Lipid nanostructure complex comprising antibody segment binding to surface protein of lipid nanostructure, and use thereof

Abstract

The present invention relates to a lipid nanostructure complex comprising a lipid nanostructure, an antibody segment that binds to a surface protein of the lipid nanostructure, a linker that binds to one end of the antibody segment, and a ligand that binds to the other end of the linker, a drug delivery vehicle in which a drug is encapsulated in the lipid nanostructure complex, a pharmaceutical composition for cancer treatment comprising the drug delivery vehicle, and a method for manufacturing the lipid nanostructure complex. More specifically, the lipid nanostructure complex of the present invention is an extracellular vesicle in which an antibody segment that binds to a surface protein of the extracellular vesicle binds to a target ligand through a linker, and possesses excellent cell and / or tissue targeting ability, thereby exhibiting an excellent drug delivery effect. In particular, since the extracellular vesicle equipped with the target ligand of the present invention has been experimentally proven and confirmed to have excellent drug efficacy when a drug is encapsulated, the lipid nanostructure complex of the present invention can be developed as an active ingredient in pharmaceutical compositions for the treatment of various types of cancer.

Description

Technology Field

[0001] The present invention relates to a lipid nanostructure complex, and more specifically, to a lipid nanostructure complex comprising a lipid nanostructure, an antibody segment that binds to a surface protein of the lipid nanostructure, a linker that binds to one end of the antibody segment, and a ligand that binds to the other end of the linker, a drug delivery vehicle in which a drug is encapsulated in the lipid nanostructure complex, a pharmaceutical composition for cancer treatment comprising the drug delivery vehicle, and a method for manufacturing a lipid nanostructure complex. Background Technology

[0003] Drug delivery systems currently in use have been reported to cause high-dose administration of therapeutic drugs, frequent repeated administration, and various side effects through non-specific methods. Although several 'targeted therapies' have recently been developed, their indications and targeting capabilities are extremely limited, which limits their ability to solve the fundamental problems of drug delivery systems.

[0004] In particular, the direction of research in the development of anticancer drugs and cancer treatment is shifting from existing 'tumor-nonspecific' treatment methods to 'tumor-specific' treatment methods. This represents a significant change that can overcome the various limitations of existing nonspecific treatment methods, such as issues regarding indications, targeting ability, and side effects.

[0005] In the present invention, exosomes, referred to as extracellular vesicles, are vesicles with a size of 30 to 200 nm that transport essential substances necessary for the body, such as lipids, proteins, and RNA, and transmit signals between cells. Since they are secreted from all cell types, they are utilized as drug delivery systems through their ability to perform early diagnosis of diseases and deliver drugs.

[0006] By loading anticancer drugs onto extracellular vesicles that target specific cancer cells and administering them so that only specific cancer cells are accurately attacked without affecting normal cells, a way may be opened to reuse existing anticancer substances that had excellent anticancer effects but could not be used due to their strong toxicity.

[0007] As such, by switching from a 'nonspecific' treatment method to a 'specific' treatment method, it is possible to develop an effective drug delivery system that can significantly reduce treatment side effects and significantly decrease the dosage.

[0009] The patent documents and references mentioned in this specification are incorporated by reference into this specification to the same extent that each document is individually and clearly identified by reference. Prior art literature

[0011] Republic of Korea Published Patent No. 10-2019-0132977 (Date of Publication: Nov. 29, 2019) The problem to be solved

[0012] The inventors have made research efforts to develop a drug delivery system with enhanced targeting ability of extracellular vesicles toward cancer cells and disease targets related to drug delivery capacity. As a result, the present invention was completed by experimentally demonstrating and confirming that an extracellular vesicle comprising an antibody fragment that binds to an extracellular vesicle surface protein, wherein the antibody fragment binds to a target ligand via a linker, possesses excellent targeting ability and thereby significantly enhances drug efficacy.

[0013] Accordingly, the object of the present invention is to provide a lipid nanostructure complex comprising a lipid nanostructure, an antibody segment that binds to a surface protein of the lipid nanostructure, a linker that binds to one end of the antibody segment, and a ligand that binds to the other end of the linker.

[0014] Another objective of the present invention is to provide a drug delivery system in which a drug is encapsulated in the lipid nanostructure complex.

[0015] Another objective of the present invention is to provide a pharmaceutical composition for cancer treatment comprising a therapeutically effective amount of the drug delivery system and a pharmaceutically acceptable carrier.

[0016] Another objective of the present invention is to provide a method for manufacturing the lipid nanostructure composite.

[0017] Other objects and technical features of the present invention are more specifically presented by the following detailed description of the invention, claims, and drawings. means of solving the problem

[0019] According to one aspect of the present invention, the present invention provides a lipid nanostructure complex comprising (i) a lipid nanostructure; (ii) an antibody segment that binds to a surface protein of the lipid nanostructure; (iii) a linker, one end of which binds to the antibody segment; and (iv) a ligand that binds to the other end of the linker.

[0020] In the present invention, the term “ligand” refers to a structure capable of binding to a receptor on the surface of a target cell.

[0021] According to one embodiment of the present invention, the lipid nanostructure may be extracellular vesicles, liposomes, or lipid nanoparticles, but is not limited thereto.

[0022] In the present invention, the term “extracellular vesicle” refers to small vesicles in the form of a phospholipid bilayer (Exosomes 30–100 nm; Microvisicles 100–1,000 nm) produced in most eukaryotic cells (bacteria, archaea, or eukaryotes, etc.), and is called by various names (Exosomes, Microvesicles, Ectosomes, Membrane vesicles, Nanovesicles, or Outer membrane vesicles, etc.) depending on the mechanism of secretion and its size.

[0023] In the present invention, the exosome referred to as an extracellular vesicle is used interchangeably with extracellular vesicle throughout the entire specification.

[0024] According to one embodiment of the present invention, the surface protein may be a surface protein derived from an extracellular vesicle.

[0025] The above extracellular vesicle-derived surface proteins may be selected from the group consisting of Table 1 below.

[0026] [Table 1]

[0027]

[0029] In one embodiment of the present invention, the extracellular vesicle-derived surface protein may be CD81.

[0030] The above CD81 is used as an extracellular vesicle (exosome) marker and is a representative exosome-derived surface protein that is involved in cell motility. It is used as an extracellular vesicle (exosome) marker and forms TEM with α5β1 / α3β1 integrins via EWI-2 to regulate cell adhesion-related properties, promote integrin activation and outside-in signaling, and facilitate integrin-dependent cell migration. Additionally, it activates α5β1 and α3β1 integrins to increase binding affinity to fibronectin and simultaneously amplifies adhesion outside-in signaling by these integrins.

[0031] According to one embodiment of the present invention, the antibody fragment may be selected from the group consisting of scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0032] In the present invention, the term “antibody” refers to an intact immunoglobulin, or its antigen-binding portion that competes with the intact antibody for specific binding. The antigen-binding portion may be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. The antigen-binding portion particularly includes Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), and complementarity determining region (CDR) fragments, variable region fragments, scFv, chimeric antibodies, diabodies, triabdies, tetrabodies, and polypeptides containing at least a portion of immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0033] The above Fab fragment is a monovalent fragment having VL, VH, CL, and CH1 domains; the F(ab')2 fragment is a divalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; the Fd fragment has VH and CH1 domains; the Fv fragment has VL and VH domains on one arm of the antibody; and the dAb fragment has an antigen-binding fragment of a VH domain, a VL domain, or a VH or VL domain.

[0034] In the present invention, the term "scFv (single chain variable fragment)" refers to an antibody in which VL and VH regions are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the linker is long enough to cause the protein chain to fold on the chain itself to form a monovalent antigen-binding site (see, e.g., [Bird et al., 1988, Science 242:423-26] and [Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83]). A diabody is a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain contains VH and VL domains connected by a linker that causes each domain to pair with a complementary domain on another polypeptide chain, as each polypeptide chain is too short to form a pair between two domains on the same chain (see, e.g., [Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48] and [Poljak et al., 1994, Structure 2:1121-23]). If the two polypeptide chains of the diabody are identical, the resulting diabody will have two identical antigen-binding sites. Polypeptide chains of different sequences can be used to produce a diabody with two different antigen-binding sites. Similarly, triabadies and tetraabadies are antibodies that contain three and four polypeptide chains, respectively, and form three and four antigen-binding sites, respectively (which may be the same or different).

[0035] According to one embodiment of the present invention, the ligand may be one or more selected from the group consisting of small molecule compounds, aptamers, peptides, proteins, and antibodies.

[0036] In the present invention, the term “aptamer” refers to an oligonucleotide having a specific binding affinity for only one target.

[0037] According to another aspect of the present invention, a drug delivery system is provided in which a drug is encapsulated in the lipid nanostructure complex.

[0038] According to another aspect of the present invention, a pharmaceutical composition for cancer treatment is provided, comprising (i) a therapeutically effective amount of the drug delivery system; and (ii) a pharmaceutically acceptable carrier.

[0039] In the present invention, the term “therapeutic effective amount” refers to an amount of the drug delivery system administered to a patient that is sufficient and suitable for cancer treatment in vivo.

[0040] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, the “drug delivery vehicle,” and such carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, menthol, and mineral oil.

[0041] The pharmaceutical composition of the present invention may additionally include, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0042] Suitable dosages of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity.

[0043] The pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, it may be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and a person skilled in the art may select a suitable and effective method from among various known methods of administration.

[0044] The concentration of the drug delivery agent, which is an active ingredient included in the pharmaceutical composition of the present invention, can be determined by considering the therapeutic purpose, the patient's condition, the required duration, etc., and is not limited to a concentration within a specific range.

[0045] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0046] According to one embodiment of the present invention, the cancer treatment may be for treating a cancer selected from the group consisting of cervical cancer, uterine cancer, pancreatic cancer, breast cancer, bile duct cancer, gallbladder cancer, colorectal cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, lung cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, skin cancer, blood cancer, thyroid cancer, parathyroid cancer, bone cancer, small intestine cancer, endocrine gland cancer, adrenal cancer, urethral cancer, and liver cancer.

[0047] According to another aspect of the present invention, a method for preparing the lipid nanostructure complex is provided, comprising the following steps: (a) transforming a cell with a vector containing a gene encoding an extracellular vesicle-derived surface protein; (b) isolating an extracellular vesicle from the transformed cell; (c) preparing a complex by binding an antibody segment that binds to the extracellular vesicle-derived surface protein to a ligand via a linker; and (d) preparing a lipid nanostructure complex by binding the prepared complex to the extracellular vesicle isolated in step (b).

[0048] In relation to step (a) above, the “coding” refers to a polynucleotide referred to as “coding” for a polypeptide, where it can be transcribed and / or translated to produce mRNA for a polypeptide and / or its fragments when manipulated in its natural state or by a method well known to those skilled in the art.

[0049] The term “polynucleotide” is used interchangeably and refers to a polymeric form of a nucleotide of any length of ribonucleotide or deoxyribonucleotide. The term polynucleotide includes, but is not limited to, polymers comprising single, double, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or purine and pyrimidine bases or other naturally, chemically or biochemically modified, unnatural, or derivatized nucleotide bases.

[0050] In the present invention, the polynucleotides encoding the proteins intended for expression may undergo various modifications to the coding region within a range that does not alter the amino acid sequence of the protein expressed from the coding region, due to codon degeneracy or considering the codons preferred by the organism intended to express the protein; and various modifications may also be made to parts excluding the coding region within a range that does not affect gene expression, and those skilled in the art will understand that such modified genes are also included within the scope of the present invention. That is, as long as the polynucleotides of the present invention code for a protein having equivalent activity, one or more nucleic acid bases may be modified by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention.

[0051] In the present invention, the recombinant vector containing the polynucleotide may be any vector known in the art, and depending on the type of host cell to be used to produce the protein, expression-regulating sequences such as promoters, terminators, and enhancers, sequences for membrane targeting or secretion, etc., may be appropriately selected and combined in various ways according to the purpose.

[0052] The vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable vectors may be prepared in various ways depending on the purpose, including signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers.

[0053] In the present invention, the vector includes antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes for ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, furomycin, and tetracycline.

[0054] In the present invention, the terms “transformation” or “transfection” refer to the process of introducing a foreign nucleotide sequence into a cell. Known transfection methods may be used for transformation, such as microinjection (Capecchi, MR, Cell 22, 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology 52, 456 (1973)), electroporation (Neumann, E. et al., EMBO J. 1, 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10, 87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol. 5, 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci. USA 87, 9568-9572 (1990)), but are not limited thereto.

[0055] In the present invention, the step of selecting transformed cells can be easily carried out by utilizing the phenotype expressed by the selection marker of the vector described above. For example, if the selection marker is a specific antibiotic resistance gene, transformed cells can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0056] In relation to step (b) above, the method for isolating extracellular vesicles from the transformed cells is not particularly limited, and can be isolated using, for example, a TFF system.

[0057] In relation to step (c) above, the linker is not particularly limited and, preferably, can be a peptide-type linker.

[0058] In an embodiment of the present invention, a conjugate is prepared in which anti-CD81scFv, which binds to the surface protein CD81 of the extracellular vesicle, is linked to anti-GPC3scFv, a liver cancer target ligand, by a linker peptide. More specifically, the gene encoding the amino acid sequence of anti-CD81scFv, the linker peptide, and anti-GPC3scFv constituting the conjugate is linked to synthesize a single nucleotide sequence, which is then cloned into an expression vector, and the vector is transfected into HEK293 cells to transform them. Subsequently, the conjugate expressed in the transformed HEK293 cells is purified to obtain the result.

[0059] In relation to step (d) above, the binding of the surface protein of the extracellular vesicle to the conjugate prepared in step (c) above is an 'antigen-antibody response' binding, which is the strongest binding reaction occurring in the human body after covalent bonding; electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions all work together to form a tight, lock-and-key bond.

[0060] According to one embodiment of the present invention, the method for manufacturing the lipid nanostructure complex may further include the step of encapsulating a drug in the separated extracellular vesicle.

[0061] The above drug encapsulation can be performed by encapsulating the drug in extracellular vesicles using an electrophoration method with a NEPA21 GENE electrophorator at 200V and 50ms.

[0062] In the method for manufacturing a lipid nanostructure composite of the present invention, the details regarding the lipid nanostructure composite are identical to those described in the “lipid nanostructure composite,” which is another embodiment of the present invention described above; therefore, they are referred to by reference and are not described redundantly. Effects of the invention

[0064] More specifically, the lipid nanostructure complex of the present invention is an extracellular vesicle in which an antibody fragment binding to an extracellular vesicle surface protein binds to a target ligand via a linker, and possesses excellent cell and / or tissue targeting ability, thereby exhibiting an excellent drug delivery effect. In particular, since the extracellular vesicle equipped with the target ligand of the present invention has been experimentally proven and confirmed to have excellent drug efficacy when a drug is encapsulated, the lipid nanostructure complex of the present invention can be developed as an active ingredient in pharmaceutical compositions for the treatment of various types of cancer. Brief explanation of the drawing

[0066] Figure 1 shows the detailed structure of a lipid nanostructure complex containing a protein (e.g., antibody fragment) that binds to a surface protein of the lipid nanostructure. Figure 2 is a schematic diagram of the production of a vector capable of overexpressing CD81 using a lentivirus vector. Figure 3 shows the results of confirming the size of extracellular vesicles isolated from HEK293 cells overexpressing CD81, an extracellular vesicle-derived surface protein. Figure 4 shows the results of a Western blot analysis of surface markers on extracellular vesicles repeatedly isolated from HEK293 cells overexpressing CD81. Figure 5 is the result of confirming the liver cancer cell targeting rate of extracellular vesicles equipped with the liver cancer target anti-GPC3scFv of the present invention. Figure 6 is the result of confirming the liver cancer tissue targeting rate of extracellular vesicles equipped with the liver cancer target anti-GPC3scFv of the present invention. Figure 7 shows the results of analyzing the tumor size in a liver cancer mouse model after administering a drug encapsulated in an extracellular vesicle equipped with the liver cancer target anti-GPC3scFv of the present invention to a liver cancer mouse model. Figure 8 shows the results of immunohistochemical staining for BCL-XL and BIM expression in liver tissue of a liver cancer mouse model after administering a drug encapsulated in an extracellular vesicle loaded with the liver cancer target anti-GPC3scFv of the present invention to a liver cancer mouse model. Specific details for implementing the invention

[0067] Examples

[0068] Experimental method

[0069] 1. Construction of a cell line overexpressing CD81, an exosome-derived surface protein

[0070] HEK293 cells were plated in a culture flask and cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U / mL penicillin (Thermo Fisher Scientific), and 0.1 mg / mL streptomycin (Thermo Fisher Scientific).

[0071] CD81, a surface protein derived from extracellular vesicles To produce an overexpressing cell line, the gene encoding CD81 was cloned into a lentivirus vector, and HEK293 cells were transfected with the lentivirus vector containing the CD81 gene (Fig. 2).

[0072] In summary, HEK293 cells were transformed into cells that overexpress CD81 to create a cell line capable of producing extracellular vesicles with a large amount of CD81 mounted on their surface.

[0074] 2. Isolation and Concentration of Extracellular Vesicles

[0075] After culturing HEK293 cells transformed to overexpress CD81 for 24 hours, the medium was replaced with serum-free (FBS-free) DMEM medium. After 24 hours, the HEK293 cell culture medium was removed from the plate and centrifuged at 1000 RPM to remove the cells. To isolate extracellular vesicles, extracellular vesicles were isolated from the recovered cell culture medium using a TFF system (300 kDa, 10 psi, Repligen) and concentrated 10-fold. More specifically, to remove the cell culture medium contained in the isolated extracellular vesicles, dilution and concentration were performed simultaneously using a TFF system (300 kDa, 10 psi) with PBS at 20 times the volume of the culture medium.

[0077] 3. Analysis of extracellular vesicles

[0078] The above-described separated and concentrated extracellular vesicles were diluted 100-fold with PBS and injected into the Zetaview DLS instrument using a syringe to confirm the size and number of extracellular vesicles.

[0080] 4. Preparation of extracellular vesicles loaded with the liver cancer-targeted anti-GPC3scFv

[0081] 4.1 Preparation of a complex binding to the surface protein CD81 of extracellular vesicles

[0082] The “antibody fragment-linker-target ligand” conjugate that binds to the surface protein CD81 of the extracellular vesicle is a conjugate in which the antibody fragment anti-CD81scFv (amino acid sequence of SEQ ID NO. 1) is linked to the liver cancer target ligand anti-GPC3scFv by a linker peptide (amino acid sequence of SEQ ID NO. 2), and the anti-GPC3scFv may be selected from the group consisting of the amino acid sequences of SEQ ID NOs. 3 to 8.

[0083] More specifically, in an embodiment of the present invention, the “antibody fragment-linker-target ligand” conjugate that binds to the surface protein CD81 of the extracellular vesicle is a conjugate in which the antibody fragment anti-CD81scFv (amino acid sequence of SEQ ID NO. 1) is linked to the liver cancer target ligand anti-GPC3scFv (amino acid sequence of SEQ ID NO. 3) by a linker peptide (amino acid sequence of SEQ ID NO. 2).

[0084] The amino acid sequences of anti-CD81scFv, linker peptide, and anti-GPC3scFv constituting the above conjugate are shown in Table 2 below.

[0086] [Table 2]

[0087]

[0089] The genes encoding the amino acid sequences of anti-CD81scFv, linker peptide, and anti-GPC3scFv constituting the above conjugate were linked to synthesize a single nucleotide sequence, cloned into an expression vector, and then transfected HEK293 cells with the vector. The above conjugate expressed in the transfected HEK293 cells was purified and obtained.

[0091] 4.2 Preparation of Extracellular Vesicles Loaded with Liver Cancer Target Anti-GPC3scFv

[0092] An extracellular vesicle equipped with the liver cancer target anti-GPC3scFv was prepared by combining a conjugate containing the liver cancer target ligand anti-GPC3scFv with the isolated extracellular vesicle. More specifically, the isolated extracellular vesicle was prepared by reacting the conjugate containing the liver cancer target anti-GPC3scFv and anti-CD81scFv, which binds to CD81, a surface protein of the extracellular vesicle, at 4°C overnight.

[0094] 5. Production of a liver cancer mouse model

[0095] A liver cancer model was conceived after 2 weeks by injecting the liver cancer cell line, Huh7 cell line, into the livers of mice. In the conceived liver cancer mouse model, extracellular vesicles loaded with the liver cancer-targeting anti-GPC3scFv were injected into the tail vein of the liver cancer mouse model at a rate of 10 times twice a week. 8 Changes in tumor size were measured by injecting into dogs for 3 weeks.

[0097] 6. Immunohistochemical staining

[0098] For immunohistochemical staining, formalin-fixed and paraffin-embedded tissue sections were deparaffinized, rehydrated with ethanol, and epitope retrieval was performed according to standard procedures.

[0099] Antibodies against BCL-XL and BIM (Cell Signaling Technology, MA) were used for immunohistochemical staining. Tissue samples were analyzed under a laser-scanning microscope (Eclipse TE300; Nikon).

[0101] Experimental results

[0102] 1. Analysis of extracellular vesicles isolated from CD81-overexpressing HEK293 cells

[0103] As a result of analyzing extracellular vesicles isolated from HEK293 cells overexpressing CD81, an extracellular vesicle-derived surface protein, using Zetaview DLS equipment, the size of the extracellular vesicles was 110 nm and the number was 1.2 x 10⁶ 6 It was confirmed as / ml.

[0105] 2. Identification of surface markers on extracellular vesicles isolated from CD81-overexpressing HEK293 cells

[0106] Western blot analysis of surface markers of extracellular vesicles repeatedly isolated from HEK293 cells overexpressing CD81 confirmed the expression of CD81 and CD9, which are surface markers of extracellular vesicles (Fig. 4).

[0108] 3. Confirmation of liver cancer cell targeting rate according to the present invention

[0109] Extracellular vesicles (Exo-CD81) loaded with the liver cancer targeting anti-GPC3scFv of the present invention were loaded into Huh7 cell lines at a rate of 5 x 10 6 The movement into liver cancer cells was confirmed using an evos5000 fluorescence microscope after treatment. As a result of the analysis, it was confirmed that extracellular vesicles (Exo-CD81) loaded with the liver cancer target anti-GPC3scFv showed significantly increased movement into liver cancer cells compared to non-target extracellular vesicles (Exo ct) (Fig. 5).

[0111] 4. Confirmation of liver cancer tissue targeting rate according to the present invention

[0112] The migration of extracellular vesicles was confirmed by intravenously injecting extracellular vesicles loaded with the liver cancer target anti-GPC3scFv of the present invention, stained with exoglow, into the tail of a mouse model of liver cancer prepared by injecting a liver cancer cell line into the mouse liver. As a result of the analysis, it was confirmed that the extracellular vesicles loaded with the liver cancer target anti-GPC3scFv of the present invention migrated more to the liver cancer tissue than non-target extracellular vesicles (Exosome only) (Fig. 6).

[0114] 5. Confirmation of drug efficacy in a liver cancer orthotopic mouse model according to the present invention

[0115] To confirm the drug efficacy of the extracellular vesicles (target extracellular vesicles) equipped with the liver cancer target anti-GPC3scFv of the present invention, doxorubicin was encapsulated in each of the non-target extracellular vesicles and target extracellular vesicles by electrophoration using a NEPA21 GENE electrophorator at 200V and 50ms.

[0116] When the tumor size was checked by intravenously injecting non-target extracellular vesicles containing doxorubicin (Exosome Doxorubicin) and target extracellular vesicles containing doxorubicin (Exosome CD81-GPC3 Doxorubicin) into an orthotopic mouse model of liver cancer, it was experimentally confirmed that the tumor size was significantly reduced when target extracellular vesicles containing doxorubicin (Exosome CD81-GPC3 Doxorubicin) were administered compared to when non-target extracellular vesicles containing doxorubicin (Exosome Doxorubicin) were administered (Fig. 7).

[0117] In addition, liver tissue was obtained from the above-mentioned liver cancer mouse model, and the expression of BCL-XL and BIM, which are associated with the mechanism of apoptosis induced by doxorubicin, was confirmed by immunohistochemical staining. As a result of the analysis, it was confirmed that when a targeted extracellular vesicle encapsulated with doxorubicin (Targeted Exosome (CD81-GPC3) + Doxorubicin) was administered to the liver tissue of the liver cancer mouse model, the expression of BCL-XL decreased further and the expression of BIM increased further compared to when a non-targeted extracellular vesicle encapsulated with doxorubicin (Exosome + Doxorubicin) was administered (Fig. 8).

[0118] In summary, it was confirmed that in a liver cancer mouse model, administering a drug encapsulated in extracellular vesicles equipped with the liver cancer target anti-GPC3scFv of the present invention resulted in significantly superior drug efficacy compared to administering the drug encapsulated in non-target extracellular vesicles.

[0120] The specific embodiments described herein represent preferred embodiments or examples of the invention and do not limit the scope of the invention. It is evident to those skilled in the art that variations and other uses of the invention do not deviate from the scope of the invention as described in the claims of this specification.

Claims

Claim 1 (i) an extracellular vesicle capable of encapsulating a drug, comprising an extracellular vesicle-derived surface protein CD81; (ii) an anti-CD81 antibody fragment (scFv) composed of the amino acids of SEQ ID NO. 1, bound to the surface protein CD81; and (iii) a peptide linker composed of the amino acid sequence of SEQ ID NO. 2, having one end bound to the anti-CD81 antibody fragment (scFv) and the other end bound to an anti-GPC3 antibody scFv capable of binding to GPC3, a liver cancer target receptor; wherein the drug comprises doxorubicin. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 A method for manufacturing a lipid nanostructure complex for drug delivery according to claim 1, comprising: (a) transforming a cell with a vector containing a gene encoding an extracellular vesicle-derived surface protein CD81; (b) isolating an extracellular vesicle from the transformed cell; (c) preparing a conjugate by binding an anti-CD81 antibody fragment (scFv) consisting of the amino acids of SEQ ID NO. 1 that binds to the extracellular vesicle-derived surface protein CD81 with an anti-GPC3 antibody scFv by means of a peptide linker consisting of the amino acid sequence of SEQ ID NO. 2; and (d) preparing a lipid nanostructure complex by binding the prepared conjugate to the extracellular vesicle isolated in step (b); wherein the drug is encapsulated in the isolated extracellular vesicle. Claim 11 delete

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