Azide lipid-containing extracellular vesicle, method for producing same, and composition containing same

Abstract

Disclosed is an azide lipid-containing extracellular vesicle containing, in a lipid bilayer, an extracellular vesicle marker protein and an azide lipid derivative obtained by binding an azide group to a hydrophilic moiety of a lipid. The extracellular vesicle has an azide group on the surface thereof, and thus can easily bind various substances (for example, chemicals) via the azide group. For example, in a patient having a disease in a specific tissue, the extracellular vesicle is effective for delivering chemicals to the specific tissue.

Description

Azidolipid-containing extracellular vesicles, their preparation method, and compositions containing them

[0001] The present invention relates to azidolipid-containing extracellular vesicles, methods for their preparation, and compositions containing them.

[0002] It is generally known that when a pharmacologically effective drug is administered to the human body, it inevitably produces undesirable side effects in addition to the intended efficacy of the drug. In recent years, drug delivery systems (DDS) have been proposed to reduce such side effects and enhance drug efficacy. By using DDS, drugs of interest can be selectively delivered to the lesion, thereby achieving efficacy at lower doses and reducing the risk of the drug being delivered to body parts other than the lesion, causing undesired side effects. Regarding the application of organic polymer nanostructures to drug delivery systems, the key to their development lies in how to impart controlled release (sustained release) and targeting (cell specificity and affinity) functions to drug-encapsulating capsules (called "carriers").

[0003] Liposomes, which are nanoparticles made of phospholipids, have been shown to be highly useful as carriers for such drug delivery systems. Liposomes encapsulating drugs have the advantage of being relatively easy to form in a test tube by mixing a drug and phospholipids, making them a widely used technology in the field of drug delivery systems. However, because liposomes are a system that is reconstituted in a test tube, it is quite difficult to impart targeting capabilities to the liposomes themselves.

[0004] Recently, it has become known that cells release a type of vesicle called exosomes. Exosomes are lipid bilayer vesicles with a diameter of 20 to 150 nm. Exosomes encapsulate proteins and nucleic acids such as miRNA and mRNA, and also possess proteins on their surface. Exosomes are typically formed in multivesicular bodies formed within cells by budding from the cytoplasm toward the lumen of the multivesicular bodies. They are then released extracellularly upon fusion of the multivesicular bodies with the cell membrane. Exosomes contain marker molecules derived from their cell of origin, have directional properties consistent with the characteristics of the cell of origin, and are believed to be involved in physiological functions such as blood coagulation and intercellular signaling. Upon contact with target cells, such exosomes are internalized by endocytosis, and the lipid bilayer membranes of the endosome and exosome fuse, releasing the proteins and nucleic acids encapsulated in the exosomes into the cytoplasm.

[0005] Attempts have been made to apply exosomes to drug delivery systems, utilizing the targeting properties of exosomes to selectively deliver drugs to specific cells or tissues. For example, Patent Document 1 discloses a hybrid liposome-exosome, which is a complex of liposomes encapsulating a physiologically active substance and exosomes, where the exosomes are vesicles released from cells, have a diameter of 30 to 200 nm, and contain phospholipids, cholesterol, proteins, and nucleic acids. According to the invention described in Patent Document 1, by complexing liposomes and exosomes, it is possible to encapsulate the substance encapsulated in the liposomes in the exosomes as well. Furthermore, Patent Document 2 discloses exosomes prepared by a method for isolating exosomes, comprising the steps of: providing a sample containing exosomes; contacting the sample with a binder having affinity for a target protein selected from PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment or variant thereof; and isolating the exosomes based on the binding between the target protein and the binder. The invention described in Patent Document 2 is said to have the purpose of improving the efficiency of exosome isolation and purification methods and thereby improving the efficiency of treatments and the like that use exosomes.

[0006] Patent No. 6137894 International Publication No. 2019 / 040920

[0007] The present inventors have found that by culturing cells in the presence of azidolipid derivatives, which are lipids with azide groups attached to their hydrophilic sites, it is possible to produce extracellular vesicles with azide groups on their surfaces, and to easily attach drugs via these azide groups. The present invention is based on this finding.

[0008] Thus, the present invention provides extracellular vesicles having azide groups on their surfaces, methods for producing the extracellular vesicles, and compositions containing the extracellular vesicles.

[0009] According to one aspect of the present invention, there is provided an azide lipid-containing extracellular vesicle comprising an extracellular vesicle marker protein and an azide lipid derivative in a lipid bilayer, the azide group being bound to the hydrophilic portion of the lipid.

[0010] According to another aspect of the present invention, there is provided a method for producing azido lipid-containing extracellular vesicles, the method comprising the steps of (a) culturing cells in the presence of an azido lipid derivative in which an azide group is bound to the hydrophilic moiety of a lipid, and (b) recovering the extracellular vesicles from the cells.

[0011] According to another aspect of the present invention, there are provided azidolipid-containing extracellular vesicles produced by the method of the present invention.

[0012] According to another aspect of the present invention, there is provided an extracellular vesicle composition comprising the azido lipid-containing extracellular vesicles of the present invention and a pharmaceutically acceptable carrier.

[0013] According to the present invention, extracellular vesicles having azide groups on their surface are provided. Various substances (e.g., drugs) can be easily bound to the extracellular vesicles of the present invention via the azide groups on their surfaces. In particular, according to the present invention, extracellular vesicles that are effective in delivering drugs to specific tissues in patients with diseases of the specific tissues can be provided.

[0014] Figure 1 shows the viability of HEK293T cells in media containing 16:0 azidocaproyl PE (a lipid containing an azide group) and distearoylphosphatidylcholine (DSPC) at various concentrations. The horizontal axis of the graph represents lipid concentration, and the vertical axis represents cell viability (%) (cell viability at a lipid concentration of 0 μM is set to 100%). Figure 2 shows the inhibitory effect of exosomes bearing anti-CD19 antibody (AntiCD19) or anti-CD69 antibody (AntiCD69) on the surface via PEG4 or PEG12 on the proliferation of Namalwa and Reh cells, comparing the group containing exosomes conjugated via azide groups on the surface (Azide-Exo) with the group containing no azide groups (Control Exo). The vertical axis of this graph represents cell viability (%) (cell viability when using normal exosomes without antibody conjugation (control) is set to 100%). Figure 3 shows the viability of HEK293T cells in media containing the azide-containing lipids 16:0 azidocaproyl PE, 16:0 azidoethyl SM (d18:1 / 16:0), and 18:0-16:0(16-azido)PC at the indicated concentrations. The horizontal axis of this graph represents the lipid concentration, and the vertical axis represents cell viability (%) (cell viability in the sample without added lipid is set to 100%). Figure 4 is a graph comparing the inhibitory effect of exosomes bearing anti-CD69 antibodies on the surface via PEG4 on Reh cell proliferation when an azidolipid (16:0 azidoethyl SM (d18:1 / 16:0)) was used as a reagent to provide azide groups on the exosome surface, with an azidosugar (Ac4ManNAz) used. The vertical axis of this graph represents cell viability (%) (cell viability when 16:0 Az-PE was used as the azidolipid is set at 100%). Specific Description of the Invention

[0015] The azido lipid-containing extracellular vesicles of the present invention contain an extracellular vesicle marker protein and an azido lipid derivative in which an azide group is bound to the hydrophilic portion of a lipid in a lipid bilayer. Such extracellular vesicles can be produced by culturing cells in the presence of an azido lipid derivative in which an azide group is bound to the hydrophilic portion of a lipid and recovering the extracellular vesicles from the cells. The azido lipid-containing extracellular vesicles of the present invention can present a certain number of azide groups on their surface, and various substances (such as therapeutic or diagnostic drugs) can be bound via these azide groups by simple procedures such as click chemistry.

[0016] In the present invention, "extracellular vesicles" refer to vesicles with a heterogeneous lipid bilayer structure secreted from living cells. Extracellular vesicles are broadly classified into three types: exosomes, microvesicles (MVs), and apoptotic bodies, based on differences in their intracellular production mechanisms. Exosomes are formed by inward budding of late endosomal membranes, then fuse with the plasma membrane to form complete particles, which are secreted extracellularly by exocytosis. The approximate size of exosomes is 30-150 nm in diameter. Microvesicles (MVs) are generated by outward budding and separation of the plasma membrane. The size of microvesicles (MVs) varies widely (approximately 100-1000 nm in diameter). Apoptotic bodies are generated when cells undergo organized cell death (apoptosis). The size of apoptotic bodies varies widely (approximately 50-5000 nm in diameter), overlapping with that of exosomes and MVs. The extracellular vesicles of the present invention may be exosomes, microvesicles (MVs), or apoptotic bodies, but are preferably exosomes or microvesicles (MVs), and more preferably exosomes. Hereinafter, the present invention will be described in detail, focusing on exosomes, but those skilled in the art will understand that the present invention can also be applied to microvesicles (MVs) and apoptotic bodies.

[0017] According to a preferred embodiment of the present invention, the extracellular vesicle marker protein contained in the azide lipid-containing extracellular vesicles of the present invention is an exosome marker protein. Known exosome marker proteins include CD63, CD81, CD9, etc., and according to a further preferred embodiment of the present invention, the exosome marker protein is selected from CD63, CD81, and CD9.

[0018] CD63, CD81, and CD9 are membrane proteins that are localized in the lipid bilayer membrane of exosomes and are specifically recognized in exosomes. The properties of each of CD63, CD81, and CD9 are briefly described below.

[0019] CD63, also known as LAMP-3 (Lysosomal-associated membrane protein 3), is a 30-60 kDa lysosomal membrane protein belonging to the tetraspanin family that plays many important roles in immunophysiological functions. CD63 is known to mediate signal transduction related to the regulation of cell development, activation, proliferation, and motility. Because CD63 is expressed on activated platelets, it has been suggested that it may function as a platelet activation marker. CD63 is a lysosomal membrane glycoprotein that is known to translocate to the plasma membrane after platelet activation. The amino acid sequence of human CD63 is disclosed online under Accession No. P08962.2 in Genbank, a sequence information database provided by NCBI. The contents of this webpage are incorporated herein by reference.

[0020] CD81 is a single-chain protein belonging to the tetraspan family of proteins with four transmembrane domains. On the cell membrane, both the N- and C-termini of CD81 are cytoplasmic, with two peptide loops exposed extracellularly. CD81 is glycosylated and has a molecular weight of 26 kDa. CD81 has a wide tissue distribution and is known to exist in association with other tetraspan family members. Immune B cells express relatively high levels of CD81 throughout their differentiation stages. The amino acid sequence of human CD81 is disclosed online under Accession No. P60033.1 in the Genbank sequence database provided by NCBI. The contents of this webpage are incorporated herein by reference.

[0021] CD9 is a single-chain membrane protein with a molecular weight of 24 kDa and belongs to the tetraspan family. The CD9 antigen has four transmembrane domains, with both the N- and C-termini present intracellularly. CD9 is found in platelet alpha granules, monocytes, pre-B cells, eosinophils, basophils, and activated T cells. CD9 associates with molecules such as VLA (Very Late Activation) integrin molecules and HLA-DR, and is thought to be involved in cell-cell adhesion, signal transduction, and cell motility. The amino acid sequence of human CD9 is disclosed online under Accession No. P21926.4 in the sequence information database Genbank provided by NCBI. The contents of this webpage are incorporated herein by reference.

[0022] The exosomes of the present invention may contain any one or any two of the three exosome marker proteins, CD63, CD81, and CD9, but preferably contain CD63 as an essential exosome marker protein, and more preferably contain all three exosome marker proteins, CD63, CD81, and CD9.

[0023] In addition to exosome marker proteins localized in the lipid bilayer membrane of exosomes, exosomes can also be identified using exosome marker proteins encapsulated in exosomes as labels. Examples of such exosome marker proteins encapsulated in exosomes include Alix.

[0024] Alix (ALG-2-interacting protein X), also known as AIP1 (ALG-2-interacting protein 1) or Hp95, is a protein encoded by the PDCD6IP gene. Alix is ​​known to be involved in apoptosis (cell death) through a mechanism involving ALG-2 (apoptosis linked gene 2 product). ALG-2 is a 22 kDa protein with five repeated EF-hand structures and is known as a regulator of calcium-induced apoptosis following endoplasmic reticulum (ER) stress. Alix is ​​known to interact with ALG-2 via a proline-rich region at its C-terminus and to be involved in the formation of multivesicular bodies. The amino acid sequence of human Alix is ​​available from Genbank, a sequence information database provided by NCBI, under Accession No. It is disclosed on the Internet under Q8WUM4.1, the contents of which are incorporated herein by reference.

[0025] In the azido lipid-containing extracellular vesicles of the present invention, the azido lipid derivative contained in the lipid bilayer has an azide group bound to the hydrophilic site of the lipid.

[0026] The lipids constituting the azide lipid derivatives can be either simple lipids or complex lipids, or a combination of these. According to a preferred embodiment of the present invention, the lipids constituting the azide lipid derivatives are complex lipids.

[0027] Simple lipids are lipids composed only of alcohols and fatty acids, and specific examples include acylglycerol, sterol esters (cholesterol esters, etc.), wax esters, tocopherol esters, etc. The saturated aliphatic groups and unsaturated aliphatic groups (such as the acyl group of acylglycerol and the partner of the ester bond of sterol in the ester) that constitute the hydrophobic group contained in the simple lipid are typically saturated aliphatic groups and unsaturated aliphatic groups derived from saturated or unsaturated fatty acids having from 12 to 20 carbon atoms. Among these, examples of saturated fatty acids include lauric acid, myristic acid, pentadecylic acid, palmitic acid, heptadecanoic acid, stearic acid, and arachidic acid, while examples of unsaturated fatty acids include monounsaturated fatty acids (myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, paccenic acid, gadoleic acid, and eicosenoic acid), diunsaturated fatty acids (linoleic acid and eicosadienoic acid), triunsaturated fatty acids (linolenic acid and pinolenic acid), tetraunsaturated fatty acids (stearidonic acid and arachidonic acid), pentaunsaturated fatty acids (eicosapentaenoic acid), and hexaunsaturated fatty acids (hexadocosaenoic acid). The aliphatic groups constituting the glycerophospholipids and glyceroglycolipids may be a mixture of saturated and unsaturated aliphatic groups, or may be a mixture of aliphatic groups with different numbers of carbon atoms or different numbers of unsaturated groups.

[0028] According to one embodiment of the present invention, the simple lipid constituting the azido lipid derivative is at least one lipid selected from cholesterol esters, cholesterol ester derivatives, acylglycerols and acylglycerol derivatives.

[0029] According to a preferred embodiment of the present invention, the lipid constituting the azide lipid derivative is a complex lipid.The complex lipid constituting the azide lipid derivative can be a complex lipid known as a lipid bilayer membrane-forming, non-polar solvent-soluble, biologically derived substance, more specifically, a complex lipid having any reactive group selected from the group consisting of a primary amino group, a carboxyl group, a hydroxyl group, a phosphate group, and a thiol group at the end.These complex lipids can be selected from, for example, sphingophospholipids, sphingoglycolipids, glycerophospholipids, glyceroglycolipids, ether-type phospholipids, ether-type glycolipids, etc.

[0030] Examples of sphingophospholipids include sphingomyelin, and examples of sphingoglycolipids include cerebrosides (galactocerebroside, sulfatide, glucocerebroside, etc.), gangliosides, globosides, sulfatides, etc. Examples of glycerophospholipids include phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, etc. Examples of glyceroglycolipids include condensates of any monosaccharide or oligosaccharide with diacylglycerol, such as monogalactosyldiacylglycerol. In addition, the saturated and unsaturated aliphatic groups constituting the hydrophobic group of the diacylglycerol moiety constituting glycerophospholipids and glyceroglycolipids typically include saturated and unsaturated aliphatic groups derived from saturated or unsaturated fatty acids having 12 to 20 carbon atoms. Among these, examples of saturated fatty acids include lauric acid, myristic acid, pentadecylic acid, palmitic acid, heptadecanoic acid, stearic acid, and arachidic acid, while examples of unsaturated fatty acids include monounsaturated fatty acids (myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, paccenic acid, gadoleic acid, and eicosenoic acid), diunsaturated fatty acids (linoleic acid and eicosadienoic acid), triunsaturated fatty acids (linolenic acid and pinolenic acid), tetraunsaturated fatty acids (stearidonic acid and arachidonic acid), pentaunsaturated fatty acids (eicosapentaenoic acid), and hexaunsaturated fatty acids (hexadocosaenoic acid). The aliphatic groups constituting the above-mentioned glycerophospholipids and glyceroglycolipids may be a mixture of saturated and unsaturated aliphatic groups, or may be a mixture of aliphatic groups with different numbers of carbon atoms or different numbers of unsaturated groups.

[0031] Ether-type phospholipids and ether-type glycolipids are complex lipids in which one or two long-chain hydrocarbon groups are ether-bonded to glycerol and which have a phosphate group or sugar as a polar group; they are widely found in thermophilic bacteria, and platelet-activating factor and the like are known to have a similar structure. In this case, the two hydrocarbon groups bonded to glycerol are preferably saturated or unsaturated hydrocarbon groups having 12 to 20 carbon atoms. That is, ether-type phospholipids and ether-type glycolipids preferably have a structure in which one or two molecules of a higher saturated or unsaturated alcohol having 12 to 20 carbon atoms are bonded to glycerol via an ether bond, and a phosphate group is bonded to the remaining hydroxyl group of the glycerol.

[0032] Complex lipids are described in detail in Int. J. Mol. Sci. 2019, 20, 2167 and Nat. Rev. Mol. Cell Biol. , 2018, 19, 281, the contents of which are incorporated herein by reference.

[0033] The azide lipid derivatives used in the present invention can be produced by any chemical reaction method that covalently bonds an azide group to a phosphate group, amino group, hydroxyl group, or the like, which a lipid has in its hydrophilic portion.

[0034] According to a preferred embodiment of the present invention, the conjugated lipid constituting the azide lipid derivative is a sphingophospholipid, a glycerophospholipid, or a combination thereof, more preferably at least one lipid selected from the group consisting of sphingomyelin (SM), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and even more preferably sphingomyelin (SM).

[0035] According to a preferred embodiment of the present invention, the azido lipid derivative is at least one selected from the group consisting of azidoethylsphingomyelin, azidoethylphosphatidylcholine, and azidocaproylphosphatidylethanolamine, and more preferably azidoethylsphingomyelin. Specific examples of the azido lipid derivative include those having the following structure:

[0036]

[0037] The azide lipid-containing exosomes of the present invention may contain integrins. Integrins are cell membrane proteins known as cell adhesion molecules, and are known to play an important role in cell-to-extracellular matrix adhesion and cell-to-cell adhesion as receptors for the extracellular matrix. As protein molecules, they are heterodimers consisting of two subunits, an α chain and a β chain. The α chain is also called integrin α, and the β chain is also called integrin β. Numerous subclasses exist for each, allowing for diverse combinations between the α and β chain subclasses. These proteins form the integrin superfamily. They are known to bind to cytoskeletal microfilaments via adaptor proteins within cells and transmit intracellular signals.

[0038] By including integrin α and / or integrin β in the exosomes, the exosomes of the present invention can be conferred with targeting properties to cells constituting specific tissues. When the exosomes of the present invention contain both integrin α and integrin β, the total amount of each is preferably 0.01 ng to 100 ng per 1 ng of the total amount of the exosome marker proteins (i.e., one, two, or all of CD63, CD81, and CD9). The upper limit of the total amount of each of the integrin α and integrin β is preferably 50 ng and 10 ng, and the lower limit of the total amount of each of the integrin α and integrin β is preferably 0.05 ng and 0.1 ng. By ensuring that the total amount of each of the integrin α and integrin β falls within the above ranges, the targeting properties of the exosomes of the present invention to cells constituting specific tissues are enhanced, while nonspecific delivery to other tissues is effectively suppressed.

[0039] The targeting ability of the exosomes of the present invention generally varies depending on the type of integrin expressed in the exosomes. For example, when only integrin α is expressed in the exosomes, targeting ability toward vascular endothelial cells is thought to be enhanced, and when only integrin β is expressed in the exosomes, targeting ability toward cells that constitute lung tissue is thought to be enhanced. On the other hand, when both integrin α and integrin β are expressed in the exosomes, targeting ability toward cells that constitute lung tissue is thought to be enhanced.

[0040] The mass of proteins contained in extracellular vesicles such as exosomes can be determined by general proteome analysis, and methods known to those skilled in the art can be used for such analysis. General protocols for proteome analysis of membrane proteins and the like are described in Pharmaceutical Research, Vol. 25, No. 6, pp. 1469-1483, 2008 and Journal of Proteome Research, Vol. 2, No. 1, pp. 43-50, 2003, etc., the contents of which are incorporated herein by reference.

[0041] The azide lipid-containing exosomes of the present invention can contain protein disulfide isomerase (PDI). While the present invention is not bound by any theory, it is believed that when the exosomes of the present invention contain protein disulfide isomerase in the lipid bilayer in addition to integrin α and integrin β, the protein disulfide isomerase induces isomerization of disulfide bonds in integrin α and integrin β, and more preferably integrin β, thereby inducing conformational changes in integrin α and integrin β and altering the binding ability of the integrin complex to its target. Therefore, it is presumed that when exosomes of the present invention contain protein disulfide isomerase in the lipid bilayer, the targeting induced by integrin α, integrin β, etc., as described above, is enhanced, facilitating delivery of the desired drug to the appropriate target cell.

[0042] In the present invention, the extracellular vesicles are obtained from any cell selected from the group consisting of pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesoderm-derived cells, and endoderm-derived cells. Therefore, in the present invention, extracellular vesicles derived from cells with diverse properties can be used, and the properties of various cells can be imparted to the extracellular vesicles according to the properties of the extracellular vesicles.

[0043] Examples of pluripotent stem cells, mesenchymal stem cells, ectoderm-derived cells, mesoderm-derived cells, and endoderm-derived cells that can be used to produce the extracellular vesicles of the present invention include the cells exemplified below.

[0044] Pluripotent stem cells are cells that have the potential to differentiate into a variety of tissues in the body, specifically, cells that can differentiate into all of the endoderm, mesoderm, and ectoderm. Examples of such cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0045] Mesenchymal stem cells are stem cells that have the ability to differentiate into tissues derived primarily from the mesoderm, as well as some ectodermal and endodermal tissues. Known markers for mesenchymal stem cells include adhesion molecules CD106, CD166, and CD29, as well as CD105, CD73, CD44, CD90, and CD71, and known negative markers include adhesion molecules CD31, CD18, and CD56, hematopoietic markers CD45, CD34, CD14, and CD11, and costimulatory molecules CD80, CD86, and CD40.

[0046] Ectoderm-derived cells include keratinizing epithelial cells (epidermal keratinocytes, epidermal basal cells, nail keratinocytes, nail bed basal cells, medullary hair stem cells, cortical hair stem cells, cuticle hair stem cells, cuticle root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells, and hair follicle cells), stratified epithelial cells (simple squamous epithelial cells of the cornea, tongue, oral cavity, esophagus, anal canal, urethra, and vagina; basal cells of the cornea, tongue, oral cavity, esophagus, anal canal, urethra, and vagina; and bladder epithelial cells), inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, basal cells of the olfactory epithelium, cold sensory neurons, heat sensory neurons, Merkel cells of the epidermis, olfactory receptor neurons, pain sensory neurons, and retinal photoreceptor cells (rod cells, blue cone cells, green cone cells, and red cone cells). vesicles), deep sensory neurons, tactile sensory neurons, type I carotid body cells, type II carotid body cells, type I hair cells of the vestibular system, type II hair cells of the vestibular system, type I taste bud cells, autonomic neurons (cholinergic neurons, adrenergic neurons, peptidergic neurons), inner pillar cells of the organ of Corti, outer pillar cells of the organ of Corti, inner phalangeal cells of the organ of Corti, outer phalangeal cells of the organ of Corti, border cells of the organ of Corti, Hensen's cells of the organ of Corti, vestibular supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, enteric glial cells, central nervous system neurons and glial cells (astrocytes, neurons, oligodendrocytes, spindle neurons), lens cells (anterior lens epithelial cells, crystalline lens fiber cells).

[0047] Mesoderm-derived cells include hepatocytes, adipocytes (white adipocytes, brown adipocytes), Ito cells, renal juxtacellular cells, glomerular epithelial cells, proximal tubule brush border cells, cells of the thin segment of the loop of Henle, distal tubule cells, collecting duct cells, type I alveolar epithelial cells, central acinar cells, smooth muscle duct cells (chief cells, interstitial cells), duct cells, intestinal brush border cells, exocrine gland striated duct cells, gallbladder epithelial cells, non-ciliated cells of the testicular efferent duct, and testis. Epithelial principal cells, epididymal basal cells, ameloblasts, semilunar epithelial cells, interdental epithelial cells of the organ of Corti, loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular connective tissue fibroblasts, other non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts, odontoblasts, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts, osteoprogenitor cells, vitreous cells, stellate cells of the ear Examples of the cells include pancreatic stellate cells, contractile cells, skeletal muscle cells (slow twitch muscle cells, fast twitch muscle cells, intermediate muscle cells, muscle spindle nuclear bag fibers, muscle spindle nuclear chain fibers), muscle satellite cells, cardiac muscle cells (cardiomyocytes, segmented cardiomyocytes, Purkinje fiber cells), smooth muscle cells, iris myoepithelial cells, exocrine gland myoepithelial cells, megakaryocytes, monocytes, connective tissue macrophages, epithelial Langerhans cells, osteoclasts, dendritic cells, microglia, neutrophils, eosinophils, basophils, hybridoma cells, mast cells, Th2 cells, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem and progenitor cells of the blood and immune system, erythroblasts, myelocytes, oocytes, spermatids, spermatocytes, spermatogonia, nurse cells, ovarian follicular cells, Sertoli cells, thymic epithelial cells, interstitial kidney cells, and any cultured cells derived therefrom.

[0048] Endoderm-derived cells include salivary gland mucous cells, salivary gland n1 cells, von Ebner's gland cells in taste buds, mammary gland cells, lacrimal gland cells, auditory canal gland cells, eccrine glands, dark gland cells, eccrine gland clear cells, apocrine gland cells, eyelash gland cells, sebaceous gland cells, Bowman's gland cells in nasal epithelial cells, Brunner's gland cells in the duodenum, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin's gland cells, urethral gland cells, endometrial cells, goblet cells, gastric mucosal cells, gastric chief cells, parietal cells, pancreatic acinar cells, Paneth cells in the small intestine, type II pneumocytes in the lungs, Clara cells in the lungs, and anterior pituitary cells ( Examples of such cells include growth hormone-producing cells, prolactin-producing cells, pituitary thyrotrophs, gonadotrophs, and corticotrophs, melanocyte-stimulating hormone-producing cells, large neurosecretory neurons, thyroid cells (follicular cells, parafollicular cells), parathyroid cells (chief cells, eosinophilic cells), adrenal cells (chromaffin cells, etc.), Leydig cells, inner theca cells, luteal cells (granulosa lutein cells, theca lutein cells), juxtaglomerular cells, macula densa cells, peripolar cells, mesangial cells, and any cultured cells derived therefrom.

[0049] The azido lipid-containing extracellular vesicles of the present invention can be produced by recovering extracellular vesicles from cells cultured in the presence of an azido lipid derivative. In the recovery step, to obtain extracellular vesicles such as exosomes from the above-mentioned cells, the culture medium is recovered, and the extracellular vesicles can be separated and concentrated, as necessary, by conventional column chromatography such as gel filtration chromatography, affinity column chromatography using an antibody against an antigen selected from the above-mentioned marker proteins, size exclusion chromatography, phosphatidylserine affinity chromatography, or flow cytometry.

[0050] The medium that can be used in cell culture to obtain extracellular vesicles is not particularly limited and may be a normal cell culture medium.

[0051] According to another aspect of the present invention, there is provided an extracellular vesicle composition comprising the extracellular vesicles of the present invention. Such a composition can be used to deliver a therapeutically effective drug (active ingredient) for a given disease to a specific tissue, since the extracellular vesicles contained therein have targeting properties directed to cells constituting a specific tissue.

[0052] According to one embodiment of the present invention, the extracellular vesicle composition of the present invention comprises the azide lipid-containing extracellular vesicles of the present invention and a pharmaceutically acceptable carrier. Furthermore, according to another embodiment of the present invention, the extracellular vesicle composition of the present invention further comprises a drug (e.g., a therapeutic or diagnostic drug) bound to the azide lipid-containing extracellular vesicles via the azide group.

[0053] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0054] 1. Cytotoxicity of Azido-Lipids 11-1. Methods: HEK293T cells were selected as a model cell line for exosome production. They were seeded at 10,000 cells / 100 μl / well in a 96-well plate. 10% FBS DMEM was used as the culture medium for HEK293T cells. After overnight incubation, 0, 2.5, 50, 125, 250, 500, and 750 μM azidocaproyl PE were added to each well, and 16:0 azidocaproyl PE was added to each well to evaluate its toxicity. Distearoylphosphatidylcholine was used as a control. After adding 16:0 azidocaproyl PE to the culture medium, HEK293T cells were cultured for 48 hours, and cell viability was evaluated using the Cell Counting Kit-8 method.

[0055] 1-2. Results The results are shown in Figure 1. This study did not reveal any significant cytotoxicity. It was shown that adding azide lipids to cell culture medium for exosome modification did not have any significant adverse effects on cells.

[0056] 2. Recovery of exosomes from cells cultured in medium containing azidolipids and labeling analysis of azidolipid-containing exosomes with AF 488 DBCO, 5-isomer. 2-1. Methods: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidocaproyl PE was added to the culture medium at a concentration of 25 μM. Distearoylphosphatidylcholine was used as a control. 16:0 azidocaproyl PE was added to the HEK293T cell culture medium and the cells were cultured for 24 hours. After 24 hours, the lipid-containing culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Furthermore, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were mixed with AF488 DBCO, 5-isomer solution, incubated at 37°C for 1 hour, and labeled using click chemistry. The labeled exosomes were then transferred to PS Capture Tube. TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TM The exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0057] 2-2. Results Flow cytometry analysis confirmed the recovery of exosomes from both control and azide-lipid-added samples using a labeled anti-CD63 antibody. Furthermore, AF488 signal acquisition and analysis revealed that more particles with a higher fluorescent signal were detected in the azide-lipid-added sample. This indicates that more azide lipids were incorporated into exosomes derived from cells with azide-lipid addition, which reacted with more AF488 DBCO, 5-isomer, resulting in the detection of more particles with a higher fluorescent signal.

[0058] 3. Recovery of exosomes derived from cells cultured in a medium containing azide lipids and labeling analysis of azide lipid-containing exosomes with 5-FAM-PEG3-BCN (exo) 3-1. Method Previous studies suggested that AF488 DBCO, 5-isomer may have reacted with cysteine ​​in the exosome membrane, resulting in relatively large fluorescent signals being detected even in control samples.

[0059] Therefore, the reactive functional group on the label side was changed from DBCO to BCN and subjected to a similar experiment. The procedure is as follows: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidocaproyl PE was added to the culture medium at concentrations of 25, 50, or 100 μM. Distearoylphosphatidylcholine was used as a control. 16:0 azidocaproyl PE was added to the HEK293T cell culture medium and the cells were cultured for 24 hours. After 24 hours, the lipid-supplemented culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Furthermore, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were mixed with a 5-FAM-PEG3-BCN (exo) solution and incubated at 37°C for 1 hour, followed by a click chemistry labeling reaction. The labeled exosomes were then transferred to a PS Capture™ system. TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TM The exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0060] In addition, derived cells stored at -80°C were also mixed with 5-FAM-PEG3-BCN (exo) solution and incubated at 37°C for 1 hour, and a labeling reaction was performed using click chemistry. Using the same method as for exosomes, modification of azide lipids on the cell membrane was also analyzed.

[0061] 3-2. Results Flow cytometry analysis confirmed the recovery of exosomes from both the control sample and the azide lipid-added sample using labeled anti-CD63 antibodies. Furthermore, FAM signal acquisition and analysis revealed that the azide lipid-added sample contained more particles with a stronger fluorescent signal. Nonspecific binding to cysteines on the control exosome membrane was reduced. Furthermore, the labeling efficiency increased with increasing azide lipid concentration in the medium.

[0062] Furthermore, in cell experiments, the azide lipid-added sample detected more cells with larger fluorescent signals. As the concentration of azide lipid in the medium increased, the efficiency of labeling the cell membrane increased.

[0063] 4. Recovery of exosomes derived from cells cultured in a medium containing azide lipids and examination of the concentration of the labeling reagent 5-FAM-PEG3-BCN (exo) for azide lipid-containing exosomes 4-1. Methods In the previous study, we confirmed the labeling effect of 5-FAM-PEG3-BCN (exo) on azide lipid-containing exosomes. However, the effect of the labeling reagent concentration was unclear.

[0064] Therefore, we investigated multiple concentrations of 5-FAM-PEG3-BCN (exo) during labeling. The procedure is as follows: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidocaproyl PE was added to the culture medium at a concentration of 25 μM. Distearoylphosphatidylcholine was used as a control. 16:0 azidocaproyl PE was added to the HEK293T cell culture medium and the cells were cultured for 24 hours. After 24 hours, the lipid-supplemented culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Further, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were mixed with 5-FAM-PEG3-BCN (exo) solution, incubated at 37°C for 1 hour, and labeled using click chemistry. The concentrations of the 5-FAM-PEG3-BCN (exo) solution were set to 100nM, 200nM, 500nM, 2μM, and 250μM. The labeled exosomes were then transferred to the PS Capture TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TM The exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0065] 4-2. Results Flow cytometry analysis confirmed the recovery of exosomes from both the control sample and the azide-lipid-added sample using labeled anti-CD63 antibodies. Furthermore, FAM signal acquisition and analysis revealed that the azide-lipid-added sample contained more particles with a stronger fluorescent signal. In a concentration study of 5-FAM-PEG3-BCN, the detected fluorescent signal increased in the azide-lipid exosome sample with increasing concentration. Furthermore, the azide-lipid exosomes labeled at 250 μM yielded the strongest fluorescent signal. In contrast, no increase was observed in the control exosome sample. Thus, we obtained information about the concentration of the labeling reagent.

[0066] 5. Recovery of exosomes derived from cells cultured in a medium containing azide lipids and examination of the blocking effect of nonspecific modification reactions of azide lipid-containing exosomes by MeO-PEG-Mal 5-1. Methods In the previous study, we confirmed the labeling effect of multiple concentrations of 5-FAM-PEG3-BCN (exo) on azide lipid-containing exosomes. However, nonspecific binding reactions of the azide with the labeling reagent were still observed.

[0067] Therefore, to block the reactive sites, we first reacted exosomes with MeO-PEG-Mal and then attempted labeling with 5-FAM-PEG3-BCN (exo). The procedure was as follows: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidocaproyl PE was added to the culture medium at a concentration of 25 μM. Distearoylphosphatidylcholine was used as a control. 16:0 azidocaproyl PE was added to the HEK293T cell culture medium and the cells were cultured for 24 hours. After 24 hours, the lipid-supplemented culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Further, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were reacted with MeO-PEG-Mal (PEG average Mn 750) for 4 hours at room temperature. After the reaction, the solution was replaced using a spin column. Furthermore, 250 μM of 5-FAM-PEG3-BCN (exo) was added and incubated at room temperature for 16 hours to carry out the labeling reaction by click chemistry. The concentration of the 5-FAM-PEG3-BCN (exo) solution was set to 250 μM. The labeled exosomes were then transferred to the PS Capture TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TMThe exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0068] In addition, derived cells stored at -80°C were treated with MeO-PEG-Mal, then mixed with 5-FAM-PEG3-BCN (exo) solution and incubated at 37°C for 1 hour, followed by a click chemistry labeling reaction. Using the same method as for exosomes, we also analyzed the modification of azide lipids on the cell membrane.

[0069] 5-2. Results Flow cytometry analysis confirmed the recovery of exosomes from the control sample and the azide-lipid-added sample using a labeled anti-CD63 antibody. Furthermore, FAM signal acquisition and analysis revealed that the azide-lipid-added sample contained more particles with a stronger fluorescent signal. However, no nonspecific fluorescent signals were observed in the MeO-PEG-Mal-added group. A similar trend was observed in cells. These results demonstrate the blocking effect of nonspecific modification reactions on exosomes due to MeO-PEG-Mal treatment.

[0070] 6. Recovery of exosomes derived from cells containing azide-modified lipids, administration of antibodies, and investigation of their effect on cancer cell proliferation. 6-1. Methods: In the previous study, we discovered the possibility of modifying exosomes. In practical applications, we anticipate modifying the exosome surface with the target substance via reaction with the azide group.

[0071] Therefore, we investigated whether the effect of exosomes on specific cells could be enhanced by administering antibodies to them as a model system. In preliminary studies, we confirmed that adding large amounts of HEK293T cell-derived exosomes to the culture medium of Namalwa cells (lymphoma-derived) and Reh cells (leukemia-derived) had a growth-suppressing effect. The experimental procedure was as follows: HEK293T cells were seeded in 25 ml of 10% FBS DMEM in a T175 flask. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidocaproyl PE was added to the culture medium at a concentration of 25 μM. Distearoylphosphatidylcholine was used as a control. 16:0 azidocaproyl PE was added to the HEK293T cell medium and cultured for 24 hours. After 24 hours, the lipid-supplemented culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Further, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until use. Linkers were conjugated to the exosomes, followed by antibody conjugation. For the first reaction, exosomes were mixed with endo-BCN-PEG4-NHS ester or endo-BCN-PEG12-NHS ester and then incubated at room temperature for 2 hours. A solution containing 0.1 mg / ml endo-BCN-PEG4-NHS was prepared, and 15 μl was added to 100 μl of the exosome solution. Alternatively, a solution containing 0.1 mg / ml endo-o-BCN-PEG12-NHS was prepared, and 24 μl was added to 100 μl of the exosome solution.After purification using a spin column (100 kDa MWCO, 0.5 ml capacity, Amicon), the linker-conjugated exosomes were washed and resuspended in 100 μl of PBS. Then, 1 μl of anti-CD19 or anti-CD69 antibody solution was added. To examine the function of the exosome-antibody complexes, CD19- and CD69-positive Rel and Namalwa cells were dispersed in 6-well plates at a cell density of 1.5 x 106 cells / 3 ml / well. The antibody-conjugated exosomes were added to the culture medium for 24 hours. The cells were then harvested and washed with PBS to remove any remaining exosomes. The cells were resuspended in 96-well plates, and cell proliferation was measured using the Cell Counting Kit-8 according to the manufacturer's protocol.

[0072] 6-2. Results The results are shown in Figure 2. As shown in Figure 2, a significant cell proliferation inhibitory effect was observed, particularly when endo-BCN-PEG4-NHS and anti-CD69 antibody were bound to the exosome surface. Furthermore, in Reh cells, a significant cell proliferation inhibitory effect was also observed when endo-BCN-PEG12-NHS and anti-CD19 antibody were combined. This indicates that modifying the exosome surface with an antibody via an azide group makes it possible to target specific cells and enhance the effect.

[0073] 7. Cytotoxicity of azidolipids 27-1. Methods In the previous study, the azidolipid was fixed to 16:0 azidocaproyl PE. However, there are several types of azidolipids.

[0074] Therefore, we decided to increase the variety of azidolipids added to cell culture media. To examine differences in molecular structure, we selected 16:0 azidocaproyl PE, 16:0 azidoethyl SM (d18:1 / 16:0), and 18:0-16:0 (16-azido) PC as azidolipids. First, we examined the cytotoxicity of the added azidolipids. The process is as follows: HEK293T cells were selected as a model cell line for exosome production. They were seeded at 10,000 cells / 100 μl / well in a 96-well plate. 10% FBS DMEM was used as the culture medium for HEK293T cells. After overnight culture, 20, 100, 200, or 400 μg / ml of each azidolipid was added to each well. After 48 hours, cell viability was assessed using the Cell Counting Kit-8 method.

[0075] 7-2. Results This study showed that none of the azide lipids had significant cytotoxicity. Adding various azide lipids to cell culture media for exosome modification did not have a significant adverse effect on cells.

[0076] 8. Comparison of exosome modification efficiency by adding multiple azide lipids 8-1. Method In the previous study, the azide lipid was fixed to 16:0 azidocaproyl PE. However, there are multiple types of azide lipids.

[0077] Therefore, we added various types of azidolipids to cell culture medium and compared their exosome modification effects. The procedure is as follows: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidocaproyl PE, 18:0 azidoethyl PC, and 16:0 azidoethyl SM (d18:1 / 16:0) were added to the culture medium at concentrations of 100 μM or 200 μM. Distearoylphosphatidylcholine was used as a control. Each azidolipid was added to HEK293T cell culture medium and the cells were cultured for 24 hours. After 24 hours, the lipid-containing culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Furthermore, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were mixed with a 5-FAM-PEG3-BCN (exo) solution and incubated at 37°C for 1 hour, followed by a click chemistry labeling reaction. The labeled exosomes were then transferred to a PS Capture™ system. TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TM The exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0078] 8-2. Results Flow cytometry analysis confirmed the recovery of exosomes from both control and azide-lipid-added samples using labeled anti-CD63 antibodies. Furthermore, FAM signal acquisition and analysis revealed that more particles with a higher fluorescent signal were detected in the azide-lipid-added sample. Furthermore, among 16:0 azidocaproyl PE, 18:0 azidoethyl PC, and 16:0 azidoethyl SM (d18:1 / 16:0), more particles with a higher fluorescent signal were detected with 16:0 azidoethyl SM (d18:1 / 16:0). Furthermore, increasing the concentration of each azide lipid from 100 μM to 200 μM when added to the culture medium resulted in the detection of more particles with a higher fluorescent signal during flow cytometry analysis after exosome labeling. In this study, exosome samples treated with 16:0 azidoethyl SM (d18:1 / 16:0) at 100 μM showed labeling efficiency comparable to that of exosome samples treated with 16:0 azidocaproyl PE or 18:0-16:0 (16-azido) PC at 200 μM, demonstrating that 16:0 azidoethyl SM (d18:1 / 16:0) was the most efficient modification agent.

[0079] 9. Recovery of exosomes derived from cells cultured in a medium containing azide sugars and labeling analysis of azide sugar-containing exosomes with 5-FAM-PEG3-BCN (exo) 9-1. Methods A similar method to this one could be to modify exosomes by adding azide sugars to cell culture medium, in addition to azide lipids.

[0080] Therefore, we also performed a similar experiment using azide sugars. The procedure was as follows: HEK293T cells were seeded in 25 ml of 10% FBS DMEM in a T175 flask. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. Ac4GlcNAz, Ac4GalNAz, and Ac4ManNAz were added to the culture medium at a concentration of 100 μM. Each azide sugar was added to the HEK293T cell culture medium and cultured for 48 hours. After 48 hours, the lipid-supplemented culture medium was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 xg for 10 minutes to remove contaminants such as cell fragments. Furthermore, contaminants were removed by centrifugation at 4000 xg for 30 minutes at 4°C and filtration through a 0.2 μm filter. The processed solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were mixed with a 5-FAM-PEG3-BCN (exo) solution and incubated at 37°C for 1 hour, followed by a click chemistry labeling reaction. The labeled exosomes were then transferred to the PS Capture TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TM The exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0081] 9-2. Results Flow cytometry analysis confirmed the recovery of exosomes from the control sample and the azide lipid-added sample using labeled anti-CD63 antibodies. Furthermore, FAM signal acquisition and analysis revealed that more particles with a higher fluorescent signal were detected in the azide sugar-added sample. In particular, more particles with a higher fluorescent signal were detected in the Ac4ManNAz sample. This suggests that the use of Ac4ManNAz may enable efficient exosome modification.

[0082] 10. Comparison of exosome recovery from cells cultured in media containing either azidolipids or azidosugars and labeling efficiency with 5-FAM-PEG3-BCN (exo) 10-1. Methods Previous studies suggested the possibility of using azidosugars.

[0083] Therefore, we attempted to directly compare the exosome modification efficiency of azidolipids and azidosugars. The procedure is as follows: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidoethyl SM (d18:1 / 16:0) or Ac4ManNAz was added to the culture medium at a concentration of 100 μM. Each azido molecule sample was added to the HEK293T cell culture medium and cultured for 24 hours. After 24 hours, the culture medium containing the azido molecule sample was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Furthermore, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until analysis. The exosomes were mixed with a 5-FAM-PEG3-BCN (exo) solution and incubated at 37°C for 1 hour, followed by a click chemistry labeling reaction. The labeled exosomes were then transferred to a PS Capture™ system. TM The exosomes were adsorbed onto beads using an exosome flow cytometry kit (WAKO) and analyzed by flow cytometry (BD FACSymphony TM The exosomes were identified using an anti-CD63 monoclonal antibody (H5C6) eFluorescent Immunosorbent Assay (EMA) and a flow cytometer (BD Biosciences). TM 660 eBioscience TM (Invitrogen) was added and the analysis was carried out.

[0084] 10-2. Results Flow cytometry analysis confirmed the recovery of exosomes from samples containing 16:0 azidoethyl SM (d18:1 / 16:0) and Ac4ManNAz media using a labeled anti-CD63 antibody. Furthermore, FAM signal acquisition and analysis revealed that more particles with a stronger fluorescent signal were detected in the sample containing 16:0 azidoethyl SM (d18:1 / 16:0) media. This suggests that azidolipids may be more efficient than azidosugars for modification.

[0085] 11. Recovery of exosomes derived from cells cultured in media containing either azidolipids or azidosugars, antibody administration, and direct comparison of the effects of inhibiting cancer cell proliferation. 11-1. Methods: Previous studies have demonstrated the possibility of exosome modification. In practical applications, it is anticipated that the target substance will be modified onto the exosome surface via reaction with the azide group. The potential use of azidosugars has also been suggested.

[0086] Therefore, we attempted to modify exosomes with azidolipids and azidosugars and examine their effects on cells in a model system. The procedure is as follows: HEK293T cells were seeded in a T175 flask with 25 ml of 10% FBS DMEM. At approximately 80% confluence, the culture medium was changed to 20 ml of EX-CELL® 293 Serum-Free Medium for HEK 293 cells. 16:0 azidoethyl SM (d18:1 / 16:0) or Ac4ManNAz was added to the culture medium at a concentration of 100 μM. The azido molecule sample was added to the HEK293T cell culture medium and then cultured for 24 hours. After 24 hours, the culture medium containing the azido molecule sample was removed, and the cells were washed with 10 ml of PBS. Next, HEK293T cells were cultured in EX-CELL® 293 Serum-Free Medium for 48 hours to allow the cells to secrete exosomes, and the conditioned medium was collected. The conditioned medium was centrifuged at 300 x g for 10 minutes to remove contaminants such as cell debris. Further, contaminants were removed by centrifugation at 4000 x g for 30 minutes at 4°C and filtered through a 0.2 μm filter. The treated solution was stored at -80°C until exosome collection. Exosomes were purified from the centrifuged solution using a column-based exosome isolation kit (exoEasy Maxi Kit, QIAGEN) and stored at -80°C until use. A linker was conjugated to the exosomes, followed by antibody conjugation. The first reaction involved mixing exosomes with endo-BCN-PEG4-NHS ester and then incubating at room temperature for 2 hours. A solution containing 0.1 mg / ml of endo-BCN-PEG4-NHS was prepared, and 15 μl was added to 100 μl of the exosome solution. After purification using a spin column (100 kDa MWCO, 0.5 ml volume, Amicon), the linker-bound exosomes were washed and resuspended in 100 μl of PBS. Then, 1 μl of anti-CD69 antibody solution was added. To examine the function of the exosome-antibody complex, Reh cells were dispersed in a 6-well plate at a cell density of 1.5 x 106 cells / 3 ml / well. The antibody-bound exosomes were added to the culture medium for 24 hours. Afterwards, the cells were harvested and washed with PBS to remove any remaining exosomes.The cells were redispersed in 96-well plates, and cell proliferation was measured by the Cell Counting Kit-8 method according to the manufacturer's protocol.

[0087] 11-2. Results The results are shown in Figure 4. As shown in Figure 4, when endo-BCN-PEG4-NHS and anti-CD69 antibody were conjugated to exosomes containing the azido lipid 16:0 azidoethyl SM (d18:1 / 16:0), a significant cell proliferation inhibitory effect was observed. In other words, modifying the exosome surface with an antibody via the azide group derived from the lipid molecule demonstrated the potential for targeting specific cells and enhancing the effect.

Claims

1. Azide lipid-containing extracellular vesicles comprising an extracellular vesicle marker protein and an azido lipid derivative in which an azide group is bound to the hydrophilic portion of a lipid, in a lipid bilayer.

2. The azide lipid-containing extracellular vesicles according to claim 1, wherein the lipid is at least one type of lipid selected from the group consisting of complex lipids and simple lipids.

3. The azide lipid-containing extracellular vesicles described in claim 2, wherein the complex lipid is at least one lipid selected from sphingophospholipids, sphingoglycolipids, glycerophospholipids, glyceroglycolipids, ether-type phospholipids, and ether-type glycolipids.

4. The azido lipid-containing extracellular vesicles described in claim 2, wherein the complex lipid is at least one lipid selected from the group consisting of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine.

5. The azide lipid-containing extracellular vesicles of claim 2, wherein the simple lipid is at least one lipid selected from the group consisting of cholesterol esters, cholesterol ester derivatives, acylglycerols, and acylglycerol derivatives.

6. The azidolipid-containing extracellular vesicle of claim 1, wherein the extracellular vesicle marker protein is an exosome marker protein.

7. The azidolipid-containing extracellular vesicle of claim 6, wherein the exosome marker protein is selected from CD63, CD81, and CD9.

8. A method for producing azido lipid-containing extracellular vesicles, comprising: (a) culturing cells in the presence of an azido lipid derivative in which an azide group is bound to the hydrophilic moiety of a lipid; and (b) recovering the extracellular vesicles from the cells.

9. The method of claim 8, wherein the lipid is at least one lipid selected from the group consisting of complex lipids and simple lipids.

10. The method according to claim 9, wherein the complex lipid is at least one lipid selected from sphingophospholipids, sphingoglycolipids, glycerophospholipids, glyceroglycolipids, ether-type phospholipids, and ether-type glycolipids.

11. The method of claim 9, wherein the complex lipid is at least one lipid selected from the group consisting of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine.

12. The method of claim 9, wherein the simple lipid is at least one lipid selected from the group consisting of cholesterol esters, cholesterol ester derivatives, acylglycerols, and acylglycerol derivatives.

13. The method according to claim 8, wherein the extracellular vesicles contain an exosome marker protein in the lipid bilayer as the extracellular vesicle marker protein.

14. The method of claim 13, wherein the exosome marker protein is selected from CD63, CD81, and CD9.

15. The method of claim 8, wherein the cells are at least one type of cells selected from the group consisting of pluripotent stem cells, mesenchymal stem cells, ectodermal-derived cells, mesodermal-derived cells, and endodermal-derived cells.

16. Azidolipid-containing extracellular vesicles produced by the method according to any one of claims 8 to 15.

17. An extracellular vesicle composition comprising the azidolipid-containing extracellular vesicles according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

18. The extracellular vesicle composition of claim 17, further comprising a drug bound to the azido-lipid-containing extracellular vesicles via an azide group.

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