶向分子可以特异性结合癌细胞上过表达的表面蛋白，例如抗体或其抗原结合片段。在一些实施方案中，靶向分子可以选自抗体或其抗原结合片段；整合素；细胞因子、趋化因子和/或细胞因子、趋化因子受体；多糖；找到我/吃我信号(find-me/eat me signal)，例如PAMP和DAMP；和不要吃我信号(don’t-eat-me signal)，例如CD47和CD24。在一些实施方案中，抗体或其抗原结合片段可以通过结合其相应的抗原，将装载有抗体或其抗原结合片段的迁移体递送至表达相应抗原的靶细胞。在一些实施方案中，整合素或其他靶向分子可以通过与组织特异的细胞外基质配对，从而实现装载有整合素或其他靶向分子的迁移体对特定器官的靶向性。
如本文所用的术语“找到我/吃我信号(find-me/eat me signal)”是指由凋亡细胞暴露或释放以引发吞噬性摄取的信号，这种吞噬性摄取转而激活耐受性途径以防止针对自身抗原的免疫 应答。在本文中，将找到我/吃我信号装载在迁移体上，该找到我/吃我信号能被巨噬细胞等识别，从而使迁移体被特定细胞吞噬。示例性的找到我/吃我信号包括例如病原体相关分子模式(PAMP)和损伤相关分子模式(DAMP)。“病原体相关分子模式(PAMP)”是指病原微生物表面存在的一些人体宿主所没有的，但可为许多相关微生物所共享、结构恒定且进化保守的分子结构。先天性免疫识别的PAMP，往往是病原体赖以生存，因而变化较少的主要部分，如病毒的双链RNA和细菌的脂多糖，对此，病原体很难产生突变而逃脱先天性免疫的作用。PAMP可以表达在病原体表面或游离于免疫细胞之外，也可以出现在免疫细胞的胞质溶胶，以及溶胶中各种携带病原体的胞内区室，如内体和吞噬溶酶体内。PAMP主要包括两类。第一类是以糖类和脂类为主的细菌胞壁成分，例如如脂多糖、肽聚糖、脂磷壁酸、甘露糖、类脂、脂阿拉伯甘露聚糖、脂蛋白和鞭毛素等。其中最为常见且具有代表性的包括革兰阴性菌产生的脂多糖(LPS)；革兰阳性菌产生的肽聚糖(proteoglycan)；分枝杆菌产生的糖脂(glycolipid)和酵母菌产生的甘露糖。第一类是病毒产物及细菌胞核成分，例如非甲基化寡核苷酸CpGDNA、单链RNA、双链RNA。“损伤相关分子模式(DAMP)”是组织或细胞受到损伤、缺氧、应激等因素刺激后释放到细胞间隙或血液循环中的一类物质，其可通过Toll样受体、RIG-1样受体或NOD样受体等模式识别受体诱导自身免疫或免疫耐受，在关节炎、动脉粥样硬化、肿瘤、系统性红斑狼疮等疾病发生和发展过程中发挥重要作用。DAMP存在于细胞核、细胞质(例如，高速泳动族蛋白盒(HMGB)1、S100蛋白)、细胞外基质(例如透明质酸)和血浆中(例如补体C3a、C4a、C5a)或作为外来体(例如热休克蛋白)。非蛋白形式的DAMP包括腺苷三磷酸、尿酸、硫酸肝素、RNA和DNA。这些蛋白质和非蛋白质在健康情况下被限制在细胞内，当细胞破坏时则被释放到细胞外。
如本文所用的术语“不要吃我信号(don’t-eat-me signal)”是指肿瘤细胞在其表面表达的用于与免疫细胞表面的配体结合从而抑制免疫细胞对肿瘤细胞的杀伤作用的信号。示例性的不要吃我信号包括例如CD47和CD24。在本文中，通过基因编辑、膜融合等方式将不要吃我信号装载在迁移体上，使得迁移体逃避免疫系统的清除，从而延长在血液中的循环时间，实现更好的组织浸润。
例如，迁移体可以具有单层膜结构，其膜来源为细胞膜和细胞胞内囊泡。与细胞膜相比，迁移体的膜特异地富集了一些蛋白(如Tetraspanin)和脂质(如胆固醇、鞘磷脂)。
本申请的荷载可以通过本领域已知的任何方式直接或间接连接或嵌入至迁移体的膜和/或迁移体内部。
在一些实施方案中，荷载可以通过液态固态转换、膜融合、电荷吸附、物理吸附或化学连接而连接或嵌入至迁移体的膜和/或迁移体内部。
在其他实施方案中，荷载可以通过选自以下组的方式连接或嵌入至所述迁移体的膜和/或迁移体内部：(1)与迁移体的膜和/或迁移体内部组分例如膜蛋白、胆固醇、磷脂、糖蛋白上的糖链或多糖连接；(2)与连接或嵌入至迁移体膜和/或迁移体内部的蛋白或肽、糖蛋白上的糖链或多糖、磷脂、胆固醇等连接，优选所述蛋白或肽、糖蛋白上的糖链或多糖、磷脂、胆固醇等通过点击化学连接或嵌入至迁移体的膜和/或迁移体内部；和(3)与结合对的第一成员连接，其中所述迁移体的膜和/或迁移体内部包含所述结合对的第二成员，所述荷载通过所述第一成员和第二成员的结合连接或嵌入至迁移体的膜和/或迁移体内部。
在一些实施方案中，荷载通过与迁移体的膜和/或迁移体内部组分例如膜蛋白或脂质连接而连接或嵌入至迁移体的膜和/或迁移体内部。在一些实施方案中，荷载通过与迁移体的膜蛋白例如Tetraspanin连接而连接或嵌入至迁移体的膜和/或迁移体内部。在一些实施方案中，荷载通过与Tetraspanin蛋白连接而连接或嵌入至迁移体的膜和/或迁移体内部。在一些实施方案中，荷载通过与迁移体膜脂质(例如胆固醇和鞘磷脂)、蛋白或肽、糖蛋白上的糖链或多糖连接而连接或嵌入至迁移体的膜和/或迁移体内部。
在一些实施方案中，结合对包括抗原-抗体、受体-配体、生物素-亲和素、HaloTag及其配体等结合对。例如，所述结合对为HaloTag及其配体，或CP05和CD63。
在一些实施方案中，荷载为mRNA，并且迁移体的膜和/或迁移体内部包含mRNA结合蛋白，所述mRNA通过其蛋白结合位点与所述mRNA结合蛋白的结合连接或嵌入至迁移体的膜和/或迁移体内部。mRNA结合蛋白及其蛋白结合位点可以是本领域已知的那些。例如，在一些实施方案中，mRNA结合蛋白为L7Ae，蛋白结合位点为C/D Box。在另一些实施方案中，mRNA结合蛋白为MS2BP，蛋白结合位点为MS2茎环(MS2SL)。
在一些实施方案中，荷载可以作为膜蛋白表达在迁移体膜的内表面或外表面。在另一些实施方案中，荷载可以作为与膜蛋白或其部分融合的融合蛋白表达在迁移体膜的内表面或外表面。本领域已知的任何膜蛋白都可用作与可溶性蛋白融合的膜锚定蛋白。作为可溶性蛋白融合的膜蛋白的实例包括但不限于细胞受体、离子通道、转运蛋白等，例如Tspan-4、CD81、CD9、CD63、PDGFR、Lamp2b、突触融合蛋白2(STX2)等。在一些实施方案中，作为可溶性蛋白融合的膜蛋白可以是STX2。在一些实施方案中，作为可溶性蛋白融合的膜蛋白可以是截短的STX2(t-STX2)。例如，可以对STX2的N端进行改造，去除其胞内端功能，以得到 t-STX2。然后可以将可溶性蛋白连接到t-STX2处于胞外的C端，以形成可溶蛋白-t-STX2融合蛋白，从而使可溶性蛋白作为人造的质膜定位融合蛋白在迁移体膜上表达。
在另一方面，本申请涉及产生递送系统的方法，所述递送系统包含分离或纯化的迁移体和荷载，所述荷载选自蛋白质、肽、核酸(例如DNA和RNA)、脂质、小分子化合物、多糖、复合物、纳米/微米颗粒、细胞器或同时装载一种以上所述载荷等，其中所述方法包括：从细胞分离或纯化迁移体，所述迁移体是天然产生的迁移体或经人工诱导产生的工程化迁移体；和将所述荷载直接或间接连接或嵌入至所述迁移体的膜和/或迁移体内部，从而产生所述递送系统。在本申请递送系统产生方法的一些实施方案中，将所述荷载通过液态固态转换、膜融合、电荷吸附、物理吸附或化学连接而连接或嵌入至迁移体的膜和/或迁移体内部。
在本申请递送系统产生方法的另一些实施方案中，将所述荷载通过选自以下的方式连接至或嵌入所述迁移体的膜和/或迁移体内部：(1)将所述荷载与迁移体的膜和/或迁移体内部组分例如膜蛋白、胆固醇、磷脂、糖蛋白上的糖链或多糖连接；和(2)将所述荷载与连接至迁移体膜和/或迁移体内部的蛋白或肽、糖蛋白上的糖链或多糖、磷脂、胆固醇等连接，优选所述蛋白或肽、糖蛋白上的糖链或多糖、磷脂、胆固醇等通过点击化学连接至迁移体的膜和/或迁移体内部。
在一些实施方案中，天然产生的迁移体可以通过本领域已知的方法从细胞分离或纯化。在一些实施方案中，经人工诱导产生的工程化迁移体由本文公开的工程化迁移体的制备方法产生。在一些实施方案中，荷载可以是本文其他地方所述的荷载的一种或多种。
在另一方面，本申请涉及产生递送系统的方法，所述递送系统包含工程化迁移体和荷载，所述荷载选自蛋白质、肽、核酸(例如DNA和RNA)、脂质、小分子化合物、多糖、复合物、纳米/微米颗粒、细胞器或同时装载一种以上所述载荷等，其中所述荷载与结合对的第一成员连接，并且所述迁移体的膜和/或迁移体内部包含所述结合对的第二成员，其中所述方法包括：使所述细胞在细胞膜上表达所述结合对的第二成员；由所述细胞产生在膜上包含所述结合对的第二成员的工程化迁移体；和将所述荷载与所述结合对的第一成员的复合物与所述工程化迁移体接触，从而通过所述第一成员与第二成员的结合产生所述递送系统。
在本申请递送系统产生方法的一些实施方案中，所述方法包括向细胞引入包含结合对的第二成员的编码序列的核苷酸序列，以使所述细胞在细胞膜上表达结合对的第二成员。在一些实施方案中，所述方法包括向细胞引入包含结合对的第二成员的编码序列的核苷酸序列，并在允许所述细胞表达结合对的第二成员的条件下培养所述细胞，以使所述细胞在细胞膜上 表达结合对的第二成员。
在一些实施方案中，所述方法包括：a)向细胞引入包含结合对的第二成员的编码序列的核苷酸序列；b)在允许所述细胞表达所述结合对的第二成员的条件下培养所述细胞；c)由所述细胞产生在膜上包含所述结合对的第二成员的工程化迁移体；d)将所述荷载与结合对的第一成员连接以形成复合物；和e)使所述工程化迁移体与所述复合物接触从而产生所述递送系统。在一些实施方案中，上述步骤d)在步骤a)之前、之后或同时进行。
在一些实施方案中，结合对的第二成员为膜蛋白或可溶性蛋白。在一些实施方案中，结合对的第二成员为膜蛋白并且表达在工程化迁移体膜的内表面或外表面。在一些实施方案中，结合对的第二成员为可溶性蛋白，并且作为与膜蛋白或其部分融合的融合蛋白表达在工程化迁移体膜的内表面或外表面。
在一些实施方案中，所述结合对包括抗原-抗体、受体-配体、生物素-亲和素、HaloTag及其配体等结合对。在一些实施方案中，所述结合对为HaloTag及其配体，或CP05和CD63。
在一些实施方案中，工程化迁移体由本文公开的工程化迁移体的制备方法产生。在一些实施方案中，荷载可以是本文其他地方所述的荷载的一种或多种。
在又一方面，本申请涉及产生递送系统的方法，所述递送系统包含工程化迁移体和荷载，所述荷载为mRNA，并且所述迁移体的膜和/或迁移体内部包含mRNA结合蛋白，所述mRNA通过其蛋白结合位点与所述mRNA结合蛋白结合，其中所述方法包括：使所述细胞表达所述mRNA；在上述步骤之前、之后或同时，使所述细胞在细胞膜上表达所述mRNA结合蛋白；和由所述细胞产生包含工程化迁移体和mRNA的递送系统，其中所述mRNA通过与所述mRNA结合蛋白的结合连接至所述工程化迁移体的膜和/或迁移体内部。
在本申请递送系统产生方法的一些实施方案中，所述方法包括向细胞引入在包含蛋白结合位点的mRNA的编码序列，以使所述细胞表达所述mRNA。
在一些实施方案中，所述方法包括向细胞引入包含mRNA结合蛋白的编码序列的核苷酸序列，以使所述细胞在细胞膜上表达所述mRNA结合蛋白。
在一些实施方案中，所述方法包括向细胞引入在包含蛋白结合位点的mRNA的编码序列和包含mRNA结合蛋白的编码序列的核苷酸序列，以使所述细胞表达所述mRNA和所述mRNA结合蛋白。
在一些实施方案中，mRNA结合蛋白为膜蛋白或可溶性蛋白。在一些实施方案中，mRNA结合蛋白为膜蛋白并且表达在工程化迁移体膜的内表面或外表面。在一些实施方案中，mRNA 结合蛋白为可溶性蛋白，并且作为与膜蛋白或其部分融合的融合蛋白表达在工程化迁移体膜的内表面或外表面。
mRNA结合蛋白及其蛋白结合位点可以是本领域已知的那些。例如，在一些实施方案中，mRNA结合蛋白为L7Ae，蛋白结合位点为C/D Box。在另一些实施方案中，mRNA结合蛋白为MS2BP，蛋白结合位点为MS2茎环(MS2SL)。
在一些实施方案中，工程化迁移体由本文公开的工程化迁移体的制备方法产生。
在另一方面，本申请涉及产生递送系统的方法，所述递送系统包含工程化迁移体和荷载，所述荷载为蛋白质并且表达在工程化迁移体膜的内表面或外表面，其中所述方法包括：使所述细胞在细胞膜上表达所述蛋白质；和由所述细胞产生包含工程化迁移体和表达在工程化迁移体膜的内表面或外表面的蛋白质的递送系统。
在一些实施方案中，所述方法包括向细胞引入包含蛋白质编码序列的核苷酸序列，以使所述细胞在细胞膜上表达所述蛋白质。在一些实施方案中，所述方法包括向细胞引入包含蛋白质编码序列的核苷酸序列，并在允许所述细胞表达所述蛋白质的条件下培养细胞，以使所述细胞在细胞膜上表达所述蛋白质。
在一些实施方案中，所述方法包括：向细胞引入包含目的蛋白编码序列的核苷酸序列；在允许所述细胞表达目的蛋白的条件下培养所述细胞；和由所述细胞产生包含工程化迁移体和表达在工程化迁移体膜的内表面或外表面的蛋白质的递送系统。
在一些实施方案中，所述蛋白质为膜蛋白。在另一些实施方案中，所述蛋白质为可溶性蛋白，并且作为与膜蛋白或其部分融合的融合蛋白表达在迁移体膜的表面。
作为可溶性蛋白融合的膜蛋白的实例如上文所述，包括但不限于细胞受体、离子通道、转运蛋白等，例如Tspan-4、CD81、CD9、CD63、PDGFR、Lamp2b、突触融合蛋白2(STX2)等。
在一些实施方案中，工程化迁移体由本文公开的工程化迁移体制备方法产生。
本申请还提供了一种药物组合物或诊断组合物，其包含本申请的工程化迁移体，以及一种或多种药学上可接受的赋形剂、稀释剂或载体组合。因此，本申请还提供了本申请的工程化迁移体在制备药物组合物中的用途。
本申请还提供了一种用于制备药物或诊断组合物的方法，其包括添加和混合本申请的工程化迁移体连同一种或多种药学上可接受的赋形剂、稀释剂或载体。所述工程化迁移体可作为药物或诊断组合物中的唯一活性成分，也可伴随有其他活性成分诸如类固醇或其他药物分 子。所述组合物可以个别施用于患者或可与其他药剂、药物或激素组合施用(例如同时、连续或分开)。
药物组合物可以包含治疗有效量的本申请的工程化迁移体。在本申请中，术语“治疗有效量”是指治疗、改善或预防靶向疾病或病况或展现可检测的治疗或预防效果所需要的治疗剂的量。对于任何公开的工程化迁移体，可最初在细胞培养测定中或在动物模型中，通常在啮齿动物、兔、狗、猪或灵长类动物中估计治疗有效量。动物模型还可用于确定施用的适当浓度范围及途径。所述信息可随后用于确定适用于施用人的剂量及途径。人受试者的精确治疗有效量将视疾病病况的严重程度、受试者的整体健康、受试者的年龄、重量及性别、饮食、施用的时间及频率、药物组合、反应敏感性及对疗法的耐受性/反应而定。
本申请的药物组合物可通过多种途径施用，包括但不限于经口、静脉内、肌肉内、动脉内、髓内、鞘内、室内、透皮、经皮、皮下、腹膜内、鼻内、经肠、局部、舌下、阴道内或直肠途径。还可使用无针注射器施用本申请的药物组合物。通常，治疗组合物可制备成作为液体溶液或悬浮液的可注射剂。还可制备适合于在注射前溶解或悬浮于液体媒介物中的固体形式。包含本申请的工程化迁移体的治疗剂量的药物组合物不显示体内明显毒理学作用。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的工程化迁移体、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
通过下面的具体实施例进一步阐述本申请。应当理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。以下实施例中未注明具体条件的实验方法，通过按照本领域的常规条件，例如，Sambrook和Russeii等人，分子克隆：实验室手册(第三版)(2001)，CSHL出版社中所述的条件，或按照制造商所建议的条件。除非另有说明，否则以下实施例中所用的实验材料和试剂均可商购获得。
试剂
ddH2O(solarbio)、KCl(sigma)、KH2PO4(sangon)、Na2HPO4-7H2O(sangon)、BSA(VWR)、Latrunculin A(cayman)、人纤连蛋白(invitrogen或sigma)、PBS(Gibco)、RPMI 1640(Gibco)、FBS(BI)、WGA-AF488/AF594/AF647(invitrogen)、BCA试剂盒(invitrogen)。
10xKDPBS(500ml)的配制：
组分 含量(g) 浓度(mM)
KCl 52.4 1405.7
KH 2PO 4 1 14.7
Na 2HPO 4-7H 2O 10.8 80.6
ddH2O 至500ml  
100xBSA(50ml)的配制：称取5g BSA，加入ddH2O至50ml，用0.45 um滤膜过滤。
实施例1
诱导细胞产生工程化迁移体
实施例1 通过低渗透压处理产生工程化迁移体
发明人发现对细胞进行低渗压处理导致细胞胞体膨胀、底面收缩并产生大量丝状结构(收缩丝)，在收缩丝上生长出迁移体样结构。为了直观地观察工程化迁移体，建立了稳定表达Tspan4-GFP的NRK细胞系，并通过迁移体的标志物之一Tspan4的信号来观察工程化迁移体的形成。将过表达Tspan4-GFP的NRK细胞用25％DPBS(对应渗透压76.3mOsmol/L)处理，结果显示于图1a中。结果显示，低渗透压处理后30秒，Tspan4-GFP信号开始在收缩丝上富集，然后形成微米级囊泡；在达到其峰值强度后，Tspan4-GFP信号开始从囊泡扩散，同时伴随着囊泡的缩小；低渗透压处理后460秒，大部分由低渗透压诱导产生的囊泡结构消失。这些微米级囊泡的产生过程以及在收缩丝上的附着类似于天然的迁移体。
为了进一步确认低渗透压诱导产生的囊泡结构是否为工程化迁移体。用荧光标记的小麦胚芽凝集素(WGA)对细胞进行染色。WGA是与唾液酸和N-乙酰-D-葡糖胺特异性结合的凝集素。荧光标记的WGA能够用于标记细胞中的迁移体，是用于检测迁移体的特异性探针(Chen et.al.,WGA is a probe for migrosomes,Cell Discovery(2019)5:13)。
对过表达Tspan4-GFP的NRK细胞分别进行等渗处理(100％DPBS，305mOsmol/L)和25％DPBS的低渗透压处理(76.3mOsmol/L)，然后用四甲基罗丹明标记的WGA对细胞进行染色，并进行激光共聚焦显微镜观察，结果如图26c所示。结果显示，过表达Tspan4的NRK细胞经诱导产生的囊泡结构被WGA特异性染色，表明所产生的囊泡结构是工程化迁移体。
为了确定渗透压的大小对工程化迁移体形成的影响，对表达Tspan4-GFP的NRK细胞进行等渗透压(100％DPBS，305mOsmol/L)或不同强度低渗透压(50％、25％和17％DPBS，分别对应渗透压152.5、76.3和50.8 mOsmol/L)处理，结果如图1b所示，工程化迁移体直径的 统计结果如图1c所示。可以发现渗透压的高低与工程化迁移体的尺寸呈负相关，渗透压越低，诱导产生的工程化迁移体尺寸越大。
基于上述结果，发明人建立了逐步降低渗透压的方案，并且发现逐步应用低渗透压显著增加了工程化迁移体的持续时间，在经历5步低渗处理的细胞中，诱导产生的工程化迁移体在低渗透压处理20分钟后仍然存在。
实施例2 通过latrunculin A处理产生工程化迁移体
由于低渗透压诱导产生的工程化迁移体的数量取决于收缩丝的数量，并且作为工程化迁移体膜来源的大部分质膜位于细胞体上，因此推测如果通过破坏细胞骨架使细胞收缩，收缩会导致细胞边缘向中心回缩，同时细胞通过粘着斑粘附在培养板底部的各个点，从而使质膜在原位粘附，作为膜系绳生成的锚点。如果事实却是如此，细胞的收缩会以类似于迁移过程中收缩丝形成的方式产生大量的膜管。
为了验证这一假设，用含不同浓度的微丝解聚剂latrunculin A(0、0.25、0.5和1μM)的等渗缓冲溶液DPBS孵育表达Tspan4-GFP的NRK细胞10min，然后通过逐步加水以每间隔2min降低1/6盐浓度的方式对细胞进行三次连续的低渗透压刺激。经过分步低渗透压处理后的结果如图2a所示，每个细胞产生的工程化迁移体数量的统计结果如图2b所示。
正如预期的那样，发现用latrunculin A处理细胞会导致细胞收缩，并且还观察到在收缩前被细胞占据的区域上形成大量的膜系绳，这些新形成的膜系绳导致工程化迁移体的形成显著增强。因此，Latrunculin A能够显著促进工程化迁移体数量的增加，并且这种促进作用具有剂量依赖性。这提示了通过破坏细胞骨架能够促进工程化迁移体的形成。
实施例3 通过抑制细胞体积调节功能产生工程化迁移体
细胞可以通过受调节的体积变化来对抗渗透压的变化。为了测试受调节的体积变化是否会影响工程化迁移体的形成，对体积调节阴离子通道的关键组分SWELL 1(其通过响应渗透压的变化维持恒定的细胞体积)进行了敲低或敲除。
敲低表达Tspan4-GFP的NRK细胞中编码SWELL 1的基因Lrrc8a，通过qPCR对细胞中Lrrc8a的敲低效率进行验证，结果表明SWELL 1的编码基因Lrrc8a被成功敲低至野生型细胞的约15％(图3a)。将Lrrc8a基因被敲低的细胞(Lrrc8a-KD)和未敲低的对照细胞(NC)分别置于DPBS中，通过逐步加水以每间隔2min降低1/3盐浓度的方式对细胞进行连续三次的低渗透压刺激，然后用激光共聚焦显微镜进行观察，结果如图3b所示；工程化迁移体尺寸的统 计结果如图3c所示。
敲除表达Tspan4-GFP的NRK细胞中编码SWELL 1的基因Lrrc8a，通过蛋白质免疫印迹对细胞中Lrrc8a的敲除进行验证，在敲除的细胞(Lrrc8a-KO)中未观察到SWELL 1的表达，表明SWELL 1的编码基因Lrrc8a被成功敲除(图4a)。将Lrrc8a-KO细胞(KO14#细胞系和KO18#细胞系)和未敲除的对照细胞(WT)分别置于DPBS中，通过逐步加水以每间隔1min降低1/6盐浓度的方式对细胞进行连续五次的低渗透压刺激，然后用激光共聚焦显微镜进行观察，结果如图4b所示。
结果显示，低渗透压处理后，Lrrc8a-敲低细胞所产生的工程化迁移体尺寸显著大于未敲低细胞(图3b和3c)；Lrrc8a-敲除细胞所产生的工程化迁移体尺寸也显著大于未敲除细胞(图4b)。这表明敲低或敲除SWELL 1显著增强了工程化迁移体的形成，提示了可以通过降低细胞在渗透压变化期间调节其体积的能力来增强工程化迁移体的形成。
已知阳离子可以调节细胞体积。如果不同阳离子在渗透压变化期间调节细胞体积的能力不同，则可以通过阳离子置换来减弱受调节的细胞体积变化，从而促进工程化迁移体的形成。接下来，测试了不同阳离子对工程化迁移体形成的影响。将DPBS中的氯化钠置换成等摩尔浓度的氯化钾、氯化铯或氯化胆碱以配制成含有不同阳离子的等渗缓冲液，用这些等渗缓冲液分别孵育细胞，在相应的缓冲液中通过逐步加水以每间隔2min降低1/6盐浓度的方式对细胞进行连续五次的低渗透压刺激。经过逐步低渗透压处理后的结果如图5a所示，工程化迁移体尺寸的统计结果如图5b所示。
结果显示不同的阳离子具有不同的促进工程化迁移体形成的能力，在所测试的阳离子中，钠离子促进工程化迁移体形成的能力相对较弱，而钾离子、铯离子和胆碱离子促进迁移体样结构形成的能力明显更强。
实施例4 通过使细胞过表达Tspan4产生工程化迁移体
Tspan4是促进迁移体形成的关键蛋白，为了测试Tspan4是否能促进工程化迁移体的形成，对仅过表达mCherry-Kras的NRK细胞或过表达Tspan4-GFP与mCherry-Kras两者的NRK细胞进行逐步低渗透压刺激。将NRK细胞在含2μM latrunculin A的KDPBS中孵育10min后，以每间隔2min降低1/6盐浓度的方式进行三次连续的低渗透压刺激，然后通过微分干涉相差显微镜(DIC)观察工程化迁移体的形成。经过逐步低渗透压处理后的结果如图6a所示，每个细胞产生的工程化迁移体数量的统计结果如图6b所示。
结果显示Tspan4的过表达对于工程化迁移体的数量具有显著的促进作用。
实施例5 通过培养皿温敏涂层使细胞快速脱离产生迁移体
MGC803-T4-GFP细胞培养在温敏培养皿上，经过破坏细胞骨架处理后，在室温下或37度经过低渗处理，或者没有经过低渗处理，将培养皿静止放置室温至45分钟。80％以上的细胞脱离皿底，并形成迁移体，部分迁移体随细胞脱离皿底(见图7)。收取上清后吹打并收集残留细胞和迁移体。
实施例6 在多种细胞系中产生工程化迁移体
为了测试在不同种属与遗传背景的细胞系中是否能够诱导产生工程化迁移体，针对过表达Tspan4的三种不同的啮齿类细胞系(正常大鼠肾细胞(NRK)、小鼠乳腺癌细胞系(4T1)和小鼠结肠癌细胞系(MC38))，两种常用人胚肾细胞株/系(HEK-293T和HEK-293FT)、三种不同的人细胞系(人胃癌MGC-803细胞、人T淋巴细胞瘤Jurkat细胞、人皮肤成纤维BJ细胞)进行了低渗透压诱导。将NRK细胞在含2μM latrunculin A的K-DPBS中孵育10min后，以每间隔2min降低1/6盐浓度的方式进行三次连续的低渗透压刺激；将4T1细胞在含2μM latrunculin A的K-DPBS中孵育20min后，以每间隔2min降低1/4盐浓度的方式进行三次连续的低渗透压刺激；将MC38细胞在含2μM latrunculin A的K-DPBS中孵育45min后，以每间隔2min降低1/4盐浓度的方式进行三次连续的低渗透压刺激。结果如图8所示。可以看出，在不同的细胞系中均产生了囊泡结构。
进一步用迁移体的特异性探针四甲基罗丹明标记的WGA对经上述低渗处理的细胞系进行染色，然后进行激光共聚焦显微镜观察，结果如图8b所示。也测试了人源细胞系(人胚肾细胞(HEK293T、HEK293FT；图8c)、人胃癌细胞(MGC803)、人皮肤成纤维细胞(BJ)和人外周血白血病T细胞(Jurkat)；图8d)，发现通过实施例1-5的方法均可产生工程化迁移体。结果显示，八种细胞系经诱导产生的囊泡结构被迁移体的特异性探针WGA染色，表明了工程化迁移体的产生。
以上结果表明，在不同的细胞系中均可通过实施例1-5的方法产生工程化迁移体。
实施例7 工程化迁移体的分离纯化及表征(图9a-b)
a.通过对贴壁细胞进行刺激产生工程化迁移体
I.包被培养瓶、铺细胞
用PBS配制2μg/ml人纤连蛋白，在37℃下对培养瓶底面进行包被一小时以上，然后按照1x10 7个细胞/T175培养瓶的密度铺细胞。
II.诱导工程化迁移体的产生
1.细胞培养14-16h后，弃掉培养基，用PBS洗一遍；
2.加入含2μM latrunculinA的K-DPBS，37度孵育(具体孵育时间根据细胞系对LatrunculinA的敏感程度作调整,例如对于NRK细胞处理10min，对于MC38细胞处理45-60min；处理完成后细胞应呈现皱缩的状态，胞体周围有大量类似于收缩丝的网状结构)；
3.将培养瓶置于细胞培养箱中的水平摇床上，40rpm转速，每三分钟加一次水，共操作三次(每一步加水的体积由低渗诱导梯度以及培养瓶中液体的初始体积决定；低渗诱导梯度因细胞系而异，对于MC38细胞，每一步降低1/4盐浓度,初始体积为15ml,三次加水体积分别为5ml、6.5ml和8.5ml；对于NRK细胞，初始体积为15ml，三次加水体积为3ml、3.6ml和4.4ml)；
4.将转速调至60rpm，摇5min。
b.通过对悬浮细胞进行刺激产生工程化迁移体
取细胞(数目根据不同细胞系而定)，PBS洗一次，弃上清
加入含2μM latrunculinA的K-DPBS将细胞重悬于培养瓶，置于水平摇床，40rpm转速孵育(时间根据不同细胞系而定)
向培养瓶中加入无菌水以降低渗透压(降低比例根据不同细胞系而定)
将转速调至60rpm，摇5min。
为了测试在悬浮状态的细胞系中是否能够诱导产生工程化迁移体，针对过表达Tspan4的两种不同的啮齿类细胞系(正常大鼠肾细胞(NRK)、和小鼠结肠癌细胞系(MC38))进行了低渗透压诱导。结果如图9a和9b所示。悬浮状态的两种细胞系中均产生了囊泡结构，免疫印迹检测显示纯化自两个细胞的囊泡均有工程化迁移体特征性的膜蛋白的富集和细胞内物质的缺失。
III.分离纯化工程化迁移体(图10)
a.贴壁细胞产生的工程化迁移体纯化方法
1.弃上清，用低渗KDPBS(h-KDPBS；其盐浓度与低渗诱导完成时溶液体系中的盐浓度相近；对于MC38细胞，h-KDPBS为40％KDPBS；对于NRK细胞，h-KDPBS为60％KDPBS)，将细胞洗两遍；
2.加入含1mg/ml BSA的h-KDPBS(h-KDPBS-BSA),130rpm摇3min；
3.将上清液收集至50ml离心管中；
4.加入h-KDPBS-BSA,用移液器轻柔吹打培养瓶底面，将所收集的液体与第3步的上清液合并；
5. 4℃、300xg离心10min,保留上清；
6. 4℃、500xg离心10min,保留上清；
7.用8um孔径的parylene滤膜过滤上清至50ml低吸附管中；
8. 4℃、17000xg离心45-60min,弃上清；
9.用h-KDPBS-BSA重悬沉淀，转移至EP管中(离心管1)，加入等体积PBS-BSA；此时取一小部分液体(约总体积的1/50)于另一个离心管(离心管2)，用于测量蛋白浓度；
10. 4℃、17000xg离心15-20min，弃上清后加PBS，于注射前重悬沉淀，得到重悬于PBS中的工程化迁移体。
b.悬浮细胞产生的工程化迁移体纯化方法
1.将上清液收集至15ml离心管中，用移液器轻柔吹打；
5. 4℃、300xg离心10min,保留上清；
6. 4℃、500xg离心10min,保留上清；
7.用8μm孔径的parylene滤膜过滤上清至低吸附管中；
8. 4℃、17000xg离心45-60min,弃上清；
9.用PBS重悬沉淀，转移至EP管中，得到重悬于PBS中的工程化迁移体。
IV.工程化迁移体的表征
1.总蛋白的测定
离心管2中的液体4℃、17000xg离心5min，弃上清后用PBS洗一遍沉淀，弃上清，用30μl 2％SDS裂解沉淀，于95℃金属浴煮样。用BCA法测定蛋白浓度，可换算出离心管1中的总蛋白量，并按照注射剂量换算出相应的重悬体积。
2.形态观察
取1μl步骤III得到的工程化迁移体样品，稀释至10μl后(为观察小泡的形态和膜表面相分离情况，可按照1:500-1:1000的比例在稀释液中加入WGA染料)滴加在预先用10μg/ml纤连蛋白包被的共聚焦小皿上，室温静置1h以上，在激光共聚焦显微镜下观察，结果如图11a所示。
另外，将步骤III得到的工程化迁移体用负染透射电镜和冷冻电镜观察，结果分别如图11b和11c所示。
3.流式计数
于步骤III-9或10时取1μl液体，稀释至100μl。如小泡呈现双色荧光，可直接用PBS稀释；如小泡只有单色荧光，可用1：500WGA染料进行染色，以提高分群的效果。
4.蛋白免疫印迹
于步骤III-1上清中取少量液体，100xg离心后沉淀用2％SDS裂解，作为细胞胞体样品；步骤IV-1得到的样品作为工程化迁移体样品。将细胞胞体(cell)样品与工程化迁移体(eMig)样品等蛋白量上样，进行western-blot，对多种细胞器的经典标志物进行检测：细胞核(组蛋白H3)，线粒体(Tim23)，内质网(钙联蛋白)，溶酶体(Lamp2)，细胞质(GAPDH)，细胞膜(Na+-K+-ATP酶)，细胞膜黏着斑(整合素α5)，Tspan4-GFP(GFP)，结果如图12所示。
结果显示，分离纯化得到的工程化迁移体上高度富集了Tspan4、整合素a5、Na+-K+-ATP酶等细胞膜蛋白，而几乎没有或少有来自胞内细胞器与可溶性蛋白的污染。
5.通透性评估
将分离纯化得到的工程化迁移体滴加在共聚焦小皿中，并在缓冲液中加入Cy5(一种无法通过完整膜的荧光染料)和右旋糖苷-TMR(Dex-TMR)以指示囊泡的通透性。运用激光共聚焦显微镜对液滴进行长时程拍摄，结果如图13a所示。可观察到在室温下工程化迁移体对Cy5(MW<1kDa)在一开始就几乎完全通透，对分子量更大的右旋糖苷-TMR(MW＝40kDa)则表现出缓慢通透的性质，工程化迁移体室温放置1.5h、6h、12h、24h、48h后对右旋糖苷-TMR通透比例的统计结果如图13b所示。
6.稳定性评估
为探究工程迁移体在室温下的稳定性，将分离纯化得到的工程化迁移体滴加在共聚焦小皿中，分别在第0、1、2、3、5和7天进行形态观察，结果如图14a所示。将平行保留的6份样品分别在第0、3、7、14天用以蛋白印迹检测所装载的OVA和mCherry(14b)，并用于免疫小鼠，检测血清中OVA-特异的抗体浓度(图14c)。
结果显示工程化迁移体在室温放置7-14天的时间内，囊泡形态完好，所负载蛋白量稳定，且作为疫苗免疫原性基本没有变化，表明工程化迁移体非常稳定。
7.胆固醇对工程化迁移体稳定性的影响
先前报道过胆固醇对迁移体的形成至关重要(Huang Y,Zucker B,Zhang S,Elias S,Zhu Y, Chen H,Ding T,Li Y,Sun Y,Lou J,Kozlov MM,Yu L.Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains.Nat Cell Biol.2019 Aug；21(8):991-1002)。为了探究胆固醇对工程化迁移体形成的影响，用可选择性地从质膜中提取胆固醇的甲基-β-环糊精(MβCD)处理分离纯化的工程化迁移体。在液滴中加入胆固醇抽提试剂MβCD(10mM)，然后进行激光共聚焦显微镜检查，结果如图15所示。发现大部分工程化迁移体在10min内就发生严重的形变与损坏，这说明胆固醇对于工程化迁移体的稳定性具有至关重要的作用。
工程化迁移体的药物装载
实施例8 膜蛋白在工程化迁移体上的装载
工程化迁移体的制备过程中可以将部分细胞膜转化为迁移体膜，因此，膜蛋白的递送可通过直接在生产细胞中转入编码目的基因及Tspan4的质粒来实现(图16a)。Tspan4的过表达配合工程化迁移体制备的其他步骤可大幅增加工程化迁移体的生产效率，而过表达的膜蛋白将大量富集在产生的工程化迁移体上，从而实现膜蛋白在工程化迁移体上的装载。可装载的膜蛋白包括各种细胞受体(如各种GPCR、PD-1、VEGFR等)、细胞外酶(如CD36、CD73)、离子通道、转运蛋白以及各种抗原(如S蛋白)等。
作为示例，验证了三种典型的质膜蛋白DAG1、Tgfbr1和PDCD1、一种膜结合蛋白Kras(图17)以及SARS-CoV-2的刺突(S)蛋白(图18)在工程化迁移体上的装载。
对于膜蛋白DAG1、Tgfbr1、PDCD1和Kras，向生产细胞NRK细胞中转染含有Tspan4-GFP和目的基因序列的质粒，为方便观察目的蛋白的亚细胞定位与表达量，通过接头将目的基因片段融合至mCherry标签。各融合蛋白的氨基酸序列和载体信息如下所示。
Tspan4-接头-GFP(载体：pB-Hygro-GFP(载体图谱如图19所示)，插入位点BsrGI+BamHI)：(SEQ ID NO：1)
Figure PCTCN2022133863-appb-000001
Figure PCTCN2022133863-appb-000002
Tspan4的氨基酸序列以下划线表示；GFP的氨基酸序列以加粗的斜体表示；Tspan4和GFP之间的氨基酸序列为接头序列(PG)。
DAG1-接头-mCherry(载体：pmCherry-N1(载体图谱如图20所示)，插入位点：EcoRI+KpnI)：(SEQ ID NO：2)
Figure PCTCN2022133863-appb-000003
DAG1的氨基酸序列以下划线表示；mCherry的氨基酸序列以加粗的斜体表示；DAG1和mCherry之间的氨基酸序列为接头序列(GDPPVAT)。
PDCD1-接头-mCherry(载体：pB-Hygro-mCherry(载体图谱如图21所示)，插入位点：BsrGI+MluI)：(SEQ ID NO：3)
Figure PCTCN2022133863-appb-000004
Figure PCTCN2022133863-appb-000005
PDCD1的氨基酸序列以下划线表示；mCherry的氨基酸序列以加粗的斜体表示；PDCD1和mCherry之间的氨基酸序列为接头序列(TVPRARDPPVAT)。
Tgfbr1-接头-mCherry(载体：pmCherry-N1(载体图谱如图20所示)，插入位点：EcoRI+KpnI)：(SEQ ID NO：4)
Figure PCTCN2022133863-appb-000006
Tgfbr1的氨基酸序列以下划线表示；mCherry的氨基酸序列以加粗的斜体表示；Tgfbr1和mCherry之间的氨基酸序列为接头序列(TVPRARDPPVAT)。
mCherry-接头-Kras(载体：pB-Hygro-mCherry(载体图谱如图21所示)，插入位点：BsrGI+MluI)：(SEQ ID NO：5)
Figure PCTCN2022133863-appb-000007
mCherry的氨基酸序列以下划线表示；Kras的氨基酸序列以加粗的斜体表示；mCherry和Kras之间的氨基酸序列为接头序列(SGLRSRG)。
转染后针对质粒上所带的抗性对细胞进行药杀筛选，建立稳定表达各种经mCherry标记的膜蛋白的细胞系。然后用实施例5中针对NRK细胞的低渗处理条件诱导细胞系产生已转载相应膜蛋白的工程化迁移体，然后进行激光共聚焦显微镜观察。结果显示，四种目的膜蛋白都在工程化迁移体上正确定位(图17)，表明膜蛋白可以整合到工程化迁移体上。
对于S蛋白，向生产细胞MC38细胞中转染含有Tspan4-GFP和目的基因序列的质粒，为方便观察目的蛋白的亚细胞定位与表达量，在目的基因片段后融合mCherry标签。转染后针对质粒上所带的抗性对细胞进行药杀筛选，建立稳定表达mCherry标记的S蛋白的细胞系。然后用实施例5中针对MC38细胞的低渗处理条件诱导细胞系产生已转载S蛋白的工程化迁移体，然后进行激光共聚焦显微镜观察。结果显示，S蛋白在工程化迁移体上正确定位(图18)，表明S蛋白可以有效整合到工程化迁移体上。
实施例9 可溶性蛋白在工程化迁移体上的装载
由于工程化迁移体是渗漏的，因此其可能不用于直接递送胞质可溶性蛋白。为了实现可溶性蛋白的递送，可通过使生产细胞表达目的蛋白-膜蛋白的融合蛋白将可溶性蛋白锚定在膜上，从而防止可溶性蛋白的渗漏(图16a及图16b)。例如，可对膜蛋白突触融合蛋白(syntaxin-2，STX2)的N端进行改造，去除其胞内端功能，以得到截短的STX2(t-STX2)。然后将可溶性蛋白连接到t-STX2处于胞外的C端，以形成可溶蛋白-t-STX2融合蛋白(图16b)，从而使可溶性蛋白作为人造的质膜定位融合蛋白进行表达。t-STX2自身的拓扑学使得可溶性目的蛋白最终悬挂在细胞膜外侧，从而例如更利于该蛋白作为完整抗原被免疫系统识别。
除STX2之外，多种其他膜蛋白也常被用做膜锚定蛋白，例如Tetraspanin-4，CD81,CD9,CD63,PDGFR,Lamp2b等。
作为示例，验证了可溶性蛋白OVA在工程化迁移体上的装载。向生产细胞MC38细胞中转染含有Tspan4-GFP和t-STX2-OVA-mCherry编码序列的质粒。t-STX2和OVA的融合蛋白的氨基酸序列和载体信息如下所示。
t-STX2-接头-OVA-接头-mCherry(载体pmCherry-N1(载体图谱如图20所示)，插入位点：HindIII+BamHI)：(SEQ ID NO：6)
Figure PCTCN2022133863-appb-000008
t-STX2的氨基酸序列以粗体表示；OVA的氨基酸序列以下划线表示；mCherry的氨基酸序列以加粗的斜体表示。
转染后针对质粒上所带的抗性对细胞进行药杀筛选，建立稳定表达mCherry标记的t-STX2-OVA融合蛋白的细胞系。然后用实施例5中针对MC38细胞的低渗处理条件诱导细胞系产生已转载t-STX2-OVA融合蛋白的工程化迁移体，然后进行激光共聚焦显微镜观察，结果如图22所示。
结果显示，t-STX2-OVA融合蛋白在工程化迁移体上正确定位，表明可溶性蛋白OVA成功装载到工程化迁移体上。
实施例10 其他目的分子在工程化迁移体上的装载
小分子药物、小核酸药物、肽段等目的分子在工程化迁移体中的装载可通过抗原-抗体、 受体-配体、生物素-亲和素结合系统对目的分子和生产细胞的改造来实现(图16a)。例如，可利用HaloTag与其人工合成配体的受体-配体相互作用(Los,G.V.,Encell,L.P.,McDougall,M.G.,Hartzell,D.D.,Karassina,N.,Zimprich,C.,Wood,M.G.,Learish,R.,Ohana,R.F.,Urh,M.,Simpson,D.,Mendez,J.,Zimmerman,K.,Otto,P.,Vidugiris,G.,Zhu,J.,Darzins,A.,Klaubert,D.H.,Bulleit,R.F.,&Wood,K.V.(2008).HaloTag:a novel protein labeling technology for cell imaging and protein analysis.ACS chemical biology,3(6),373–382.)来实现目的分子在工程化迁移体上的装载。首先，构建编码膜蛋白Tspan4和受体蛋白HaloTag的融合蛋白Tspan4-HaloTag的质粒。然后，将该质粒转染到工程化迁移体生产细胞中，以实现对生产细胞的改造。改造后的生产细胞经诱导产生工程化迁移体，分离纯化后的工程化迁移体在膜上包含受体蛋白HaloTag。通过将目的分子与HaloTag的配体进行偶联，形成目的分子-配体偶联物，从而实现对目的分子的改造。然后，将目的分子-配体偶联物与在膜上包含HaloTag的工程化迁移体在体外共孵育，通过HaloTag与其配体的相互作用将目的分子固定到工程化迁移体的膜和/或迁移体内部。HaloTag与其配体的共价结合特异、高效且不可逆。
除了Tspan4之外，其他膜蛋白，例如本文其他地方所述的膜蛋白也可以作为膜锚定蛋白用于目的分子在工程化迁移体上的装载。除了HaloTag及其配体，其他抗原-抗体、受体-配体、生物素-亲和素等结合系统也可用于目的分子在工程化迁移体上的装载，例如CP05与CD63结合系统(X.Gao,N.Ran,X.Dong,B.Zuo,R.Yang,Q.Zhou,H.M.Moulton,Y.Seow,H.Yin,Anchor peptide captures,targets,and loads exosomes of diverse origins for diagnostics and therapy,Sci.Transl.Med.10(2018))，先将受体CD63加载到工程化迁移体膜上，然后利用CD63与其配体CP05的结合，将目的分子-CP05偶联物装载到含有CD63的工程化迁移体膜上。选择抗原-抗体、受体-配体、生物素-亲和素结合系统时可以尽量避免使用拥有大量体内自然配体的受体(例如PD-1)。
为了对该装载方法进行概念性验证，向生产细胞NRK细胞中转染含有Tspan4-HaloTag-GFP编码序列的质粒，转染含有Tspan4-GFP编码序列的质粒作为对照。Tspan4和HaloTag的融合蛋白的氨基酸序列和载体信息如下所示。
Tspan4-接头-GFP-接头-Halo(载体：pB-Hygro-GFP(载体图谱如图19所示)，插入位点：BsrGI+MluI)：(SEQ ID NO：7)
Figure PCTCN2022133863-appb-000009
Figure PCTCN2022133863-appb-000010
Tspan4的氨基酸序列以粗体表示；GFP的氨基酸序列以下划线表示；Halo的氨基酸序列以加粗的斜体表示。
转染后针对质粒上所带的抗性对细胞进行药杀筛选，建立稳定表达的细胞系。然后用实施例5中针对NRK细胞的低渗处理条件诱导细胞系产生工程化迁移体。向工程化迁移体添加带荧光的HaloTag配体-TMR染料，室温共孵育15min后用不含染料的缓冲液洗两遍，滴加于共聚焦小皿中室温静置5h后进行激光共聚焦显微镜观察，结果如图23所示。
结果显示，HaloTag配体-TMR与Tspan4-HaloTag-GFP共定位在工程化迁移体上，表明目的分子可以通过HaloTag及其配体的相互作用装载到工程化迁移体上，并且这种装载高效、特异且不可逆。
小核酸药物除了可以通过上述抗原-抗体、受体-配体、生物素-亲和素偶联方式装载到工程化迁移体之外，还可通过多种其他修饰方式装载到工程化迁移体膜上。例如，可以将化学修饰后的siRNA或反义寡核苷酸(ASO)与胆固醇偶联，通过胆固醇与细胞膜的相容性将小核酸药物装载到工程化迁移体的膜上(S.S.Yerneni,S.Lathwal,P.Shrestha,H.Shirwan,K.Matyjaszewski,L.Weiss,E.S.Yolcu,P.G.Campbell,S.R.Das,Rapid on-demand extracellular vesicle augmentation with versatile oligonucleotide tethers,ACS Nano 13(2019)10555–10565)。该方式不需要对迁移体进行额外修饰，是一种快速经济的装载方式。还可以通过点击化学的方式将siRNA偶联物固定到膜上(T.Tian,H.X.Zhang,C.P.He,S.Fan,Y.L.Zhu,C.Qi,N.P.Huang, Z.D.Xiao,Z.H.Lu,B.A.Tannous,J.Gao,Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy,Biomaterials 150(2018)137–149)。该方式需要先将siRNA与肽段偶联，再通过点击化学的方式将肽段偶联到膜上。
mRNA药物在工程化迁移体中的装载可以通过mRNA结合蛋白来实现。常用的mRNA结合蛋白有L7Ae(Kojima,R.,Bojar,D.,Rizzi,G.,Hamri,G.C.,El-Baba,M.D.,Saxena,P.,
Figure PCTCN2022133863-appb-000011
S.,Tan,K.R.,&Fussenegger,M.(2018).Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.Nature communications,9(1),1305；Zhitnyuk,Y.,Gee,P.,Lung,M.,Sasakawa,N.,Xu,H.,Saito,H.,&Hotta,A.(2018).Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles.Biochemical and biophysical research communications,505(4),1097–1102)和MS2BP(Prel,A.,Caval,V.,Gayon,R.,Ravassard,P.,Duthoit,C.,Payen,E.,Maouche-Chretien,L.,Creneguy,A.,Nguyen,T.H.,Martin,N.,Piver,E.,Sevrain,R.,Lamouroux,L.,Leboulch,P.,Deschaseaux,F.,Bouillé,P.,Sensébé,L.,&Pagès,J.C.,Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles,Mol.Ther.-Meth.Clin.Dev.2(2015),15039)。首先，构建编码膜蛋白-mRNA结合蛋白融合蛋白的质粒，例如Tspan4-L7Ae或Tspan4-MS2。接下来，构建mRNA表达质粒，包含蛋白结合位点，L7Ae的蛋白结合位点为C/D Box，MS2BP蛋白结合位点为MS2 Stem Loop(MS2SL)。然后，将融合蛋白质粒和mRNA质粒共同转入生产细胞，诱导生产细胞产生工程化迁移体，分离纯化后的迁移体在其膜上包含目的mRNA分子。
实施例11 装载SARS-CoV-2刺突蛋白的工程化迁移体在小鼠体内诱导免疫应答
实验流程如图24a所示。实验动物为C57BL/6品系的8周龄雌鼠，每组5只。具体分组为：
I.50μg S1重组蛋白(SinoBiological)+铝佐剂(thermo scientific)，腹腔注射(i.p.)；
II.10μg S1重组蛋白，尾静脉注射(i.v.)；
III.总蛋白20μg的仅表达Tspan4-GFP的工程化迁移体(NC-eMig)，尾静脉注射，作为阴性对照；
IV.总蛋白20μg的共表达Tspan4-GFP与S蛋白-mCherry的工程化迁移体(S-eMig)，尾静脉注射。
利用蛋白免疫印迹对S-eMig组中S1蛋白含量进行半定量分析(图24b)。左起前6个泳道分别为1、2、5、10、20和50ng的S1重组蛋白，第7-10泳道分别为总蛋白量1μg的NC-eMig或S-eMig(eMig-1和eMig-2代表两次独立实验的样品)。对比各泳道条带亮度，可以换算出一剂(20μg)S-eMig中S1蛋白为约100ng，远小于铝佐剂组所用的S1重组蛋白量(50μg)。
小鼠在第0天接受1次免疫，在第14天被处死后取外周血，用ELISA检测血清中S蛋白特异性IgG抗体的浓度，结果如图24c所示。发现单独注射S1蛋白组(第II组)和NC-eMig阴性对照组(第III组)都无法促进抗体产生；S1蛋白+铝佐剂组(第I组)与S-eMig组(第IV组)都能有效地促进抗体产生，两者的免疫效果大致相似。这表明与传统的佐剂加免疫原性蛋白的免疫方式相比，工程化迁移体在没有佐剂且蛋白量很小的情况下引起相似的抗体反应，是更为高效的递送载体，能够有效地促进免疫应答。
工程化迁移体与已知的细胞外囊泡的比较
实施例12 工程化迁移体与天然迁移体的比较
对同一细胞来源的对照迁移体(生产纯化流程详见Ma et al,Cell Res.2015)和工程化迁移体(生产纯化流程见图9)进行生产和纯化，进行多方面的比较。
过表达Tspan4-GFP的NRK细胞在低渗处理后，用2.5％戊二醛固定并用扫描电镜观察(图25a)。运用转盘共聚焦显微镜对过表达Tspan4-GFP的NRK细胞产生工程化迁移体的过程进行4D拍摄，可以观察到在低渗处理的过程中，细胞胞体膨胀、底面收缩并产生大量丝状结构；在丝状结构上又生长出工程化迁移体(图25b)。
天然产生的迁移体是由于细胞中心点发生位移，迁移体所附着的收缩丝只位于细胞迁移的尾端，且迁移体数量较少(图26a)；工程化迁移体是通过不同方式诱导细胞膜的相对位移，所附着的收缩丝可以在细胞的各个方位都有分布，每条收缩丝上和单个细胞的迁移体数量相比对照迁移体可以有显著的升高(图26b)。过表达Tspan4-GFP的NRK细胞，用四甲基罗丹明标记的WGA染色后，可清晰看到对照迁移体(图26c上图)和工程化迁移体(图26c下图)的差别。电镜下天然迁移体内部有不同数量的分泌囊泡(图26d；摘自Ma et al,Cell Res.2015)，而工程化迁移体电镜下内容物有限或缺乏(图26e)。同一细胞来源的工程化迁移体和对照迁移体(对照迁移体又可称为非低渗迁移体或天然迁移体)分别做质谱分析，相比天然迁移体，工程化迁移体中近4000个蛋白的缺失(图26f)，其中部分蛋白在天然迁移体中相比 于胞体有明显富集(图26g)；另外工程化迁移体中1350个天然迁移体中缺失的蛋白的表达，一些工程化迁移体膜上有更多表达的包括Ezrin在内的ERM家族成员蛋白和Fxyd5,Atp1β1蛋白(图26h)。(参考文献：Ma L,Li Y,Peng J,Wu D,Zhao X,Cui Y,Chen L,Yan X,Du Y,Yu L.Discovery of the migrasome,an organelle mediating release of cytoplasmic contents during cell migration.Cell Res.2015 Jan；25(1):24-38.doi:10.1038/cr.2014.135.)
实施例13 工程化迁移体区别于其他低渗囊泡
扫描电镜下NRK细胞产生的工程化迁移体(图25a)有别于报导的低渗囊泡(图25c)。工艺和特性上主要区别有以下几个方面：
1)工程化迁移体产生于细胞周围的丝上，而非Cohen等人的实验数据所显示的产生于细胞上表面。
2)工程化迁移体的尺寸为微米级，很少有直径超过5μm的。而Cohen等人利用低渗透压制作的囊泡可大至20μm。
3)工程化迁移体的低渗诱导过程为温和地逐级降低(目前几种细胞系中，最终渗透压不低于40％)，而Cohen等人采用的是非常剧烈的5％PBS处理，在vesiculation中又使用了118％的高渗透压缓冲液。(参考文献:Cohen S,Ushiro H,Stoscheck C,Chinkers MA native 170 000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.J Biol Chem 257:1523-1531.)
实施例14 工程化迁移体区别于细胞外小囊泡/外泌体
迁移体和外泌体同是细胞外囊泡，但产生的机制以及囊泡大小(外泌体一般为50-150nm等特性不同。对同一细胞来源的细胞外小囊泡(sEVs)/外泌体(生产纯化流程见图27a)和工程化迁移体(生产纯化流程见图9)进行生产、纯化和不同方面的比较。纯化后外泌体的NTA检测(图27b)和透射电镜(TEM)观察(图27c)证实外泌体的大小和形态符合文献描述。此外免疫印迹实验证实纯化的外泌体富集公认的细胞外小囊泡/外泌体标志物，例如tsg101，Alix，CD63，CD81，而工程化迁移体中并不富集这些细胞外小囊泡/外泌体标志物。与此同时，工程化迁移体则更加富集钠/钾ATP酶，Lamp2等迁移体标记物(图27d)，表明有效的生产纯化了同一细胞来源的迁移体和外泌体，而两者的蛋白组成和标记物是有着明显的不同的。
工程化迁移体生产方式：
提前一天将过表达Tspan4-GFP的MC-38细胞铺在纤连蛋白处理后的细胞培养皿中培养；
实验当天，弃上清，用40％KDPBS清洗细胞；加入含1mg/ml BSA的h-KDPBS(h-KDPBS-BSA),130rpm摇3min；收集上清液；加入h-KDPBS-BSA吹打培养瓶底面，将所收集的液体与上一步的上清液合并；4℃、300xg离心10min,保留上清；4℃、500xg离心10min,保留上清；用8um孔径的parylene滤膜过滤上清至50ml低吸附管中；4℃、17000xg离心45-60min,弃上清；用h-KDPBS-BSA重悬沉淀，转移至EP管中(离心管1)，加入等体积PBS-BSA；此时取约总体积的1/50于另一个离心管(离心管2)，用于测量蛋白浓度；4℃、17000xg离心15-20min，弃上清后加PBS，于注射前重悬沉淀，得到重悬于PBS中的工程化迁移体。
细胞外小囊泡的生产和纯化：
以～20％的细胞汇合度种下过表达小鼠Tspan4-GFP蛋白的MC-38细胞系，培养72小时；收集培养基，4000x rcf离心10min去除胞体，收集上清；使用0.45微米滤膜过滤上清；过滤后的上清使用70kD超滤管进行浓缩；向浓缩后的上清加入1mM MgCl2和20U/mL的Benzonase，室温处理过夜；使用0.22um滤膜过滤；过滤后的液体使用超速离心机在4℃以140,000xg离心1小时；弃上清，收集沉淀，以PBS重悬；将重悬后的沉淀与60％碘克沙醇混合混合，并加入超离管底部；向超离管内依次加入9mL 30％碘克沙醇、5mL 23％碘克沙醇、6mL 18％碘克沙醇；150,000xg离心16h，轻轻吸取收集各密度梯度间组分(共4个组分，F1-F4)；将收集到的外泌体组分与30mL PBS混合，16,000xg离心1小时，保留上清；将上清用0.2um滤膜过滤后，4℃下150000xg离心2hr，将pellet重悬至1x10 13颗粒/mL冻存待用。
通过在同一细胞系中对细胞外小囊泡/外泌体和工程化迁移体的生产和纯化，本申请人发现工程化迁移体的单位产量要远超细胞外小囊泡，且无需超速离心、超速密度梯度离心等方式进行纯化，生产速度大幅增加。使用等量细胞进行培养时，工程化迁移体的产量约为细胞外小囊泡/外泌体产量的35倍(图27e)。
同时，在使用等染料量的细胞外小囊泡和工程化迁移体在C57BL/6小鼠进行尾静脉注射5分钟后，工程化迁移体和细胞外小囊泡在各器官中的积累相差无几。而随着时间增加，在注射后的4小时和24小时，工程化迁移体比起细胞外小囊泡在肝、脾、肺中的积累有着明显增加，这也说明两者在体内的代谢和组织积累不同，在携带同等染料的情况下，工程化迁移体的递送效果更好(图27f)。
对同一细胞系中对细胞外小囊泡/外泌体和工程化迁移体进行质谱分析，PCA分析显示3个工程化迁移体样本高度相似，与3个细胞外小囊泡/外泌体样本明显不同(图28a)，信号通 路分析热图(图28b)和最为富集的10个蛋白(图28c)显示细胞外小囊泡/外泌体和工程化迁移体之间差别明显。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (113)

  1. 一种制备迁移体的方法，所述方法包括使细胞产生相对位移继而产生源自所述细胞的迁移体。
  2. 根据权利要求1所述的方法，其还包括分离所述细胞所产生的迁移体。
  3. 根据权利要求1-2中任一项所述的方法，其包括对所述细胞进行低渗透压处理。
  4. 根据权利要求3所述的方法，其中所述低渗透压处理包括使所述细胞置于低渗缓冲溶液中。
  5. 根据权利要求3-4中任一项所述的方法，其中所述低渗透压处理包括使所述细胞置于缓冲溶液中，并将所述缓冲溶液的渗透压降低成为低渗缓冲液。
  6. 根据权利要求5所述的方法，其中所述降低包括线性降低和/或阶梯式降低。
  7. 根据权利要求5-6中任一项所述的方法，其中所述低渗缓冲溶液的渗透压低于约305mOsmol/L。
  8. 根据权利要求5-7中任一项所述的方法，其中所述低渗缓冲溶液的渗透压为约10mOsmol/L至约274.5mOsmol/L。
  9. 根据权利要求1-8中任一项所述的方法，其包括破坏所述细胞的细胞骨架。
  10. 根据权利要求9所述的方法，其中破坏所述细胞的细胞骨架包括使所述细胞与破坏细胞骨架的试剂接触。
  11. 根据权利要求10所述的方法，其中所述破坏细胞骨架的试剂包括微丝和/或微管解聚剂。
  12. 根据权利要求10-11中任一项所述的方法，其中所述破坏细胞骨架的试剂包括Latrunculin A，Latrunculin B，细胞松弛素A，细胞松弛素B，细胞松弛素C，细胞松弛素D和/或细胞松弛素E。
  13. 根据权利要求1-12中任一项所述的方法，其包括抑制所述细胞的细胞体积调节功能。
  14. 根据权利要求13所述的方法，其中抑制所述细胞的细胞体积调节功能包括使所述细胞中体积调节蛋白的数量和/或功能下降。
  15. 根据权利要求14所述的方法，其中所述体积调节蛋白包括体积调节离子通道和/或转运蛋白。
  16. 根据权利要求15所述的方法，其中所述体积调节离子通道包括体积调节阴离子通道VRAC和/或体积调节阳离子通道VRCC。
  17. 根据权利要求16所述的方法，其中所述体积调节阴离子通道VRAC包括SWELL1或其功能活性片段。
  18. 根据权利要求16-17中任一项所述的方法，其中所述体积调节阳离子通道VRCC包括TRPV4，TRPM3和/或其功能活性片段。
  19. 根据权利要求15-18中任一项所述的方法，其中所述转运蛋白包括协同转运蛋白。
  20. 根据权利要求19所述的方法，其中所述协同转运蛋白包括KCC1、KCC3和/或KCC4。
  21. 根据权利要求13-20中任一项所述的方法，其中抑制所述细胞的细胞体积调节功能包括将所述细胞置于具有减弱的体积调节能力的缓冲溶液中。
  22. 根据权利要求21所述的方法，其中所述缓冲溶液含有增加的阳离子。
  23. 根据权利要求22所述的方法，其中所述阳离子包括K +，Na +，Cs +，Li +，Ca 2+，Mg 2+，Ba 2+，Mn 2+，Fe 2+，Ni 2+，Zn 2+，Al 3+，Fe 3+,CH 3NH 3 +，C 2H 5NH 3 +，(CH 3) 2NH 2 +，(C 2H 5) 2NH 2 +，(C 2H 5) 3N +，氨离子和/或胆碱离子。
  24. 根据权利要求21所述的方法，其中所述缓冲溶液含有增加的阴离子。
  25. 根据权利要求24所述的方法，其中所述阴离子包括Br -，Cl -，I -，F -，OH -，HCO 3 -，H 2PO 4 -，NO 2 -，NO 3 -，CN -，HPO 4 2-，CO 3 2-，SO 4 2-和/或PO 4 3-。
  26. 根据权利要求1-25中任一项所述的方法，其中所述方法包括使所述细胞脱离其粘附表面。
  27. 根据权利要求1-26中任一项所述的方法，其中所述方法包括使所述细胞的细胞膜与其粘附表面之间发生相对位移。
  28. 根据权利要求1-27中任一项所述的方法，其还包括增加所述细胞中tetraspanin蛋白，其功能性片段和/或功能性变体的数量和/或功能。
  29. 根据权利要求28所述的方法，其包括使所述细胞过表达所述tetraspanin蛋白，其功能性片段和/或功能性变体。
  30. 根据权利要求28-29中任一项所述的方法，其中所述tetraspanin蛋白选自：Tspan1、Tspan2、Tspan3、Tspan4、Tspan5、Tspan6、Tspan7、Tspan8、Tspan9、Tspan10、Tspan11、Tspan12、Tspan13、Tspan14、Tspan15、Tspan16、Tspan17、Tspan18、Tspan19、Tspan20(UPK1B)、Tspan21(UPK1A)、Tspan22(PRPH2)、Tspan23(ROM1)、Tspan24(CD151)、Tspan25(CD53)、Tspan26(CD37)、Tspan27(CD82)、Tspan28(CD81)、Tspan29(CD9)、Tspan30(CD63)、Tspan31、Tspan32和Tspan33。
  31. 根据权利要求1-30中任一项所述的方法，所述方法还包括减小所述迁移体的尺寸。
  32. 根据权利要求31所述的方法，其中所述减小所述迁移体的尺寸包括使用过滤器或挤压器挤压所述迁移体。
  33. 根据权利要求32所述的方法，其中所述过滤器或挤压器的孔径为约30nm至约10000nm。
  34. 根据权利要求31-33中任一项所述的方法，其中所述迁移体的尺寸为约50nm至约8000nm。
  35. 根据权利要求1-34中任一项所述的方法，其中所述迁移体产生于所述细胞的收缩丝。
  36. 根据权利要求1-35中任一项所述的方法，其中所述迁移体的膜上富集有钠/钾ATP酶和/或其功能性片段。
  37. 根据权利要求1-36中任一项所述的方法，其中所述迁移体的膜上富集有整合素和/或其功能性片段。
  38. 根据权利要求1-37中任一项所述的方法，其中所述迁移体的膜上富集有tetraspanin蛋白，其功能性变体和/或其功能性片段。
  39. 根据权利要求1-38中任一项所述的方法，其中所述迁移体的膜上富集有胆固醇。
  40. 根据权利要求1-39中任一项所述的方法，其中所述迁移体富集有膜微结构域。
  41. 根据权利要求1-40中任一项所述的方法，其中所述迁移体在体外产生或离体产生。
  42. 根据权利要求1-41中任一项所述的方法，其中与相应细胞产生的天然迁移体相比，所述迁移体的内容物至少部分减少或缺失。
  43. 根据权利要求42所述的方法，其中所述至少部分减少的内容物包含腔内囊泡。
  44. 根据权利要求1-43中任一项所述的方法，其为体外或离体方法。
  45. 根据权利要求1-44中任一项所述的方法，其中所述细胞为经体外培养的细胞。
  46. 根据权利要求1-45中任一项所述的方法，其中所述细胞为经悬浮培养或经贴壁培养的细胞。
  47. 根据权利要求1-46中任一项所述的方法，其中所述细胞包括原代细胞。
  48. 根据权利要求47所述的方法，其中所述原代细胞包括源自生物体的组织细胞，所述生物体包括人，猴，小鼠，大鼠，兔，鸡和/或昆虫。
  49. 根据权利要求47-48中任一项所述的方法，其中所述原代细胞包括肝细胞，脾细胞，肾细胞，组织巨噬细胞，脑胶质细胞，破骨细胞，骨髓细胞，白细胞，成纤维细胞和/或脂肪细胞。
  50. 根据权利要求49所述的方法，其中所述白细胞包括B细胞，T细胞，NK细胞，树突状细胞，中性粒细胞和/或巨噬细胞。
  51. 根据权利要求1-50中任一项所述的方法，其中所述细胞包括肿瘤细胞。
  52. 根据权利要求51所述的方法，其中所述肿瘤细胞包括肿瘤细胞系，源自患者的原代或经有限传代的肿瘤细胞，肿瘤基质细胞，和/或肿瘤类器官。
  53. 根据权利要求1-52中任一项所述的方法，其中所述细胞包括CHO细胞、CHO-K1细胞、HEK293细胞、HEK293T细胞、HEK293FT细胞、HEK293F细胞、Vero细胞、NRK细胞、L929细胞、MC38细胞、4T1细胞、DC2.4细胞、MGC803细胞、Jurkat细胞、NK-92MI细胞、BJ细胞和/或HepG2细胞。
  54. 根据权利要求1-53中任一项所述的方法，其中所述细胞包括白细胞、干细胞和/或成纤维细胞。
  55. 根据权利要求54所述的方法，其中所述干细胞包含间充质干细胞。
  56. 根据权利要求1-55中任一项所述的方法制备的迁移体。
  57. 体外或离体制备的迁移体，其中所述迁移体的尺寸为约50nm至约8000nm。
  58. 根据权利要求57所述的迁移体，其在体外产生于细胞的收缩丝。
  59. 根据权利要求57-58中任一项所述的迁移体，其中所述迁移体的膜上富集有钠/钾ATP酶和/或其功能性片段。
  60. 根据权利要求57-59中任一项所述的迁移体，其中所述迁移体的膜上富集有整合素和/或其功能性片段。
  61. 根据权利要求57-60中任一项所述的迁移体，其中所述迁移体的膜上富集有tetraspanin蛋白，其功能性变体和/或其功能性片段。
  62. 根据权利要求57-61中任一项所述的迁移体，其中所述迁移体的膜上富集有胆固醇。
  63. 根据权利要求57-62中任一项所述的迁移体，其中所述迁移体富集有膜微结构域。
  64. 根据权利要求57-63中任一项所述的迁移体，其中与相应细胞产生的天然迁移体相比，所述迁移体的内容物至少部分减少或缺失。
  65. 根据权利要求64所述的迁移体，其中所述至少部分减少的内容物包含腔内囊泡。
  66. 一种迁移体用于递送外源荷载的用途。
  67. 根据权利要求66所述的用途，其中所述迁移体包含权利要求57-65中任一项所述的迁移体。
  68. 递送系统，其包含迁移体和一种或多种外源荷载。
  69. 根据权利要求68所述的递送系统，其中所述外源荷载直接或间接结合、连接或嵌入至所述迁移体的膜和/或内部。
  70. 根据权利要求68-69中任一项所述的递送系统，其中所述迁移体包括权利要求57-65中任一项所述的迁移体。
  71. 根据权利要求68-70中任一项所述的递送系统，其中所述迁移体源自细胞。
  72. 根据权利要求68-71中任一项所述的递送系统，其中所述外源荷载包含一种或多种靶向物质和/或治疗活性物质。
  73. 根据权利要求68-72中任一项所述的递送系统，其中所述外源荷载包含蛋白质，脂质，多核苷酸，小分子化合物，复合物、多糖、聚合物、纳米颗粒、微米颗粒和/或细胞器。
  74. 根据权利要求68-73中任一项所述的递送系统，其中所述外源荷载包括膜蛋白，可溶性 蛋白和/或多肽。
  75. 根据权利要求68-74中任一项所述的递送系统，其中所述外源荷载包括DNA和/或RNA。
  76. 根据权利要求68-75中任一项所述的递送系统，其中所述外源荷载包括抗体或其抗原结合抗体片段、整合素或其片段、免疫原性蛋白、细胞因子、趋化因子、受体蛋白或其片段、酶、抑癌基因产物、siRNA、microRNA、反义寡核苷酸ASO、mRNA、DNA、基因编辑工具和/或细胞毒性剂。
  77. 根据权利要求68-76中任一项所述的递送系统，其中所述外源荷载包括PAMP、DAMP、CD47、CD24、IL-12、IL-15、凝血因子VII、凝血因子VIII、凝血因子IX和/或它们的功能活性片段。
  78. 根据权利要求68-77中任一项所述的递送系统，其中所述外源荷载通过基因编辑、外源表达、液态固态转换、膜融合、电荷吸附、物理吸附和/或化学连接而与所述迁移体直接或间接结合。
  79. 根据权利要求68-78中任一项所述的递送系统，其中所述外源荷载通过与所述迁移体的膜组分直接或间接连接而结合或嵌入至所述迁移体。
  80. 根据权利要求79所述的递送系统，其中所述迁移体的膜组分包含膜蛋白、胆固醇、磷脂、糖蛋白上的糖链和/或多糖。
  81. 根据权利要求79-80中任一项所述的递送系统，其中所述间接连接包括通过点击化学反应连接。
  82. 根据权利要求79-81中任一项所述的递送系统，其中所述间接连接包括提供与结合对的第一成员连接的所述外源荷载，并使其与所述迁移体接触，所述迁移体的膜上包含所述结合对的第二成员，其中所述第一成员能够与所述第二成员结合。
  83. 根据权利要求82所述的递送系统，其中所述结合对的第一成员和第二成员选自抗原及其抗体；受体及其配体；生物素和亲和素；HaloTag及其配体；以及CP05和CD63。
  84. 根据权利要求68-83中任一项所述的递送系统，其中所述外源荷载作为膜蛋白表达在所述迁移体的膜的内表面或外表面。
  85. 根据权利要求68-84中任一项所述的递送系统，其中所述外源荷载作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体的膜的内表面或外表面。
  86. 根据权利要求68-85中任一项所述的递送系统，其中所述外源荷载通过基因编辑和/或外源表达而作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体的膜的内表面或外表面。
  87. 制备递送系统的方法，所述方法包括提供迁移体，和使所述迁移体携带外源荷载。
  88. 根据权利要求87所述的方法，其中所述迁移体为经分离或纯化的迁移体。
  89. 根据权利要求87-88中任一项所述的方法，其中所述使迁移体携带外源荷载包括使所述外源荷载直接或间接连接或嵌入至所述迁移体的膜和/或所述迁移体的内部。
  90. 根据权利要求87-89中任一项所述的方法，其还包括由细胞分离或纯化所述迁移体。
  91. 根据权利要求87-90中任一项所述的方法，其包括提供所述外源荷载与结合对的第一成员的复合物；使细胞产生迁移体，所述迁移体包含结合对的第二成员；以及使所述迁移体与所述复合物接触以形成所述递送系统。
  92. 根据权利要求87-91中任一项所述的方法，其中所述迁移体包括权利要求57-65中任一项所述的迁移体。
  93. 根据权利要求87-92中任一项所述的方法，其中所述迁移体源自细胞。
  94. 根据权利要求87-93中任一项所述的方法，其中所述外源荷载包含一种或多种靶向物质和/或治疗活性物质。
  95. 根据权利要求87-94中任一项所述的方法，其中所述外源荷载包含蛋白质，脂质，多核苷酸，小分子化合物，复合物、多糖、聚合物、纳米颗粒、微米颗粒和/或细胞器。
  96. 根据权利要求87-95中任一项所述的方法，其中所述外源荷载包括膜蛋白，可溶性蛋白和/或多肽。
  97. 根据权利要求87-96中任一项所述的方法，其中所述外源荷载包括DNA和/或RNA。
  98. 根据权利要求87-97中任一项所述的方法，其中所述外源荷载包括抗体或其抗原结合抗体片段、整合素或其片段、免疫原性蛋白、细胞因子、趋化因子、受体蛋白或其片段、酶、抑癌基因产物、siRNA、microRNA、反义寡核苷酸ASO、mRNA、DNA、基因编辑工具和/或细胞毒性剂。
  99. 根据权利要求87-98中任一项所述的方法，其中所述外源荷载包括PAMP、DAMP、CD47、CD24、IL-12、IL-15、凝血因子VII、凝血因子VIII、凝血因子IX和/或它们的功能活性片段。
  100. 根据权利要求87-99中任一项所述的方法，其中所述外源荷载通过基因编辑、外源表达、液态固态转换、膜融合、电荷吸附、物理吸附和/或化学连接而与所述迁移体直接或间接结合。
  101. 根据权利要求87-100中任一项所述的方法，其中所述外源荷载通过与所述迁移体的膜组分直接或间接结合而连接或嵌入至所述迁移体。
  102. 根据权利要求101所述的方法，其中所述迁移体的膜组分包含膜蛋白、胆固醇、磷脂、糖蛋白上的糖链和/或多糖。
  103. 根据权利要求101-102中任一项所述的方法，其中所述间接结合包括通过点击化学反应连接。
  104. 根据权利要求101-103中任一项所述的方法，其中所述间接结合包括提供与结合对的第一成员连接的所述外源荷载，并使其与所述迁移体接触，所述迁移体的膜上包含所述结合对的第二成员，其中所述第一成员能够与所述第二成员结合。
  105. 根据权利要求104所述的方法，其中所述结合对的第一成员和第二成员选自抗原及其抗体；受体及其配体；生物素和亲和素；HaloTag及其配体；以及CP05和CD63。
  106. 根据权利要求87-105中任一项所述的方法，其中所述外源荷载作为膜蛋白表达在所述迁移体膜的内表面或外表面。
  107. 根据权利要求87-106中任一项所述的方法，其中所述外源荷载作为与膜蛋白或其部分融合的融合蛋白表达在所述迁移体膜的内表面或外表面。
  108. 制备递送系统的方法，所述方法包括：使细胞表达mRNA；使所述细胞产生迁移体，所述迁移体包含mRNA结合蛋白，且所述mRNA通过所述mRNA结合蛋白连接至所述迁移体。
  109. 制备递送系统的方法，所述方法包括：使细胞在细胞膜上表达外源荷载；和使所述细胞产生迁移体，所述迁移体包含所述外源荷载。
  110. 根据权利要求109所述的方法，其中所述外源荷载为蛋白质。
  111. 根据权利要求110所述的方法，其中所述蛋白质为膜蛋白。
  112. 根据权利要求110-111中任一项所述的方法，其中所述蛋白质为可溶性蛋白，并且其与膜蛋白或其部分融合而形成融合蛋白。
  113. 组合物，其包含权利要求57-65中任一项所述的迁移体或权利要求68-86中任一项所述的递送系统。
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