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WO2024019441A1 - Nanoparticules hybrides de fusion de vésicules et de liposomes dérivées de cellules et utilisation associée - Google Patents

Nanoparticules hybrides de fusion de vésicules et de liposomes dérivées de cellules et utilisation associée Download PDF

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Publication number
WO2024019441A1
WO2024019441A1 PCT/KR2023/010148 KR2023010148W WO2024019441A1 WO 2024019441 A1 WO2024019441 A1 WO 2024019441A1 KR 2023010148 W KR2023010148 W KR 2023010148W WO 2024019441 A1 WO2024019441 A1 WO 2024019441A1
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cancer
cell
liposomes
hybrid
derived
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Korean (ko)
Inventor
박우람
신하은
배신규
오승욱
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Mdimune Inc
Industry Academic Cooperation Foundation of Catholic University of Korea
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Mdimune Inc
Industry Academic Cooperation Foundation of Catholic University of Korea
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, their use, and their manufacturing method.
  • Extracellular vesicles are nano-scale membrane structures that mediate intercellular communication by transferring cellular substances such as proteins between cells.
  • the endoplasmic reticulum can not only preserve the cell membrane and cytoplasmic intrinsic active substances, but also can carry new substances, thereby exerting various biological activities.
  • the targeting ability of the target site in vivo can be maximized, so it is expected to be a next-generation drug delivery vehicle.
  • CDV cell derived vesicles
  • Crude-CDV can be prepared by continuously extruding cells through increasingly smaller micron-sized pores or by repeatedly passing cells through a depth filter. Afterwards, large quantities of CDV can be easily obtained by separating and purifying impurities from Crude-CDV.
  • the membrane proteins and lipids of CDV are derived from the plasma membranes or organelle membranes of CDV parent cells, the physiologically active molecules (proteins, lipids, sugars, etc.) expressed by the cells are stored in the membrane or inside the membrane. Includes.
  • cell-derived vesicles have the advantage of maximizing therapeutic efficacy because they can additionally encapsulate or combine pharmaceuticals with various characteristics in addition to the function of active substances derived from cells.
  • liposomes are self-assembled bilayer structures made of lipids and are amphipathic particles that have both a hydrophobic part and a hydrophilic part. Liposomes have the advantage of excellent biocompatibility, simple manufacturing method, and ability to transport both water-soluble and fat-soluble drugs. In addition, liposomes can be manufactured by changing the surface properties in various ways, so they can be used in various fields such as cosmetics, adjuvants, and drug delivery. However, liposomes have the potential for non-specific drug delivery and may cause unexpected side effects. Therefore, there is a need to develop new drug carriers that can improve the target targeting ability of liposomes.
  • the present inventors developed a technology that can effectively fuse cell-derived endoplasmic reticulum and drug-encapsulated liposomes, and the hybrid nanoparticles produced through this process target specific tissues through proteins expressed in cell-derived endoplasmic reticulum. It was confirmed that targeting is possible and that the drug contained in liposomes can be delivered to target cells to exert physiological activity.
  • the purpose of the present invention is to provide a method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused.
  • Another object of the present invention is to provide hybrid nanoparticles in which cell-derived vesicles and liposomes are fused.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating various diseases, comprising the hybrid nanoparticles as an active ingredient.
  • Another object of the present invention is to provide a drug delivery system containing the hybrid nanoparticles as an active ingredient.
  • the present invention provides a method for producing hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused, comprising the step of mixing cell-derived vesicles (CDV) and liposomes to induce their fusion. .
  • the fusion may be performed in 10 to 50 (v/v)% C 1 to C 5 alcohol solvent, but is not limited thereto.
  • the cell-derived vesicle and the liposome may be mixed at a ratio of 1 to 30:1 (cell-derived vesicle:liposome), but the mixture is not limited thereto.
  • the cell-derived vesicle may be obtained by extruding a sample containing cells into micropores, but is not limited thereto.
  • the diameter of the micropores may be 0.1 to 20 ⁇ m, but is not limited thereto.
  • the cell-derived vesicle may satisfy one or more characteristics selected from the group consisting of, but is not limited to:
  • the diameter of cell-derived vesicles is 100 to 300 nm.
  • the cell-derived vesicle may include a tumor antigen-specific protein, but is not limited thereto.
  • the cell-derived vesicle may include a chimeric antigen receptor, but is not limited thereto.
  • the liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), It may include, but is not limited to, one or more selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is used in a molar ratio of 1 to 20:1. ratio %), but is not limited to this.
  • the liposome may have a surface charge of 0 to 100 mV, but is not limited thereto.
  • the liposome may contain an active ingredient, but is not limited thereto.
  • the present invention provides hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused.
  • CDV cell-derived vesicles
  • the cell-derived vesicle may be manufactured by a manufacturing method including the step of extruding a sample containing cells into micropores, but is not limited thereto.
  • the cell-derived vesicle may include a chimeric antigen receptor on its surface, but is not limited thereto.
  • the cell-derived vesicle contains a chimeric antigen receptor on the surface, and the liposome may be loaded with an active ingredient, but is not limited thereto.
  • the hybrid nanoparticle may include the chimeric antigen receptor on its surface and the active ingredient between its lipid bilayers or in its internal compartment, but is not limited thereto.
  • the chimeric antigen receptor may be a tumor antigen-specific chimeric antigen receptor, but is not limited thereto.
  • the liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), It may include, but is not limited to, one or more selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). .
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • the active ingredients include peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, and viruses. , quantum dots, fluorochromes, and toxins, but is not limited thereto.
  • the active ingredients may be photosensitizers, but are not limited thereto.
  • the hybrid nanoparticle may satisfy one or more characteristics selected from the group consisting of, but is not limited to:
  • the diameter of the hybrid nanoparticles is 100 to 300 nm
  • the surface charge of the hybrid nanoparticle is -30 to +20 mV.
  • the hybrid nanoparticles may be manufactured by the manufacturing method of claim 1, but are not limited thereto.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent. do.
  • the present invention provides the prevention of cancer, comprising the step of administering a hybrid nanoparticle in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused to an individual in need thereof. Or provide a treatment method.
  • the present invention provides a use of hybrid nanoparticles in the prevention or treatment of cancer in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused.
  • the present invention provides the use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused for the production of a drug for treating cancer.
  • the hybrid nanoparticle may include the tumor antigen-specific chimeric antigen receptor on its surface and the anticancer agent between its lipid bilayers or in its internal compartment, but is not limited thereto.
  • the anticancer agent may be one or more selected from the group consisting of a chemical anticancer agent, a targeted anticancer agent, an immunological anticancer agent, and a photosensitizer, but is not limited thereto.
  • the anticancer agent is a photosensitizer
  • the hybrid nanoparticle may generate active oxygen in response to light stimulation, but is not limited thereto.
  • the cancer is breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, and intraocular melanoma.
  • Tumor uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.
  • the present invention provides a kit for preventing or treating cancer, including the pharmaceutical composition.
  • the present invention provides a drug delivery system comprising, as an active ingredient, a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome carrying an anticancer agent.
  • the present invention includes the step of administering hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and liposomes loaded with a drug (e.g., an anticancer agent) are fused to an individual in need thereof.
  • a drug e.g., an anticancer agent
  • the present invention provides a drug delivery use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a drug-loaded liposome are fused together.
  • the present invention provides the use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a drug-loaded liposome are fused for the production of a drug delivery system.
  • the present invention relates to a fusion technology of cell-derived vesicles and liposomes and to hybrid nanoparticles manufactured through the fusion technology, wherein the hybrid nanoparticles can exert both targeting functions and drug delivery functions to target tissues.
  • the present inventors discovered the optimal conditions to effectively fuse cell-derived vesicles and liposomes, and through this, cell-derived vesicles expressing active ingredients and drug-encapsulated liposomes were fused to possess all of their functions. Since hybrid nanoparticles, that is, dual-functional hybrid nanoparticles, can be manufactured, a wide range of types of hybrid nanoparticles can be manufactured depending on the purpose.
  • hybrid nanoparticles prepared by fusing a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with a photosensitizer effectively target cancer cells through the chimeric antigen receptor, and the photosensitizer It can kill cancer cells by generating active oxygen in response to light stimulation. Therefore, the hybrid nanoparticle according to the present invention can effectively target target tissues and deliver drugs through the combination of various cell-derived vesicles and liposomes, and is expected to be a promising therapeutic platform that can be used as a drug delivery vehicle and therapeutic agent in various fields. It is expected.
  • FIG. 1a shows a schematic diagram of the structure of a chimeric antigen receptor (CAR) and a schematic diagram of HEK93T cells expressing CAR according to an embodiment.
  • CAR chimeric antigen receptor
  • Figure 1b shows the results of confirming the CAR expression level of HEK293 cells transfected with CAR (EGFR-CAR) plasmid according to an example by flow cytometry.
  • Figure 1c shows the results of Western blot confirmation of the expression of CD3zeta, a component of CAR, to confirm CAR expression in HEK293 cells transfected with EGFR-CAR plasmid.
  • FIG. 2a is a diagram showing the production process of cell-derived endoplasmic reticulum (CDV) derived from HEK293 cells expressing EGFR-CAR.
  • CDV cell-derived endoplasmic reticulum
  • Figure 2b shows the results of measuring the surface charge of control CDV and EGFR-CAR expressing CDV through nanoparticle tracking analysis.
  • Figure 2c shows the results of measuring the size of control CDV and EGFR-CAR expressing CDV.
  • Figure 2d shows the results of confirming the expression of the EGFR-CAR component (CD3zeta) and endoplasmic reticulum marker protein (CD63) of control CDV or EGFR-CAR expressing CDV using Western blot.
  • CD3zeta EGFR-CAR component
  • CD63 endoplasmic reticulum marker protein
  • Figure 2e shows the results of confirming the cytotoxicity of each CDV by measuring the cell survival rate after treating MDA-MB-231 cells with control CDV or EGFR-CAR expressing CDV, respectively.
  • Figure 3a is a schematic diagram showing FRET analysis to evaluate fusion between liposomes.
  • FRET liposomes fused with unlabeled control liposomes have a reduced surface density of fluorophores.
  • Figure 3b shows the results of measuring the surface zeta potential of control liposomes and FRET liposomes through nanoparticle tracking analysis.
  • Figure 3c shows the results of measuring the sizes of control liposomes and FRET liposomes.
  • Figure 3d shows the results of confirming the fusion rate by measuring the degree of FRET after inducing fusion by reacting control liposomes and FRET liposomes for 1 hour (Excitation/Emission wavelength: 465/520 nm).
  • Figure 3e shows the results of confirming the fusion rate by measuring the degree of FRET after inducing fusion by reacting FRET liposomes and HEK293 CDV for 1 hour (Excitation/Emission wavelength: 465/520 nm).
  • Figure 4a shows the results of measuring the sizes of liposomes with different lipid compositions through nanoparticle tracking analysis.
  • Figure 4b shows the results of measuring the surface zeta potential of liposomes with different lipid compositions.
  • Figure 4c shows the results of confirming the fusion rate by inducing fusion by reacting liposomes with different lipid compositions and HEK293 CDV for 1 hour (Excitation/Emission wavelength: 465/520 nm).
  • Figure 4d is a transmission electron microscope image of liposome B fused with HEK293 CDV.
  • Figure 4e shows the results of confirming the presence or absence of cytotoxicity after treating fibroblast or breast cancer cell lines with liposome B fused with HEK293 CDV at various concentrations.
  • Figure 5a is a diagram showing the structure of a liposome loaded with a photosensitizer according to one embodiment.
  • Figure 5b shows the results of measuring the surface zeta potential of liposomes loaded with photosensitizer through nanoparticle tracking analysis.
  • Figure 5c shows the results of measuring the size of liposomes loaded with photosensitizer.
  • Figure 5d is a TEM image of a liposome loaded with a photosensitizer.
  • Figure 5e shows the results of measuring the amount of active oxygen over time after irradiating a laser to a liposome loaded with a photosensitizer.
  • Figure 5f shows the results of measuring the degree of cancer cell death depending on whether or not laser irradiation was performed after treating breast cancer cell lines with liposomes loaded with a photosensitizer at various concentrations.
  • Figure 6a shows the results of measuring the surface zeta potential of a dual-functional hybrid nanoparticle fused with a photosensitizer-loaded liposome and an EGFR-CAR expressing CDV.
  • Figure 6b shows the results of measuring the size of the dual-functional hybrid nanoparticles.
  • Figures 6d and 6e show nanoparticles in which control liposomes and control CDV were fused to breast cancer cell lines; Alternatively, the control liposome and EGFR-CDV fused nanoparticles were treated for 4 hours each, and then the hybrid nanoparticles absorbed into the cells were obtained through fluorescence imaging ( Figure 6d), and the detected fluorescence levels were compared. ( Figure 6e).
  • Figure 6f shows the results of confirming the cancer cell death rate depending on whether or not laser irradiation was performed after treating the breast cancer cell line with dual-functional hybrid nanoparticles.
  • Figure 7a shows an experimental schedule for evaluating the function of dual-functional hybrid nanoparticles in a tumor animal model.
  • Figure 7b shows the results of measuring the levels of liver and kidney toxicity markers in the peripheral blood of a tumor animal model intravenously administered hybrid nanoparticles to evaluate the biocompatibility of the dual-functional hybrid nanoparticles.
  • Figure 7c shows the results of confirming the distribution of nanoparticles in a tumor animal model in which fluorescently labeled hybrid nanoparticles were administered intravenously to evaluate the tumor-targeting ability of the dual-functional hybrid nanoparticles.
  • Figure 7d shows the results of administering hybrid nanoparticles to a tumor animal model to evaluate the cancer cell killing ability of dual-functional hybrid nanoparticles and then confirming the degree of tumor growth with or without laser irradiation.
  • Figure 8 is a schematic diagram showing the manufacturing process, structure, and mechanism of action of the hybrid nanoparticle according to the present invention.
  • the present invention relates to a fusion technology of cell-derived vesicles and liposomes and hybrid nanoparticles produced through the same.
  • the present inventors have discovered optimal conditions for effective fusion of cell-derived vesicles and liposomes, and the It was confirmed that the hybrid nanoparticle can exert a dual function by maintaining the functions of both cell-derived vesicles and liposomes.
  • a cell vesicle carrying a targeting substance such as a chimeric antigen receptor and a liposome carrying a pharmacological/physiological active ingredient can be fused, so a hybrid is produced by fusing the two.
  • Nanoparticles can effectively exert both target tissue targeting and drug delivery functions. Therefore, through the fusion technology according to the present invention, hybrid nanoparticles with appropriate active ingredients can be manufactured according to the purpose, so a wide range of hybrid nanoparticles can be manufactured and used for the treatment of various diseases.
  • the present invention provides a method for producing hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused, which includes the step of mixing cell-derived vesicles (CDV) and liposomes to induce their fusion.
  • the purpose is to provide
  • the cell-derived vesicles according to the present invention are manufactured by a method including the step of extruding a sample containing cells into micropores, and are distinguished from natural extracellular vesicles produced by cells.
  • the step of extruding into the micropores may be performed by sequentially extruding cells using micropores with large micropores and small micropores.
  • CDV Cell derived vesicles
  • CDV can be produced by being released from the cell membrane in almost all types of cells, and is characterized by having a double phospholipid membrane form, which is the structure of the cell membrane.
  • the cell-derived vesicles of the present invention are distinct from naturally secreted vesicles, such as exosomes.
  • the term “vesicles” is distinguished between the inside and the outside by a lipid bilayer composed of the cell membrane components of the cell from which it is derived, and has the cell membrane lipids, membrane proteins, nucleic acids, and cell components, etc. It means that the size is smaller than the original cell, but is not limited to this.
  • the cell-derived vesicle according to the present invention has a size at the micrometer level.
  • the diameter of the CDV may be less than 0.2 ⁇ m. More specifically, the diameter of the CDV is 50 to 300 nm, 50 to 250 nm, 50 to 200 nm, 50 to 180 nm, 50 to 160 nm, 50 to 150 nm, 50 to 100 nm, 100 to 300 nm, 100 nm. It may be, but is limited to, 250 nm, 100 to 200 nm, 100 to 180 nm, 120 to 300 nm, 150 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 180 nm, or 130 to 170 nm. It doesn't work.
  • the cell-derived vesicle according to the present invention may have a negative surface charge ( ⁇ 0 mV). That is, the surface charge of the cell-derived vesicles is -100 to -5 mV, -80 to -5 mV, -50 to -5 mV, -40 to -5 mV, -30 to -5 mV, -50 to -10. It may be mV, -50 to -15 mV, -50 to -20 mV, -40 to -20 mV, -35 to -20 mV, or -30 to -20 mV, but is not limited thereto.
  • the “cell” can be used without limitation as long as it is a cell capable of separating cell-derived vesicles, and includes all cells isolated from natural organisms.
  • the cells are nucleated cells.
  • the cells may be of plant origin or any type of animal, including human and non-human mammals. Therefore, there is no particular limitation on the type of cells from which the cell-derived vesicle according to the present invention can be obtained, but for example, stem cells, kidney cells, embryonic kidney cells, cancer cells, acinar cells, myoepithelial cells, red blood cells, immune cells, etc.
  • the cell-derived vesicles can be obtained from cells, monocytes, dendritic cells, natural killer cells, T cells, B cells, macrophages, endothelial cells, epidermal cells, neurons, glial cells, astrocytes, muscle cells, and platelets. You can. It may be various types of immune cells, tumor cells, stem cells, acinar cells, myoepithelial cells, or platelets.
  • the stem cells include mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, and salivary gland stem cells. It may be any one or more selected from the group consisting of, and preferably may be adipocyte-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells, but is not limited thereto.
  • the vesicle is used to extrude a suspension containing nucleated cells, ultrasonic disintegration, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical disintegration, and physical stimulation by externally applying force to the cells. It can be manufactured using a method selected from the group consisting of treatments, but is not limited thereto. In the present invention, the vesicle was manufactured by extruding a suspension containing nucleated cells using an extruder.
  • the extrusion force of the extruder applied to produce the nucleated cell-derived vesicle of the present invention may be 5 to 200 psi, 10 to 150 psi, 10 to 100 psi, 10 to 50 psi, 10 to 40 psi, or 50 to 100 psi. there is.
  • the vesicle according to the present invention can be obtained by extruding a sample containing cells into micropores.
  • the cells are sequentially extruded using those with micropores from large to small. It can be obtained by doing.
  • the diameter of the micropores is 0.01 to 100 ⁇ m, 0.01 to 80 ⁇ m, 0.01 to 60 ⁇ m, 0.01 to 40 ⁇ m, 0.01 to 20 ⁇ m, 0.01 to 15 ⁇ m, 0.01 to 10 ⁇ m, 0.01 to 7 ⁇ m, 0.01 to 3 ⁇ m. , 0.01 to 1 ⁇ m, 0.01 to 0.5 ⁇ m, 0.01 to 0.1 ⁇ m, or 0.1 to 0.5 ⁇ m, but is not limited thereto.
  • the sequential extrusion may be performed using filters with pore diameters of 5 to 20 ⁇ m, 2 to 7 ⁇ m, 0.7 to 3 ⁇ m, and 0.1 to 0.5 ⁇ m.
  • the size of the micropores can be appropriately adjusted depending on the type of cell used. CDV obtained through the above process may undergo additional purification.
  • cell-derived vesicles may be prepared by removing the nucleus of the cell before extruding the cell-containing sample into the micropore.
  • the cell nuclei can be removed through centrifugation.
  • the vesicle according to the present invention can also be manufactured through a manufacturing method that includes the step of passing a sample containing cells through a depth filter and extruding it.
  • the extrusion force of the depth filter may be 5 to 200 psi, 10 to 150 psi, 10 to 100 psi, 10 to 50 psi, 10 to 40 psi, or 50 to 100 psi, but is not limited thereto.
  • CDV obtained through the above process may undergo additional purification.
  • the purification process is intended to remove other substances (vesicles smaller than the CDV of the present invention or other contaminants) in addition to the CDV of the present invention and obtain only the desired CDV, preferably by mixing cell-derived vesicles and liposomes. This can be done by adding 1 to 5 times, 1 to 4 times, or 1 to 3 times the volume of buffer solution compared to the total volume of the solution.
  • CDV produced through the extrusion step is purified through a tangential flow filtration (TFF) process.
  • the membrane used at this time preferably has a cut off of 500 kDa, 450 kDa, 400 kDa, 350 kDa, or 300 kDa or more, and a concentration step and buffer exchange step to concentrate the concentration of the CDV suspension to 5 times or more. is performed.
  • CDV is concentrated and impurities are removed.
  • size exclusion chromatography may be further performed. At this time, it is preferable to use a column with a recovery range of about 35 to 350 nm or 70 to 1,000 nm. Since CDV corresponds to relatively large particles, CDV can be obtained by collecting the fraction eluted through the column. there is. Through this purification process, impurities such as vesicles and other proteins smaller than CDV can be removed.
  • sample containing cells may be a sample containing nucleated cells or transformed cells thereof, and includes without limitation any cell capable of producing vesicles.
  • the filter used in the extrusion step can be used without limitation as long as it contains micropores and has a membrane structure capable of filtering samples, but is preferably a polycarbonate membrane filter. .
  • the cell-derived vesicle according to the present invention can be manufactured using a cell extruder including a support, an intermediate filter, and a membrane filter.
  • a cell extruder including a support, an intermediate filter, and a membrane filter.
  • the production method may include the step of obtaining cell-derived vesicles from cells transformed or transfected with a recombinant vector.
  • the cell-derived vesicle obtained at this time contains the expression product of the gene contained in the recombinant vector.
  • the recombinant vector is preferably a recombinant vector containing the gene to be expressed in the autologous cell-derived vesicle in which the present invention is to be practiced. Since the cell-derived vesicle according to the present invention is derived from the plasma membrane or organelle of the mother cell and may contain physiologically active molecules expressed by the mother cell, the mother cell is transformed/transfected with the desired gene, and then transformed/transfected. Vesicles derived from cells expressing the above genes can be obtained from infected cells.
  • the expression product is a protein targeting a specific tissue (i.e., a protein with tissue-specific binding ability). More preferably, the expression product is a protein targeting tumor tissue and/or cancer cells.
  • the cancer cell and/or tumor tissue specific protein is not limited to a specific type and includes all ligands, receptors, signaling proteins, and antibodies or fragments thereof.
  • the gene is a gene encoding a chimeric antigen receptor targeting tumor tissue or cancer cells.
  • chimeric antigen receptor refers to synthetic receptors capable of targeting specific antigens.
  • CAR according to the present invention is preferably a polypeptide comprising an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain (cytoplasmic domain).
  • antigen-binding domain refers to a protein or polypeptide domain that can specifically recognize and bind to a target antigen.
  • antigen refers to a polypeptide, compound, or substance that can specifically bind to humoral immune mediators such as antibodies or cellular immune mediators such as T cell receptors.
  • the antigen may be a cancer cell-specific protein (i.e., expressed only in cancer cells, or particularly expressed or highly active in cancer cells), and more preferably, it may be a cancer cell-specific surface protein. That is, the antigen is a tumor antigen.
  • the tumor antigens are included without limitation as long as they are specifically expressed in cancer cells or have a particularly high expression level in cancer cells, and are not limited to specific types, but include EGFR, CD19, TAG72, IL13R ⁇ 2 (Interleukin 13 receptor alpha-2 subunit), CD52, CD33, CD20, TSLPR, CD22, CD30, GD3, CD171, NCAM (Neural cell adhesion molecule), FBP (Folate binding protein), Le(Y) (Lewis-Y antigen), PSCA (Prostate stem cell antigen), PSMA ( Prostate-specific membrane antigen), CEA (Carcinoembryonic antigen), HER2 (Human epidermal growth factor receptor 2), Mesothelin, CD44v6 (Hyaluronate receptor variant 6), B7-H3, Glypican-3, ROR1 (receptor tyrosine kinase like orphan receptor 1) ), Survivin, FOLR1 (folate receptor), WT1 (Wilm'
  • the chimeric antigen receptor according to the present invention is a chimeric antigen receptor having a breast cancer tumor antigen-specific antigen binding domain, and more preferably, is a chimeric antigen receptor having an EGFR-specific antigen binding domain.
  • the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 6 or may include an antigen-binding domain consisting of the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.
  • the EGFR-specific antigen binding domain may include the base sequence of SEQ ID NO: 1 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 1, but is not limited thereto.
  • hinge region refers to a region located between the antigen-binding domain and the transmembrane domain and serves as a flexible linker.
  • the hinge region ensures that the antigen-binding domain is properly positioned when the antigen-binding domain binds to the antigen, thereby forming a stable bond with the antigen.
  • the hinge region is not limited to a specific type, but may preferably be the CD8 hinge region.
  • the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 7 or a hinge region consisting of the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.
  • the hinge region may include the base sequence of SEQ ID NO: 2 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 2, but is not limited thereto.
  • transmembrane domain refers to any polypeptide or oligopeptide that functions to connect extracellular and intracellular signaling domains across the cell membrane.
  • the transmembrane domain is not limited to a specific type, but is preferably a CD28 transmembrane domain.
  • the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 8 or may include a transmembrane domain consisting of the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.
  • the transmembrane domain may include the base sequence of SEQ ID NO: 3 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto.
  • intracellular signaling domain cytoplasmic domain
  • ED endodomain
  • intracellular signaling domain refers to a polypeptide or oligopeptide inside the cell membrane.
  • the intracellular signaling domain may include one or more intracellular co-stimulatory domains.
  • the “stimulatory signal domain” and “co-stimulatory domain” are any polypeptide or oligopeptide that transmits signals that cause activation or inhibition of biological processes within the cell. means.
  • the intracellular signaling domain is not limited to a specific type, but may preferably consist of a CD28 signaling domain and/or a CD3zeta domain.
  • the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10, or may include a signaling domain consisting of the amino acid sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10. , but is not limited to this.
  • the signaling domain may include the base sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5, or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5, but is not limited thereto. .
  • the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 12 or may be composed of the amino acid sequence of SEQ ID NO: 12, but is not limited thereto. Additionally, the chimeric antigen receptor may include the base sequence of SEQ ID NO: 11 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 11, but is not limited thereto.
  • a polypeptide consisting of an amino acid sequence (nucleic acid sequence) represented by a specific sequence number is not limited to the corresponding amino acid sequence (nucleic acid sequence), and variants of the amino acid sequence (nucleic acid sequence) are within the scope of the present invention. included within.
  • polypeptide molecule (polynucleotide molecule) of the amino acid sequence (nucleic acid sequence) of the present invention is a functional equivalent of the polypeptide molecule (polynucleotide molecule) constituting it, for example, some amino acid sequences (nucleic acid sequences) of the polypeptide molecule are deleted ( It is a concept that includes variants that have been modified by deletion, substitution, or insertion, but can have the same functional effect as the corresponding polypeptide (polynucleotide).
  • the polypeptide (polynucleotide) disclosed in the present invention contains at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 90% of the amino acid sequence (nucleic acid sequence) represented by a specific sequence number.
  • it may include an amino acid sequence (nucleic acid sequence) having more than 95% sequence homology.
  • sequence homology includes polypeptides (polynucleotides) having .
  • the “% sequence homology” for a polypeptide (polynucleotide) is determined by comparing a comparison region with two optimally aligned sequences, and the portion of the polypeptide sequence (polynucleotide sequence) in the comparison region is consistent with the optimal alignment of the two sequences. may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions).
  • the cells are cells that express tumor antigen-specific proteins, and the cell-derived vesicles obtained therefrom may contain the same tumor antigen-specific proteins as those expressed in the cells.
  • the tumor antigen-specific protein is expressed on the surface of the cell-derived vesicle. Therefore, hybrid nanoparticles obtained by fusing a liposome and a cell-derived vesicle containing a tumor antigen-specific protein contain the same tumor antigen-specific protein as that contained in the cell-derived vesicle.
  • the tumor antigen-specific protein is a chimeric antigen receptor specific for a tumor antigen.
  • liposomes refers to a structure composed of a lipid membrane surrounding an aqueous internal compartment.
  • the liposome membrane is mainly composed of phospholipids and their derivatives.
  • phospholipids and their derivatives When the phospholipids and their derivatives are dispersed in an aqueous solution, a single layer or lipid bilayers are spontaneously formed in the endoplasmic reticulum.
  • Liposomes can carry water-soluble active ingredients in an aqueous internal space and can also carry hydrophobic active ingredients in a lipid bilayer, so they can be used as carriers for various drugs.
  • the liposome according to the present invention preferably contains 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1, It may include one or more selected from the group consisting of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine
  • DOPC 2-dioleoyl-sn-glycero-3-phosphocholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is 1 to 20:1, 1 to 15:1, 1 It may be included in a molar ratio (molar ratio %) of 1 to 10:1, 1 to 8:1, 1 to 5:1, 1 to 4:1, or 2 to 5:1.
  • the liposome containing DOPC and DOTAP is characterized by a positive charge on the surface.
  • 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPE
  • DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine
  • DOPC 1,2-dioleoyl-sn-glycero -3-phosphocholine
  • 1,2-dioleoyl-sn-glycero-3-phospho-L-serine 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is 1: 1 to 20, 1: 1 It may be included in a molar ratio (molar ratio %) of 1 to 15, 1:1 to 10, 1:1 to 8, or 1:1 to 5.
  • the liposome according to the present invention may have a positive surface charge (>0 mV). That is, the liposome has a surface charge of 0 to 100 mV, 0 to 80 mV, 0 to 60 mV, 0 to 50 mV, 0 to 45 mV, 10 to 100 mV, 10 to 50 mV, 20 to 60 mV, and 20 to 50 mV. It may be 50 mV, 30 to 50 mV, or 30 to 40 mV, but is not limited thereto.
  • the present inventors confirmed that effective fusion occurred when cell-derived vesicles with a negative charge on the surface were mixed with liposomes with a positive charge on the surface.
  • the diameter of the liposome according to the present invention is 100 to 500 nm, 100 to 400 nm, 100 to 350 nm, 100 to 320 nm, 100 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 190 nm, It may be 200 to 500 nm, 200 to 400 nm, 200 to 350 nm, 250 to 500 nm, 250 to 400 nm, 250 to 350 nm, 280 to 400 nm, 280 to 350 nm, or 280 to 320 nm. It is not limited.
  • the liposome may be prepared through a production method comprising the following steps:
  • the organic solvent may be one or more selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether, but is not limited thereto.
  • the aqueous solution may be phosphate-buffered saline, but is not limited thereto.
  • the method for producing liposomes may further include (S4) treating the hydrated liposome membrane with ultrasound.
  • the liposome according to the present invention may contain an active ingredient.
  • the active ingredient is not limited to a specific type, and any one skilled in the art can be applied as long as it exerts the desired effect and can be loaded into liposomes.
  • Non-limiting examples include peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, and quantum dots. , fluorochrome, and toxin, but is not limited thereto.
  • the active ingredient may be supported inside the liposome or between lipid bilayers.
  • the hybrid nanoparticle obtained by fusing the liposome carrying the active ingredient and the cell-derived vesicle may contain the same active ingredient as that carried in the liposome.
  • the active ingredient is supported between the lipid bilayers or in the internal compartment of the hybrid nanoparticle.
  • fusion of cell-derived vesicles and liposomes is characterized in that 10 to 50 (v/v)% C 1 to C 5 alcohol solvent is used.
  • the alcohol solvent is a C 1 to C 5 , C 1 to C 3 , or C 1 to C 2 alcohol solvent.
  • the alcohol solvent is ethanol.
  • the concentration of the alcohol (ethanol) is 10 to 50 (v/v)%, 10 to 45 (v/v)%, 10 to 40 (v/v)%, 10 to 35 (v) compared to the total mixed solution.
  • the fusion can be achieved by adding alcohol to a mixed solution of cell-derived vesicles and liposomes to a final concentration of 10 to 50 (v/v)%.
  • the fusion occurs 10 minutes to 180 minutes, 10 minutes to 150 minutes, 10 minutes to 120 minutes, 10 minutes to 100 minutes, 10 minutes to 80 minutes, and 10 minutes to 65 minutes after adding alcohol to the mixed solution of cell-derived vesicles and liposomes. minutes, 20 minutes to 100 minutes, 30 minutes to 100 minutes, 40 minutes to 100 minutes, 50 minutes to 100 minutes, or 50 minutes to 80 minutes, but is not limited thereto.
  • the present inventors have found that fusion of liposomes and cell-derived vesicles occurs more effectively when using alcohol rather than using sonication or electroporation, especially at a final concentration of 10 to 50 (v/v)% compared to the total reaction solution. It was confirmed that the most effective fusion occurred under alcohol conditions.
  • the fusion technology of liposomes and cell-derived vesicles according to the present invention is more effective in fusing liposomes and cell-derived vesicles compared to the electroporation or sonication technology used for the fusion of liposomes or vesicles. It is characterized by the ability to fuse.
  • the fusion technology according to the present invention can more easily produce hybrid nanoparticles in which liposomes and cell-derived vesicles are fused using an alcohol solvent. In particular, it does not require special substances such as other catalysts and can be produced using only an alcohol solvent. Since liposomes and cell-derived vesicles can be effectively fused, it is economically and time-effective as it does not require impurity removal processes.
  • cell-derived vesicles and liposomes can be mixed at a number ratio of 1 to 30:1 (cell-derived vesicles: liposomes).
  • the cell-derived vesicle:liposome is 1 to 30:1, 1 to 25:1, 1 to 20:1, 1 to 15:1, 1 to 10:1, 5 to 30:1, 5 to 20. : 1, 5 to 15 : 1, 5 to 10 : 1, 7 to 10 : 1, or 8 to 10 : 1, but is not limited thereto.
  • 'effective fusion' means that a hybrid nanoparticle obtained by fusion of a cell-derived vesicle and a liposome is manufactured into one structure containing both the cell-derived vesicle-derived component and the liposome-derived component. do.
  • the present invention provides hybrid nanoparticles in which cell-derived vesicles and liposomes are fused.
  • the hybrid nanoparticle is a fusion of cell-derived vesicles and liposomes, and is characterized by containing both components derived from each of the cell-derived vesicles and liposomes.
  • the membrane of the hybrid nanoparticle may be composed of the membrane components of the cell-derived vesicle and the liposome.
  • the membrane of the hybrid nanoparticle is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1, It may include one or more selected from the group consisting of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), preferably 1,2- It may include dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine
  • DOPC 2-dioleoyl-s
  • hybrid nanoparticles prepared by fusing cell-derived vesicles expressing a specific protein and liposomes loaded with a specific active ingredient contain both the protein and the active ingredient, and thus can exert all of their activities and functions.
  • the protein derived from the cell-derived vesicle is located on the surface of the hybrid nanoparticle, and the active ingredient derived from the liposome is trapped between the lipid bilayers of the hybrid nanoparticle or stored in an internal compartment inside the lipid bilayer. It may be possible, but it is not limited to this.
  • the hybrid nanoparticle can be manufactured by fusing a cell-derived vesicle containing a chimeric antigen receptor and a drug-loaded liposome. Therefore, the hybrid nanoparticle can be produced by fusing the chimeric antigen receptor with a liposome loaded with the drug. and all of the above drugs.
  • the chimeric antigen receptor is located on the surface of the nanoparticle, and the drug can be captured within the lipid bilayer of the nanoparticle or encapsulated in an internal compartment inside the lipid bilayer.
  • the chimeric antigen receptor may be a tumor antigen-specific chimeric antigen receptor that specifically binds to the tumor antigen. Therefore, hybrid nanoparticles obtained by fusing a cell-derived vesicle containing the tumor antigen-specific chimeric antigen receptor with a liposome also contain the tumor antigen-specific chimeric antigen receptor, thereby killing cancer cells and tumor tissues. It can be targeted.
  • Hybrid nanoparticles targeting cancer cells through the chimeric antigen receptor can be introduced into target cells and deliver drugs (derived from liposomes) loaded on the nanoparticles to the corresponding cells.
  • the drug is preferably an anticancer agent.
  • the diameter of the hybrid nanoparticle according to the present invention is 100 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 180 nm, 100 to 150 nm, 130 to 300 nm, 130 to 250 nm, 130 to 200 nm, It may be 130 to 180 nm, 150 to 300 nm, 150 to 250 nm, 150 to 200 nm, or 150 to 180 nm, but is not limited thereto.
  • the hybrid nanoparticle according to the present invention has a surface charge of -30 to +20 mV, -25 to +20 mV, -20 to +20 mV, -15 to +20 mV, -30 to +10 mV, -25 mV. to +10 mV, -20 to +10 mV, -15 to +10 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -15 to 0 mV, -30 to -5 mV, It may be -25 to -5 mV, -20 to -5 mV, or -15 to -5 mV, but is not limited thereto.
  • the hybrid nanoparticles may be manufactured using the method for producing hybrid nanoparticles according to the present invention.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent. do.
  • the hybrid nanoparticle may contain a tumor antigen-specific chimeric antigen receptor derived from the cell-derived vesicle on the surface, and an anticancer agent derived from the liposome between lipid bilayers or in an internal compartment. Therefore, the hybrid nanoparticle can target cancer cells/tumor tissues through the chimeric antigen receptor and deliver the anticancer agent to the cancer cells/tumor tissues.
  • the anticancer agent may be one or more selected from the group consisting of chemical anticancer agents, targeted anticancer agents, immune anticancer agents, and photosensitizers, but is not limited thereto, as long as it exerts an anticancer effect and can be carried in liposomes or hybrid nanoparticles of the present invention. May be included without limitation.
  • the anticancer drugs include doxorubicin, paclitaxel, docetaxel, cisplatin, Gleevec, 5-fluorouracil (5-FU), tamoxifen, carboplatin, topotecan, belotecan, imatinib, irinotecan, floxuridine, and vinorelbine.
  • gemcitabine leuprolide, flutamide, zoledronate, methotrexate, camptothecin, vincristine, hydroxyurea, streptozocin, valubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, It may be one or more selected from the group consisting of doxyfluridine, vinblastine, mitomycin, prednisone, Armitor, and mitoxantrone, but is not limited thereto.
  • the anticancer agent is a photosensitizer.
  • photosensitizer refers to a drug that exhibits specific activity in response to light stimulation.
  • the photosensitizer refers to a substance that generates reactive oxygen by chemically reacting with light stimulation and oxygen. Therefore, hybrid nanoparticles containing the photosensitizer generate active oxygen only under light stimulation, so they do not exert cytotoxicity in the absence of light stimulation, but when exposed to light stimulation, they generate active oxygen and cause cell death. . In other words, hybrid nanoparticles containing a photosensitizer selectively exert an anticancer effect, thereby significantly lowering the risk of side effects due to non-specific anticancer activity.
  • the photosensitizer may be any agent that can generate active oxygen in response to photostimulation, but is preferably porphyrin, temoporfi (THPC), or verteporphine.
  • THPC temoporfi
  • verteporphine preferably porphyrin, temoporfi (THPC), or verteporphine.
  • THPC temoporfi
  • Verteporfin Rostaporfin
  • Talaporfin sodium Padeliporfin
  • Chlorin e6 Zinc Pthalocyanine
  • Pheophorbide a sulftalanzinc
  • the photosensitizer may generate active oxygen in response to light stimulation with a wavelength of 500 to 800 (nm), but is not limited thereto. More specifically, the photosensitizer may have a wavelength of 500 to 800, 500 to 750, 500 to 700, 500 to 680, 600 to 800, 600 to 750, 600 to 700, or 600 to 680 (nm).
  • composition according to the present invention may be administered sequentially or simultaneously with the photostimulation treatment, but is not limited thereto.
  • cancer includes both solid cancer and blood cancer.
  • the cancer is breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, and intraocular melanoma.
  • the cancer may be characterized as a cancer expressing EGFR.
  • the chimeric antigen receptor contained in the hybrid nanoparticle is characterized as being a chimeric antigen receptor specific for an antigen that is specifically expressed in the target cancer or is expressed at a particularly high level in the cancer.
  • the content of the hybrid nanoparticles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight, based on the total weight of the composition. It may be %, but is not limited to this.
  • the content ratio is a value based on the dry amount with the solvent removed.
  • the pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.
  • the excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.
  • the pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods.
  • Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid.
  • Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin.
  • binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl
  • soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.
  • Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.
  • a solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.
  • Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.
  • Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.
  • Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil.
  • solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate;
  • Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide;
  • Weak acids and their salts acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums
  • Isotonic agents such as sodium chloride
  • Stabilizers such as sodium bisulfite (NaHSO 3 ) carbon dioxide gas, sodium metabisulfite (Na 2 S 2 O 5 ), sodium sulfite (Na 2 SO 3 ), nitrogen gas (N 2
  • Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.
  • Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.
  • the pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.
  • the pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.
  • the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and body weight, and is generally administered at 0.001 to 150 mg, preferably 0.01 to 100 mg, per kg of body weight every day or every other day, or 1 It can be administered in divided doses 1 to 3 times a day.
  • the above dosage does not limit the scope of the present invention in any way.
  • “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of
  • “administration” means providing a given composition of the present invention to an individual by any appropriate method.
  • prevention refers to any action that suppresses or delays the onset of the desired disease
  • treatment refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.
  • the present invention provides a kit for preventing or treating cancer comprising the pharmaceutical composition.
  • the kit according to the present invention may include other components, compositions, solutions, devices, etc. commonly required for the prevention or treatment of cancer.
  • the kit may further include cell-derived vesicles, liposomes, etc. for producing the hybrid nanoparticles, and may further include instructions containing information related to the hybrid nanoparticles (e.g., manufacturing method, etc.). there is.
  • the present invention provides a drug delivery system comprising, as an active ingredient, a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome carrying an anticancer agent.
  • the drug delivery system is characterized in that it delivers the anticancer agent to target cells.
  • the target cell of the drug delivery system is a cancer cell or tumor tissue, and more preferably, a cancer cell or tumor tissue that expresses a tumor antigen to which the chimeric antigen receptor contained in the hybrid nanoparticle specifically binds.
  • the term "combination thereof" included in the Markushi format expression means a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of constituent elements.
  • 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPE
  • 1,2-dioleoyl-sn-glycero-3-phospho-L-serine DOPS
  • 1,2-dioleoyl-sn-glycero-3- phosphocholine DOPC
  • 1,2-dioleoyl-3-trimethylammonium-propane DOTAP
  • mini-extruder was purchased from Avanti Polar Lipids; n-Dodecyl ⁇ ULTROL grade) was purchased from Merck; Di
  • HEK293 and NIH3T3 cells were purchased from the Korean Cell Line Bank, and MDA-MB-231 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
  • HEK293 and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C and 5% CO 2 .
  • NIH3T3 cells were cultured at 37°C and 5% CO 2 in Roswell Park Memorial Institute Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
  • Liposomes were prepared by thin film hydration method. Several types of lipids were combined at various molar ratios (see Tables 1 and 2), dissolved in chloroform, and liposomes were prepared in a round bottom flask. Next, the sample was placed in a rotary evaporator and used under vacuum and 60°C conditions for 15 minutes to evaporate chloroform. Next, the lipid film was treated with 1 mL of PBS for 30 minutes to hydrate, and then subjected to ultrasound at 20% amplitude (pulse mode; 3 seconds/3 seconds) for 7 minutes and 30 seconds to obtain lipid bilayer liposomes. The obtained liposomes were stored at 4°C.
  • HEK293 cells were transfected with Geneporter3000 transfection reagent (Genlantis) according to the manufacturer's protocol. Seeded HEK293 cells showed 80% confluency on the day of transfection.
  • the plasmid DNA was diluted and mixed in GP3K diluent and the GP3K reagent was diluted in serum-free medium, and the mixed reagents were incubated at room temperature for 5 minutes to prepare lipoplexes for transfection. Complete medium was removed from the plated cells, replaced with serum-free medium, and then lipoplex was treated. After 24 hours of culture, transfected cells were collected.
  • the obtained cells were centrifuged to remove nuclei and sonicated with a tip-sonicator at 20% ultrasound amplitude (pulse mode, 3 sec/3 sec) for 30 sec.
  • Treated cells were successively extruded through polycarbonate membrane filters (Whatman, UK) with pore sizes of 10 ⁇ m, 5 ⁇ m, 1 ⁇ m, and 400 nm using a mini extruder (Avanti Polar Lipids, AL, USA).
  • CDV Cell-derived vesicles (CDV) expressing CAR were obtained.
  • the protein concentration of the prepared CDV was quantified using the BCA protein assay kit (Thermo Fisher, Korea) according to the manufacturer's protocol.
  • CDV size and shape of CDV were analyzed using a transmission electron microscope (TEM, H-7600, Hitachi). CDV size, surface zeta potential, and particle number were measured using Nanoparticle Tracking Analysis (NTA, Zetaview, Particle Metrix, USA).
  • the Cell Counting Kit-8 (CCK-8) assay kit was used according to the manufacturer's protocol to measure viability and cytotoxicity of MDA-MB-231 and NIH3T3 cells. did.
  • Various nanovesicles were treated in a number of 1 ⁇ 10 7 to 1 ⁇ 10 9 in serum-free medium in which cells were cultured for 4 hours.
  • the medium was changed to culture medium and a laser of 12 J intensity was illuminated using a 671 nm laser source (1000MWCW400F, LaserLab, Korea). did.
  • CCK-8 reagent was applied to each well. Cell viability was calculated by measuring the absorbance of each well at 450 nm for 30 minutes with a microplate reader (Synergy H1, BioTek, Winooski, USA).
  • Liposomes and CDV were mixed in a black plate maintained at 37°C in a microplate reader (Synergy H1, BioTek, USA). CDV and liposomes were mixed in PBS at a liposome/CDV ratio of 1/9 (1 ⁇ 10 11 pieces). All vesicles were counted by NTA, and the total volume of the reaction mixture was 200 ⁇ L. Triton x-100 or ethanol was added to a final concentration of 2% or 30%, respectively. Additionally, sonication or electroporation was performed at 40 kHz or bipolar (50 voltage, 10 ⁇ s, 5 pulses (ECM830, BTX Apparatus, USA)) for 20 minutes.
  • the lipid mixture was monitored by FRET. Additionally, donor fluorescent liposomes containing the same molar ratio (1.5%) of fluorescent lipids NBD-PS and Rhod-PE were prepared (see Tables 1 and 2). When NBD is excited, the fluorescence resonance energy is transferred to rhodamine in a FRET process that depends on the distance between the two fluorophores. Fusion was monitored by following NBD fluorescence intensity (excitation at 460 nm, emission at 535 nm) at 37°C.
  • the fusion reaction was stopped by adding 10 ⁇ l of DDM (25 mg/mL) to solubilize all liposomes and the NBD fluorescence intensity was measured at infinite dilution: Max(NBD).
  • the fusion curve was normalized using the following equation:
  • NBD fluorescence increase (%) [NBD-Min(NBD)]/[Max(NBD)-Min(NBD)]
  • Min(NBD) means the lowest NBD fluorescence value at all times.
  • SOSG reagent singlet oxygen sensor green reagent
  • Thermo Fisher Scientific USA
  • SOSG reagent was added to temoporphine-loaded liposomes, exposed to a 671 nm laser source (LaserLab, Korea), and monitored using a microplate reader at 504/525 nm wavelengths (excitation/emission).
  • MDA-MB-231 cells were seeded in 24-well plates and cultured overnight before experiments. After 24 h, the cells were washed with PBS, the medium was replaced with serum-free medium, and incubated with DiR-labeled CDV for 4 h. After completion of incubation, the serum-free medium was replaced with culture medium again. After 24 hours, cells were observed using a confocal laser scanning microscope (CLSM, Fluoview FV1000, Olympus, Japan). Image analysis was performed using the Fiji analysis program. The degree of fluorescence was quantified by measuring the average value of n > 3 pictures for each sample.
  • CLSM confocal laser scanning microscope
  • Proteins from cell lysates or CDV were separated by SDS-PAGE and then transferred to nitrocellulose membrane. Blots were blocked with 5% skim milk for 1 hour at room temperature, treated with diluted primary antibody, incubated overnight at 4°C, and then incubated with HRP-conjugated secondary antibody (Invitrogen, USA) for 1 hour at room temperature. . Staining of the membrane was developed using ECL detection reagent (Bio-rad, USA). CD3zeta (Abcam, USA) was used as a CAR structural marker. CD63 and TSG101 (Invitrogen, USA) were used as exosome markers. GAPDH was used as a control.
  • mice All in vivo experiments were performed in accordance with the approved protocol guidelines of the Animal Care Committee of the clergy University Animal Hospital (Republic of Korea, CUK-IACUC-2021-017-01). All mice were housed under temperature and light controlled conditions (12 h light/dark cycle). Eight-week-old female BABL/c nude mice were stabilized for one week and then used in animal experiments. To prepare the mouse tumor model, MDA-MB-231 cells (1 ⁇ 10 6 cells) were subcutaneously inoculated into the right flank of 9-week-old BALB/c nude mice. After 7 days, when the average tumor volume reached 100 mm 3 , mice were used for the experiment.
  • nanovesicles were injected intravenously into the mouse. Twenty-four hours later, the tumor was irradiated twice with a laser (laser dose, 100 J/cm 2 ) at 12-hour intervals using a 671 nm fiber-coupled laser system (LaserLab, Korea). Tumor size was measured with calipers, and tumor volume was calculated using the formula width ⁇ width ⁇ length ⁇ 0.5.
  • images were obtained via a fluorescently labeled organism bioimaging device (FOBI, Neo-Science, Suwon, Korea).
  • Chimeric antigen receptor is a targeting receptor that binds to specific proteins expressed by cancer cells.
  • CAR Chimeric antigen receptor
  • NK cells natural killer cells
  • EGFR Epidermal growth factor receptor
  • EGF epidermal growth factor
  • EGFR is associated with various diseases such as solid tumors (lung cancer, colon cancer, breast cancer, etc.) and SARS-CoV-2 infection.
  • the transfected cells were sequentially extruded in a mini extruder through membrane filters with various pore sizes to prepare anti-EGFR-CAR expressing cell-derived vesicles (CDVs) ( Figure 2A).
  • CDVs cell-derived vesicles
  • the surface charge and size of the control CDV and EGFR-CAR expressing CDV were first measured through nanoparticle tracking analysis (NTA). As a result, the surface charge and size of each CDV were determined. It was confirmed that there was no significant difference in size ( Figures 2b and 2c).
  • Fluorescence resonance energy transfer is a fluorescence-based technique that monitors interactions (such as fusion) between liposomes.
  • FRET Fluorescence resonance energy transfer
  • FRET liposomes fused with unlabeled control liposomes have a reduced surface density of fluorophores, and the molar ratio composition of the two fluorescent probes (NBD-PS and Rhod-PE) and lipid is important in FRET analysis (Table 1 and Figure 3a).
  • NBD-PS and Rhod-PE two fluorescent probes
  • lipid is important in FRET analysis (Table 1 and Figure 3a).
  • control liposomes and FRET liposomes having the lipid composition shown in Table 1 were mixed, then ethanol was added to a concentration of 30% EtOH compared to the total solution, and after 1 hour, a volume of phosphoric acid three times the total volume was added. It was purified by adding buffered saline.
  • the degree of FRET of liposomes induced by fusion by each method was measured, and the results showed that the group that used the method using a 30% EtOH solution or electroporation performed It was confirmed that fusion occurred as the fluorescence value of NBD increased.
  • the group fused with 30% EtOH showed a similar fluorescence value to Triton x-100, a positive control well known to dissolve lipid membranes, and showed a higher fluorescence value than Triton x-100 1 hour after fusion ( Figure 3d).
  • the degree of FRET after inducing fusion of FRET liposomes and HEK293 CDV with the lipid composition shown in Table 1 in the same manner, there was no change in fluorescence value, unlike the group using Triton x-100, which was the positive control, and the negative control group. Fluorescence values similar to those of the phosphoPBS group were confirmed, showing that fusion between the FRET liposome and CDV was not triggered under any conditions except triton x-100 (FIG. 3E).
  • Dual-functionalized nanoparticles that can target cancer and deliver cancer-specific drugs were created. Specifically, first, a hydrophobic photosensitizer (mTHPC), which generates reactive oxygen species (ROS) in response to laser, was encapsulated in cationic liposomes (Figure 5a). As a result of checking the size of the liposome encapsulated with the photosensitizer, it was confirmed that the diameter was about 307 nm ( Figure 5c) and that it had a positive surface charge (32 mV) on the surface ( Figure 5b). In addition, as a result of TEM imaging of the liposome encapsulated with the photosensitizer, a spherical shape was observed (Figure 5d).
  • mTHPC hydrophobic photosensitizer
  • ROS reactive oxygen species
  • hybrid nanovesicles were prepared by fusing liposomes loaded with a photosensitizer and CDV expressing EGFR-CAR, and their properties and effects were confirmed.
  • the hybrid nanovesicles were prepared by fusing liposomes and CDV with 30% EtOH as in Experimental Example 2.
  • the sizes of control CDV, EGFR-CDV, liposomes fused with control CDV, and liposomes fused with EGFR-CDV were 145, 165, 132, and 173 nm, respectively, showing no significant difference in size (Figure 6b).
  • hybrid nanovesicles fused with liposomes loaded with EGFR-CAR expressing CDV and photosensitizer were intravenously administered to a tumor mouse model, and then laser was irradiated to kill the tumor caused by the hybrid nanovesicles. The killing effect was evaluated.
  • the tumor size in the group irradiated with the laser was significantly reduced compared to the group not irradiated with the laser. According to the present invention, It was confirmed that the hybrid nanoparticles were effectively absorbed into cancer cells and then responded to the laser to generate active oxygen, effectively killing cancer cells and inhibiting tumor growth (Figure 7d).
  • the hybrid nanoparticle according to the present invention can effectively target cancer cells through cancer cell-specific CAR and kill cancer cells by effectively delivering drugs for photodynamic therapy into cancer cells.
  • the hybrid nanoparticles have excellent biocompatibility and do not induce cell death unless irradiated with a laser, so the risk of side effects due to non-specific drug activity can be significantly reduced.
  • the present inventors have discovered an effective fusion method of CDV and liposomes, and various types of CDV and liposomes can be fused without being limited to the type of active ingredient (FIG. 8).
  • those skilled in the art can prepare hybrid nanoparticles with dual functions through the fusion of liposomes loaded with various drugs and CDV expressing appropriate active ingredients according to the purpose, and the nanoparticles can be used for the treatment of various drugs. It is expected that it can be used for.
  • Table 3 below shows sequence information of the components used in the present invention.
  • the present invention relates to a fusion technology of cell-derived vesicles and liposomes and to hybrid nanoparticles produced through the fusion technology, wherein the hybrid nanoparticles can exert both targeting functions and drug delivery functions to target tissues.
  • the present inventors discovered the optimal conditions to effectively fuse cell-derived vesicles and liposomes, and through this, cell-derived vesicles expressing active ingredients and drug-encapsulated liposomes were fused to possess all of their functions. Since hybrid nanoparticles, that is, dual-functional hybrid nanoparticles, can be manufactured, a wide range of types of hybrid nanoparticles can be manufactured depending on the purpose.
  • hybrid nanoparticles prepared by fusing a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with a photosensitizer effectively target cancer cells through the chimeric antigen receptor, and the photosensitizer It can kill cancer cells by generating active oxygen in response to light stimulation. Therefore, the hybrid nanoparticle according to the present invention can effectively target target tissues and deliver drugs through a combination of various cell-derived vesicles and liposomes, and is expected to be a promising therapeutic platform that can be used as a drug delivery vehicle and therapeutic agent in various fields. It is expected.

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Abstract

La présente invention concerne la technologie de fusion de vésicules et de liposomes dérivés de cellules, ainsi que des nanoparticules hybrides fabriquées par la technologie de fusion, les nanoparticules hybrides pouvant présenter à la fois une fonction de ciblage de tissu spécifique et une fonction d'administration de médicament. En particulier, les présents inventeurs ont découvert des conditions optimales pour fusionner efficacement des vésicules et des liposomes dérivés de cellules. Dans les conditions, il est possible de fusionner des vésicules dérivées de cellules exprimant une substance active à des liposomes encapsulés dans un médicament pour produire des nanoparticules hybrides possédant les fonctions des deux, c'est-à-dire des nanoparticules hybrides à double fonction. Ainsi, la présente invention permet la fabrication d'une large gamme de nanoparticules hybrides en fonction de l'objectif. En particulier, des nanoparticules hybrides fabriquées par fusion de vésicules dérivées de cellules contenant des récepteurs antigéniques chimériques spécifiques de l'antigène tumoral à des liposomes portant des photosensibilisateurs peuvent cibler efficacement des cellules cancéreuses à travers les récepteurs antigéniques chimériques et peuvent détruire des cellules cancéreuses par génération d'espèces réactives de l'oxygène en réponse à une stimulation lumineuse par les photosensibilisateurs. Par conséquent, les nanoparticules hybrides selon la présente invention peuvent cibler efficacement les tissus visés et administrer des médicaments par l'intermédiaire de l'association de divers vésicules et liposomes dérivés de cellules et sont censées être une plateforme thérapeutique prometteuse qui peut être utilisée en tant que vecteurs de médicament et agents thérapeutiques dans divers domaines.
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