Method for introducing target molecule into lipid vesicle and cell, and antibody dispersion preparation

Abstract

The present disclosure provides a method for introducing a target molecule into a lipid vesicle (for example, an extracellular vesicle). The present disclosure also provides a lipid vesicle (for example, an extracellular vesicle) produced using the method for introducing a target molecule into a lipid vesicle (for example, an extracellular vesicle). According to the present disclosure, a complex (a first complex) of a lipid (for example, an ionic lipid; particularly a liquid lipid composition) and a target molecule is fused with a lipid vesicle (for example, an extracellular vesicle) to form a complex (a second complex). The first complex and the second complex are suitable for the introduction of the target molecule into a cell, and the second complex is particularly suitable for the introduction of the target molecule into a cell. The target molecule may be a protein (for example, an antibody), a nucleic acid, or other compounds.

Description

Lipid vesicles, method for introducing target molecules into cells, and dispersed antibody preparations

[0001] The present disclosure relates to lipid vesicles and a method for introducing a target molecule into a cell. The present disclosure relates to lipid vesicles obtained using the method for introducing a target molecule into a lipid vesicle. The present disclosure also relates to a dispersion preparation comprising a substrate and a complex containing an antibody and a lipid, wherein the complex is dispersed in the substrate.

[0002] Drug delivery systems deliver therapeutic drugs in the required amount to the required location, enabling treatment of diseases while avoiding serious adverse events, and are therefore expected to be used in the treatment of a wide variety of diseases. In particular, nanoparticles such as liposomes and lipid nanoparticles (LNPs) are small enough to penetrate intercellular spaces and membranes, and can be modified on the surface with antibodies, polyethylene glycol (PEG), etc., impart environmental responsiveness, or protect drugs from the surrounding environment within the body, so their application to drug delivery systems is progressing. Furthermore, nano-sized particles can also utilize the enhanced permeability and retention effect (EPR) to target tumor tissue.

[0003] Liposomal formulations are small vesicles with a lipid bilayer structure composed of phospholipids, capable of encapsulating water-soluble drugs within the interior and hydrophobic drugs within the lipid bilayer. Their development has been most widely pursued, and currently, in Japan, they are used as pharmaceuticals, including anti-cancer drugs (Onivyde, Doxil), anti-fungal drugs (AmBisome), and drugs for age-related macular degeneration (Visudyne). Lipid nanoparticles are typically composed of multiple lipids with different functions, encapsulating water-soluble drugs in the internal hydrophilic regions and oil-soluble drugs in the hydrophobic regions. Lipid nanoparticles encapsulating mRNA (Patent Documents 1 and 2) are used as pharmaceuticals for COVID-19 vaccines (Comirnaty, Spikebax).

[0004] In recent years, development has been progressing toward utilizing extracellular vesicles (e.g., exosomes) released by cells as novel drug delivery carriers with high biocompatibility and low immunogenicity. Exosomes are nano-sized secretory vesicles (30-200 nm) produced by all cells and present throughout the body, present in all living organisms. Exosomes have a lipid bilayer structure, encapsulate proteins and nucleic acids, and display signaling molecules on their surface. They are thought to function as intercellular signaling mechanisms within the body. Tsai et al. reported that exosomes are less toxic to cells than LNPs and that exosomes loaded with mRNA for SARS-CoV-2 spike protein or nucleocapsid protein elicit high antibody production in mice (Non-Patent Document 1). However, the type and amount of drugs that can be loaded into exosomes alone may be limited due to the state and charge state of the surface molecules. For this reason, there have been reports of fusing exosomes with liposomes and using them as drug delivery carriers. Hybrid particles of exosomes and liposomes not only overcome the above-mentioned problems associated with the use of exosomes alone, but also facilitate the enhancement of particle stability and surface modification. Lv et al. reported a method for effectively inhibiting tumorigenesis by combining drug delivery using hybrid particles of exosomes and liposomes with hyperthermic intraperitoneal chemotherapy (Non-Patent Document 2).

[0005] US8,058,069BUS8,278,036B

[0006] J Biol Chem. 2021 297(5):101266Adv Sci 2020 29;7(18):2000515

[0007] The present disclosure provides a method for introducing a molecule of interest into lipid vesicles (e.g., extracellular vesicles). The present disclosure also provides lipid vesicles (e.g., extracellular vesicles) obtained using the method for introducing a molecule of interest into lipid vesicles (e.g., extracellular vesicles). The present disclosure also provides a dispersion formulation comprising a substrate and a complex comprising an antibody and a lipid, wherein the complex is dispersed in the substrate.

[0008] The present disclosure has found that, for example, a complex (first complex) between an ionic lipid (particularly a natural or non-natural cationic lipid) or a non-ionic lipid (particularly a non-natural lipid, particularly a lipid containing a non-natural non-ionic lipid) and a molecule of interest can be obtained, and then a hybrid particle (second complex) of the first complex with a lipid vesicle (e.g., extracellular vesicle) or exosome can be formed. The present disclosure also revealed that the first complex and the second complex are useful as drug delivery carriers loaded with foreign molecules such as nucleic acids and proteins (e.g., antibodies, enzymes, etc.). The lipid constituting the first complex is an additional lipid to the lipid constituting the lipid vesicle (e.g., extracellular vesicle) or exosome in the second complex, and is preferably different from either the lipid constituting the lipid vesicle (e.g., extracellular vesicle) or exosome, e.g., a non-natural lipid.

[0009] According to the present disclosure, the following inventions can be provided. (1) A composition comprising a complex of a target molecule (e.g., a water-soluble molecule), a lipid mixture (e.g., a mixture of one or more types of lipids, or, for example, a natural lipid or a non-natural lipid, such as a lipid vesicle or lipid nanoparticle), and a lipid vesicle (preferably, an extracellular vesicle). (2) The composition according to (1) above, in which the lipid mixture and the lipid vesicle (e.g., an extracellular vesicle) form a vesicle, and the target molecule is encapsulated in the formed vesicle. (3) The lipid mixture comprises a cationic lipid (e.g., 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC)), and a lipid vesicle (preferably, an extracellular vesicle). + (4) The composition according to (3) above, which comprises a cationic lipid and an anionic lipid, preferably the cationic lipid and the anionic lipid are lipids that become liquid under atmospheric pressure and at room temperature when mixed at a charge ratio of 1:1. (5) The anionic lipid is R-COO -(6) The composition according to any one of (3) to (5) above, wherein the cationic lipid is represented by R-O-P(OR1)(OR2)(=O), wherein R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, and at least one ethylene group constituting the alkenyl group may be substituted with a vinylene group, and preferably R is a saturated fatty acid or a cis-unsaturated fatty acid. (7) The composition according to any one of (1) to (6) above, wherein the target molecule is a polypeptide. (8) The composition according to any one of (1) to (6) above, wherein the target molecule is a nucleic acid. (9) The composition according to any one of (1) to (6) above, wherein the target molecule is an antibody or an antigen-binding fragment thereof. (10) The composition according to any one of (1) to (9) above, for delivering a target molecule to a cell. (11) The composition according to any one of (1) to (10) above, wherein the complex is obtained by the following method, comprising: obtaining a complex (first complex) of a target molecule and a lipid mixture; and contacting the obtained complex with lipid vesicles (e.g., extracellular vesicles) to obtain complex particles (second complex) of the complex and lipid vesicles (e.g., extracellular vesicles), wherein the target molecule is encapsulated in the lipid vesicles (e.g., extracellular vesicles). (12) A method for introducing a target molecule into a lipid vesicle (e.g., an extracellular vesicle), comprising: obtaining a complex (first complex) between the target molecule and a lipid mixture; and contacting the obtained complex with a lipid vesicle (e.g., an extracellular vesicle) to obtain a complex particle (second complex) between the complex and the lipid vesicle (e.g., an extracellular vesicle), thereby introducing the target molecule into the complex particle.(13) A method for introducing a target molecule into a cell, comprising: obtaining a complex (first complex) of the target molecule and a lipid mixture; contacting the obtained complex with lipid vesicles (e.g., extracellular vesicles) to obtain a complex particle (second complex) of the complex and lipid vesicles (e.g., extracellular vesicles), thereby introducing the target molecule into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

[0010] (21) The composition or method according to any one of the above, wherein the lipid mixture comprises a cationic lipid. (22) The composition or method according to the above (21), wherein the cationic lipid comprises an uncharged phosphate ester moiety and a quaternary ammonium cation. (23) The composition or method according to the above (21), wherein the cationic lipid is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC). + ) or a pharmaceutically acceptable salt thereof.

[0011] (31) A target molecule (preferably a water-soluble molecule) and 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) + (32) A dispersion formulation comprising particles containing a solid complex of a lipid mixture (containing one or more lipid surfactants) containing 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) or a pharmaceutically acceptable salt thereof, and an oily base, wherein the particles are dispersed in the oily base. + (33) The dispersion formulation according to the above (31), which consists of EDMPC or a pharmaceutically acceptable salt thereof (preferably a chloride salt). + Chloride salt of (EDMPC + Cl -(34) The dispersion formulation according to any one of (31) to (33) above, wherein the lipid mixture further comprises an anionic lipid. (35) The dispersion formulation according to (34) above, wherein the anionic lipid comprises one or more selected from the group consisting of oleic acid (OA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), cardiolipin (CL), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (DOPS), and phosphatidylinositol (PI).

[0012] (41) A target molecule (e.g., a water-soluble molecule) and 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) + (42) A composition comprising a complex of a lipid mixture containing EDMPC or a pharmaceutically acceptable salt thereof with lipid vesicles (e.g., extracellular vesicles). + Chloride salt of (EDMPC + Cl - (43) The composition according to (41) above, wherein the lipid mixture is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC). +(44) The composition according to any one of (41) to (43) above, wherein the lipid mixture further comprises an anionic lipid. (45) The composition according to (44) above, wherein the anionic lipid comprises one or more selected from the group consisting of oleic acid (OA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), cardiolipin (CL), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (DOPS), and phosphatidylinositol (PI). (46) The composition according to any one of (41) to (45) above, wherein the lipid mixture and lipid vesicles (e.g., extracellular vesicles) form vesicles, and the target molecule is encapsulated in the vesicles. (47) The composition according to any one of (41) to (46) above, wherein the target molecule is a polypeptide. (48) The composition according to any one of (41) to (46) above, wherein the target molecule is a nucleic acid. (49) The composition according to any one of (41) to (46) above, wherein the target molecule is an antibody or an antigen-binding fragment thereof. (50) The composition according to any one of (41) to (49) above, for delivering a target molecule to a cell. (51) The composition according to any one of (41) to (50) above, wherein the complex is obtained by the following method: obtaining a complex (first complex) of a target molecule and a lipid mixture; and contacting the obtained complex with lipid vesicles (e.g., extracellular vesicles) to obtain a complex particle (second complex) of the complex and lipid vesicles (e.g., extracellular vesicles), wherein the target molecule is encapsulated in the lipid vesicles (e.g., extracellular vesicles).

[0013] (61) The cationic lipid is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC). + ) or a pharmaceutically acceptable salt thereof.

[0014] (71) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to the brain. (72) The composition according to any one of (1) to (9) above, for use in delivering a target molecule into brain cells. (73) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to muscle. (74) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to myocardium. (75) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to the pancreas. (76) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to the ovary. (77) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to the spleen. (78) The composition according to any one of (1) to (9) above, for use in delivering a target molecule to the kidney.

[0015] (81) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the brain. (82) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the lung. (83) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the muscle. (84) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the heart. (85) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the spleen. (86) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the kidney.

[0016] (91) A composition according to any one of (1) to (9) above for use in treating a neurological disease in a subject {wherein the target molecule provides a therapeutic benefit for the neurological disease}. (93) A composition according to any one of (1) to (9) above for use in treating a muscular disease in a subject {wherein the target molecule provides a therapeutic benefit for the muscular disease}. (94) A composition according to any one of (1) to (9) above for use in treating a cardiac disease in a subject {wherein the target molecule provides a therapeutic benefit for the cardiac disease}. (95) A composition according to any one of (1) to (9) above for use in treating a pancreatic disease in a subject {wherein the target molecule provides a therapeutic benefit for the pancreatic disease}. (96) A composition according to any one of (1) to (9) above for use in treating an ovarian disease in a subject {wherein the target molecule provides a therapeutic benefit for the ovarian disease}. (97) A composition according to any one of (1) to (9) above, for use in treating a spleen disease in a subject {wherein the target molecule provides a therapeutic benefit for the spleen disease}. (98) A composition according to any one of (1) to (9) above, for use in stimulating antigen-specific immunity in a subject {wherein the target molecule can be an antigen or a nucleic acid (e.g., DNA or RNA) encoding the antigen}. (99) A composition according to any one of (1) to (9) above, for use in treating a kidney disease in a subject {wherein the target molecule provides a therapeutic benefit for the kidney disease}.

[0017] (111) A composition according to any one of (41) to (49) above for use in treating a cranial nerve disease in a subject {wherein the target molecule provides a therapeutic benefit for the cranial nerve disease}. (112) A composition according to any one of (41) to (49) above for use in treating a lung disease in a subject {wherein the target molecule provides a therapeutic benefit for the lung disease}. (113) A composition according to any one of (41) to (49) above for use in treating a muscle disease in a subject {wherein the target molecule provides a therapeutic benefit for the muscle disease}. (114) A composition according to any one of (41) to (49) above for use in treating a heart disease in a subject {wherein the target molecule provides a therapeutic benefit for the heart disease}. (115) A composition according to any one of (41) to (49) above for use in treating a pancreatic disease in a subject {wherein the target molecule provides a therapeutic benefit for the pancreatic disease}. (116) The composition according to any one of (41) to (49) above, for use in stimulating antigen-specific immunity in a subject {wherein the target molecule may be an antigen or a nucleic acid (e.g., DNA or RNA) encoding the antigen}. (117) The composition according to any one of (41) to (49) above, for use in delivering a target molecule to the kidney {wherein the target molecule provides a therapeutic benefit for a kidney disease}.

[0018] (120) A dispersion solution comprising (i) a complex of a target molecule and a lipid and (ii) an aqueous or oily base, the complex being dispersed in the base. Here, the target molecule may preferably be an antibody. The lipid may be an ionic lipid, preferably a cationic lipid. In one embodiment, when the dispersion solution comprises an oily base, the dispersion solution is not a composition for ophthalmic application, such as for eye drops or intravitreal administration.

[0019] (121) An aqueous dispersion comprising a lipid-antibody complex and an aqueous base, the complex being dispersed in the aqueous base. (122) The aqueous dispersion according to (121), wherein the lipid comprises or consists of a cationic lipid. (123) The lipid is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC). + (124) The aqueous dispersion according to any one of (121) to (123) above, wherein the lipid further comprises an anionic lipid. (125) The aqueous dispersion according to (124) above, wherein the anionic lipid comprises linoleic acid. (126) The aqueous dispersion according to any one of (121) to (125) above, wherein the aqueous base is physiological saline. (127) The aqueous dispersion according to any one of (121) to (125) above, wherein the aqueous base is a medium suitable for introducing a reagent into cells (for example, a serum-free medium).

[0020] (131) An oil-based dispersion solution (excluding ophthalmic preparations) comprising a solid complex of a lipid and an antibody and an oil base, the complex being dispersed in the oil base. (132) The oil-based dispersion solution according to (131) above, wherein the lipid comprises or consists of a cationic lipid. (133) The lipid is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC). + (134) The oily dispersion solution according to any of (131) to (133) above, wherein the lipid further comprises an anionic lipid. (135) The oily dispersion solution according to any of (131) to (134) above, wherein the anionic lipid comprises linoleic acid. (136) The oily dispersion solution according to any of (131) to (135) above, wherein the oily base comprises or consists of isopropyl myristate.

[0021] FIG. 1A shows the particle size distribution of extracellular vesicles, lipid-anti-IL-2 antibody complexes (first complexes), and complexes of extracellular vesicles and the first complex (second complexes). The extracellular vesicles are the smallest, followed by the first complexes, which are of intermediate size, and the second complexes, which are the largest. FIG. 1B shows the results of a test in which a lipid-peptide molecule complex (first complex) was formed, a complex of extracellular vesicles and the first complex (second complex) was further formed, and the ratio of peptide-loaded extracellular vesicles to extracellular vesicles was examined. FIG. 1C shows the results of a test in which a lipid-nucleic acid molecule complex (first complex) was formed, a complex of extracellular vesicles and the first complex (second complex) was further formed, and the ratio of nucleic acid-loaded extracellular vesicles to extracellular vesicles was examined. Figure 1D shows the results of a test in which a lipid-RNP molecular complex (first complex) was formed, followed by the formation of a complex between the first complex and extracellular vesicles (second complex), and the ratio of RNP-loaded extracellular vesicles to extracellular vesicles was examined. RNP refers to an RNA-protein complex, and in this experiment, a Cas9-gRNA complex was used as the RNP. Figure 1E shows the formation of a lipid-antibody molecular complex (first complex), followed by the formation of a complex between the first complex and extracellular vesicles (second complex), and the resulting solution was treated with a protein-digesting enzyme, resulting in the antibody being encapsulated in the vesicles in the second complex. Figure 1E shows the formation of a lipid-microRNA molecular complex (first complex), followed by the formation of a complex between the first complex and extracellular vesicles (second complex), and the resulting solution was treated with an RNA-digesting enzyme, resulting in the microRNA being encapsulated in the vesicles in the second complex. FIG. 2 shows the results of immunostaining of a lipid-target molecule complex (first complex) and a complex of extracellular vesicles and the first complex (second complex). FIG. 3A shows the results of immunostaining of cells contacted with a lipid-target molecule complex (first complex) and a complex of extracellular vesicles and the first complex (second complex). FIG. 3B shows the results of immunostaining of cells contacted with a complex of one type of lipid and a target molecule (first complex) and a complex of extracellular vesicles and the first complex (second complex). FIG. 4A shows the percentage of cells in which the antibody was delivered intracellularly in cells contacted with the first complex or the second complex containing the antibody.Figure 4B shows a fluorescence microscope image of cells in which an antibody was delivered into cells contacted with a first complex or a second complex containing an antibody. Figure 4C shows the delivery of a peptide into cells contacted with a first complex or a second complex containing a peptide. Figure 5 shows the amount of siRNA introduced into cells contacted with a first complex or a second complex containing siRNA. Figure 6A shows the rate of cell invasion into the lower chamber after delivery of an anti-matrix metalloproteinase-7 antibody (anti-MMP7 antibody) or various complexes containing the antibody. Figure 6B shows stained images of cells that invaded into the lower chamber after delivery of an anti-matrix metalloproteinase-7 antibody (anti-MMP7 antibody) or various complexes containing the antibody. Figure 6C shows the effect of treatment with a first complex or a second complex containing an anti-MMP7 antibody on the migration ability of cells. Specifically, Figure 6C shows the rate of cell invasion into the lower chamber after delivery of an anti-matrix metalloproteinase-7 antibody (anti-MMP7 antibody) or various complexes containing the antibody. Figure 6D shows the effect on cell proliferation of treatment with a first or second complex containing an anti-MMP7 antibody. Figure 7 shows stained images of cells into which antisense oligonucleotides (ASOs) were introduced via a second complex containing the first complex. Figure 8 shows the particle size distribution of the prepared exosomes, first complex, and second complex as measured by dynamic light scattering. Figure 9 shows the tissue distribution after administration of the first and second complexes via tail vein injection. Figure 10A shows the distribution of the first complex in brain tissue injected via tail vein. The dotted frame indicates the area near blood vessels (CD31 positive), while the solid frame indicates the area away from blood vessels. The IgG was Cy5-labeled, and the presence of the antibody was confirmed within the dotted and solid frames. Figure 10B shows the distribution of the second complex in brain tissue injected via tail vein. The dotted and solid frames indicate areas away from blood vessels. The IgG was labeled with Cy5, and the presence of the antibody was confirmed within the dotted and solid frames. A lipid-antibody molecule complex (first complex) was formed, and a complex between extracellular vesicles and the first complex (second complex) was further formed. The resulting solution was injected via tail vein into a mouse, and the distribution of antibody molecules in the brain 48 hours later is shown. This is an enlarged view of a portion of Figure 11A.In the right column of Figure 11B, the localization of the antibody is indicated by an arrow based on Cy5 fluorescence. This is an enlarged view of a portion of Figure 11A. The cerebral cortex is shown, and the uptake of the antibody into microglial cells is indicated by an arrow.

[0022] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "having," "including," or "containing" mean including an unspecified third component, and also encompass the meaning of "consisting of," which means essentially not including a third component.

[0023] As used herein, a "lipid vesicle" is a vesicle having a lipid membrane structure. Lipid vesicles typically have a lipid bilayer membrane. Lipid vesicles may contain artificial lipids, but may not contain artificial lipids, or may be composed of natural lipids, e.g., naturally produced lipids, preferably produced by cells (e.g., extracellular vesicles). The size of lipid vesicles is not particularly limited, but may have a sub-micron diameter, for example. Lipid vesicles may be particles having a diameter of, for example, about 30 nm to about 150 nm. Lipid vesicles may be, for example, liposomes.

[0024] As used herein, "extracellular vesicles" refer to particles with a membrane structure secreted by cells. Extracellular vesicles are thought to be involved in intercellular communication. Extracellular vesicles are broadly classified into three types: exosomes, microvesicles, and apoptotic bodies. Exosomes are particles with diameters of approximately 30 nm to approximately 150 nm and are thought to be secreted from multilamellar endoplasmic reticulum (MVBs) formed inside endosomes. Exosomes are capable of transporting genetic material (e.g., mRNA and miRNA), proteins, and lipids from cells, which are thought to affect the function and phenotype of recipient cells. Apoptotic bodies are extracellular vesicles with diameters of approximately 500 nm to approximately 2000 nm that are released when cells are degraded during programmed cell death (apoptosis). Apoptotic vesicles contain genetic material, organelles, and portions of the cell membrane, and are thought to be internalized and disposed of by neighboring cells or cells of the immune system. Microvesicles are particles with diameters of approximately 100 nm to 1000 nm that are produced by a mechanism distinct from exosomes and apoptotic vesicles. The resulting microvesicles contain portions of the cell membrane and cytoplasmic components (e.g., proteins, lipoproteins, and genetic material). Microvesicles play an important role in intercellular communication, affecting the function and phenotype of neighboring or distant cells by transporting genetic material and proteins. Substances can be delivered to cells by introducing them into microvesicles or exosomes.

[0025] As used herein, "cation" refers to a molecule that has a positive charge under pH 7 conditions. As used herein, "cationic" refers to the property of having a positive charge under pH 7 conditions. As used herein, "anion" refers to a molecule that has a negative charge under pH 7 conditions. As used herein, "anionic" refers to the property of having a negative charge under pH 7 conditions.

[0026] As used herein, a "surfactant" is a molecule having a relatively hydrophilic portion and a relatively hydrophobic portion. Surfactants are broadly classified into ionic surfactants and nonionic surfactants. Ionic surfactants include cationic surfactants and anionic surfactants. Typically, an ionic surfactant may have either a cation or an anion and a hydrophobic portion. Cationic surfactants may contain a cationic portion and a fatty chain (e.g., a long-chain fatty chain) such as an alkyl, alkenyl, or alkynyl. Anionic surfactants may contain an anionic portion and a fatty acid such as a long-chain fatty acid, an unsaturated fatty acid (e.g., monovalent or divalent), or a long-chain unsaturated fatty acid (e.g., monovalent or divalent). The term "long chain" is used to refer to compounds having 14 to 22 carbon atoms. The fatty chain may be a fatty chain having 8 to 22 carbon atoms, i.e., C8 to C 22 Alkyl, C8-C 22 Alkenyl, or C8-C 22 C8-C such as alkynyl 22 The fatty chain may be a fatty chain. The fatty chain may also be a long fatty chain. Saturated means that the fatty chain has no double or triple bonds, and unsaturated means that the fatty chain has at least one double or triple bond. Fatty chains with double bonds include cis- and trans-types, with the cis-type being preferred from the viewpoint of biocompatibility. Among ionic surfactants, molecules whose hydrophobic moiety is a lipid (fatty chain) are called ionic lipids. In this specification, a lipid mixture refers to a composition containing one or more lipids.

[0027] As the surfactant (e.g., nonionic lipid or ionic lipid), a lipophilic nonionic lipid or ionic surfactant (e.g., ionic lipid, particularly cationic lipid and anionic lipid) having an HLB (Hydrophile-Lipophile Balance) value of 10 or less is preferred, as this allows the complex to be easily dispersed in the oil phase. The HLB of the nonionic lipid or ionic surfactant (e.g., ionic lipid, preferably cationic lipid and anionic lipid) is preferably 8 or less, more preferably 5 or less, and particularly preferably 3 or less. The HLB of the nonionic lipid or ionic surfactant (e.g., ionic lipid, preferably cationic lipid and anionic lipid) can be, for example, 1 to 3, 3 to 5, or 5 to 10. In one aspect, the HLB of the nonionic lipid or ionic surfactant (e.g., ionic lipid, preferably cationic lipid and anionic lipid) can be 5 to 10. In some aspects, the HLB of a nonionic lipid or cationic surfactant (e.g., cationic lipid) can be 1 to 10, e.g., 1 to 3, 3 to 5, or 5 to 10. In some aspects, the HLB of a nonionic lipid or cationic surfactant (e.g., cationic lipid) can be 5 to 10. In some aspects, the HLB of a nonionic lipid or anionic surfactant (e.g., anionic lipid) can be 1 to 10, e.g., 1 to 3, 3 to 5, or 5 to 10. In some aspects, the HLB of a nonionic lipid or anionic surfactant (e.g., anionic lipid) can be 5 to 10.

[0028] Lipids are biological molecules soluble in nonpolar solvents. Lipids can be classified into glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, and polyketides. Glycerolipids are a general term for molecules in which a fatty acid is ester-linked to the hydroxyl group of glycerol. Glycerol has three hydroxyl groups, and up to three fatty acids can be ester-linked. In glycerophospholipids, two fatty acids are ester-linked to two hydroxyl groups of glycerol, and a phosphate group is linked to one hydroxyl group. Sphingolipids can be ceramides, sphingophospholipids, or sphingoglycolipids. Sterol lipids are a subgroup of terpenoids. Sterol lipids can be sterols (e.g., cholesterol). Prenol lipids are terpenoids other than sterol lipids. Nonionic lipids contain a hydrophilic portion and a hydrophobic portion (particularly the lipid portion).

[0029] Cationic lipids are described in U.S. Patent Application Publication Nos. 2006 / 0083780 and 2006 / 0240554, U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992, and PCT Publication No. WO 96 / 10390, the disclosures of which are incorporated by reference herein in their entirety for all purposes. Cationic lipids include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), and 2,2-dilinoleyl-4-(4-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA). 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-D MA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or a mixture thereof. In a preferred embodiment, the cationic lipid is DLinDMA, DLin-K-C2-DMA ("XTC2"), or a mixture thereof. The cationic lipid is preferably 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC, + Cationic lipids can have, for example, a phosphate moiety (e.g., uncharged) and a quaternary amine.

[0030] Preferred anionic lipids include those represented by the general formula (1): R-COO - X + Examples of the anionic lipid include anionic lipids represented by the following formula:

[0031] In general formula (1), R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, and at least one ethylene group constituting the alkenyl group may be substituted with a vinylene group.

[0032] The alkyl group represented by R may be linear, branched, or cyclic. The alkyl group represented by R preferably has 8 to 22 carbon atoms, more preferably 12 to 22 carbon atoms, and even more preferably 14 to 22 carbon atoms. Examples of the alkyl group represented by R include linear alkyl groups such as dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, and docosyl groups, as well as branched alkyl groups and cyclic alkyl groups. Substituents that may be substituted on the alkyl group include amino, benzyl, and halogen atoms (e.g., fluorine atoms). These substituents may be further substituted with a substituent. When the alkyl group is substituted with a substituent, the total number of carbon atoms in the alkyl group and the substituent is preferably 8 to 22, more preferably 12 to 22, and even more preferably 14 to 22.

[0033] The alkenyl group in R may be either linear or branched. The alkenyl group in R preferably has 8 to 22 carbon atoms, more preferably 12 to 22 carbon atoms, and even more preferably 14 to 22 carbon atoms. Examples of the alkenyl group in R include linear alkenyl groups such as dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, henicosenyl, and docosenyl, as well as branched alkenyl groups thereof. Substituents that may be substituted on the alkenyl group include amino, benzyl, and halogen atoms (e.g., fluorine atoms). These substituents may be further substituted with a substituent. When the alkenyl group is substituted with a substituent, the total number of carbon atoms of the alkenyl group and the substituent is preferably 8 to 22, more preferably 12 to 22, and even more preferably 14 to 22.

[0034] Furthermore, at least one ethylene group of the alkenyl group in R may be substituted with a vinylene group to form a polyene structure. The number of double bonds in the polyene structure is preferably 2 to 6, more preferably 2 to 4. The position of the double bonds is not particularly limited, but it is preferable that the double bonds are separated by at least two single bonds. Among these, R is preferably a group having an unsaturated bond, and more preferably a substituted or unsubstituted alkenyl group or a group having a substituted or unsubstituted polyene structure.

[0035] Furthermore, the alkyl group, alkenyl group, and group having a polyene structure in R may be substituted with a substituent, but even in this case, it is preferable that R consists only of carbon atoms and hydrogen atoms. That is, when the alkyl group, alkenyl group, and polyene structure are substituted with a substituent, it is preferable that the substituent also consists only of carbon atoms and hydrogen atoms.

[0036] R-COO -For example, carboxylate ions, in which a hydrogen ion dissociates from the carboxy group of a fatty acid, or derivatives thereof can be used. The fatty acid that generates the carboxylate ions may be saturated or unsaturated, but is preferably an unsaturated fatty acid, more preferably a cis-unsaturated fatty acid, and even more preferably a cis-long-chain unsaturated fatty acid. Examples of saturated fatty acids include myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), and lauric acid (C12:0). Examples of unsaturated fatty acids include oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid (C18:3), γ-linolenic acid (C18:3), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5), docosahexaenoic acid (C22:6), and erucic acid (C22:1). Here, the numbers in parentheses indicate the number of carbon atoms and the number of double bonds of each fatty acid. For example, the (C18:2) of linoleic acid indicates that the number of carbon atoms is 18 and the number of double bonds is 2. In general formula (1), X + represents a phospholipid having a cationic group. Here, "phospholipid" means a lipid containing a phosphate ester structure, and "cationic group" means a substituent group that is positively charged. X + is preferably a glycerophospholipid having a cationic group, and more preferably a phosphatidylcholine derivative. Here, the phosphatidylcholine derivative is a glycerol backbone, a substituted or unsubstituted alkanoyl group bonded to the 1st and 2nd positions of the glycerol backbone, and a phosphate ester group (-P(=O)(OH)O) bonded to the 3rd position of the glycerol backbone. - The term "phosphatidylcholine derivative" refers to a compound having a choline residue bound to the phosphate ester group. In this phosphatidylcholine derivative, at least one ethylene group of the alkanoyl group may be replaced with a vinylene group, and the hydrogen atom of the hydroxyl group of the phosphate ester group may be substituted with a substituted or unsubstituted alkyl group.

[0037] Also, X +is preferably a phospholipid having a structure represented by the following general formula (2), and more preferably a glycerophospholipid having a structure represented by the following general formula (2): CH(—CH—O—C(═O)—R 10 )(-OC(=O)-R 11 )-CH2-OP(OR 1 ) (OR 2 ) (=O) ... (2)

[0038] In general formula (2), R 1 represents an alkyl group substituted with a cationic group, and R 2 represents a hydrogen atom or a substituted or unsubstituted alkyl group. The phospholipid having the structure represented by general formula (2) has a positive charge as a whole, and preferably does not have a negatively charged portion.

[0039] R 1 The alkyl group in R may be linear, branched, or cyclic. 1 The alkyl group in the formula (I) preferably has 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. For example, R 1 Examples of the alkyl group in R include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. 1 The alkyl group in the formula (I) is preferably an ethyl group.

[0040] R 1 The cationic group in —NR is preferably an ammonium group represented by the following general formula (3): 4 R 5 R 6+ ... (3)

[0041] In general formula (3), R 4 , R 5 and R 6 R each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, and * represents the bonding position to the alkyl group. 4 , R 5 and R 6 may be the same or different. 4 , R 5 and R 6The number of substituted or unsubstituted alkyl groups among R 4 , R 5 and R 6 All of may be hydrogen atoms or substituted or unsubstituted alkyl groups, or one or two of them may be hydrogen atoms and the remaining may be substituted or unsubstituted alkyl groups.

[0042] R 4 , R 5 and R 6 The alkyl group in R may be linear, branched, or cyclic. 4 , R 5 and R 6 The alkyl group in the formula (I) preferably has 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. For example, R 4 , R 5 and R 6 Examples of the alkyl group in R include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. 4 , R 5 and R 6 The alkyl group in R is preferably a methyl group. Examples of substituents that can be substituted on the alkyl group include a benzyl group and a halogen atom (e.g., a fluorine atom). These substituents may be further substituted with a substituent. In one preferred embodiment, R 4 , R 5 and R 6 is a methyl group.

[0043] R 1 The cationic group in is preferably a quaternary ammonium group, and is preferably a quaternary ammonium group (R 4 In addition, the substitution position of the cationic group in the alkyl group is not particularly limited, but it is preferable that a hydrogen atom bonded to a carbon atom at the terminal of the alkyl group is substituted with a cationic group.

[0044] In general formula (2), R 2represents a hydrogen atom or a substituted or unsubstituted alkyl group. 2 is preferably a substituted or unsubstituted alkyl group. 2 The alkyl group in R may be linear, branched, or cyclic. 2 The alkyl group in the formula (I) preferably has 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. For example, R 2 Examples of the alkyl group in R include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. 2 The alkyl group in is preferably an ethyl group. Examples of substituents that can be substituted on the alkyl group include a benzyl group and a halogen atom (e.g., a fluorine atom). These substituents may be further substituted with a substituent.

[0045] In general formula (2), R 10 and R 11 are each independently a fatty chain having 8 to 22 carbon atoms, preferably a long fatty chain, which may be a long saturated fatty chain or a long unsaturated fatty chain. 10 and R 11 are each independently a long saturated fatty chain having 12 to 22 carbon atoms. 10 and R 11 may be a long saturated fatty chain having 12 to 14 carbon atoms (e.g., 13). In one embodiment, the glycerophospholipid represented by general formula (2) is EDMPC + It could be.

[0046] The number of alkylcarbonyloxy groups substituted on the alkyl group is preferably 2 to 8, more preferably 2 to 4, and most preferably 2. There are no particular restrictions on the positions of the alkylcarbonyloxy groups substituted on the alkyl group, but R 3 When the alkyl group as the main chain is an n-propyl group, the hydrogen atom bonded to the terminal carbon atom and the hydrogen atom bonded to the carbon atom adjacent thereto are replaced with alkylcarbonyloxy groups, which corresponds to a glycerophospholipid and is a particularly preferred phospholipid.

[0047] For the explanation, preferred range, and specific examples of the alkyl group in the alkylcarbonyloxy group, reference can be made to the explanation, preferred range, and specific examples of the alkyl group in R above.

[0048] Preferred examples of compounds having a structure represented by general formula (1) are listed below. However, the ionic surfactant according to this embodiment should not be construed as being limited by these specific examples. For example, the carboxylate ion is a long-chain fatty acid, preferably a long-chain unsaturated fatty acid, such as linoleic acid or oleic acid, or a long-chain saturated fatty acid, such as stearic acid. In the glycerophospholipid represented by general formula (2), R1 is -NR 4 R 5 R 6 and R 4 , R 5 and R 6 are each independently methyl or ethyl, preferably both are methyl, and R 10 and R 11 are each independently a long-chain fatty acid chain, preferably a long-chain fatty acid chain having 12 to 22 carbon atoms, more preferably a long-chain fatty acid chain having 12 to 14 carbon atoms. More specific examples include a carboxylate ion that is a long-chain unsaturated fatty acid, such as linoleic acid or oleic acid, and a glycerophospholipid represented by general formula (2) in which R1 is -NR 4 R 5 R 6 and R 4 , R 5 and R 6 are each independently methyl or ethyl, preferably both are methyl, and R 10 and R 11 are each independently a long-chain fatty chain having 12 to 22 carbon atoms, more preferably a long-chain fatty chain having 12 to 14 carbon atoms. A more specific example is, for example, a carboxylate ion is a long-chain unsaturated fatty acid, such as linoleic acid or oleic acid, and in the glycerophospholipid represented by general formula (2), R1 is -NR 4 R 5 R6 and R 4 , R 5 and R 6 is methyl, and R 10 and R 11 are each independently a long-chain aliphatic chain having 12 to 22 carbon atoms, more preferably a long-chain aliphatic chain having 12 to 14 carbon atoms. - represents R—COO in general formula (1). - Specific examples thereof include carboxylate ions of linoleic acid, oleic acid, acetic acid, and stearic acid.

[0049]

[0050] The above shows the ionic bond formed between the carboxylate ion and the preferred cationic surfactant. Mixtures of these cationic and anionic lipids can be liquids (especially ionic liquids) at room temperature when mixed at a 1:1 charge ratio.

[0051] Anionic surfactants include phospholipids and fatty acids, such as phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0052] Phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.

[0053] Fatty acids are carbon chains with monocarboxylic acids. Fatty acids are preferably in the cis form rather than the trans form. Fatty acids are represented by the rational formula CH3-R-CO2H {where carbon chain}, and are represented based on the number of carbon atoms and double bonds contained in the fatty acid. For example, capric acid has R = -(CH2)8- and is represented as 10:0, and lauric acid has R = -(CH2) 10 - and is represented by 12:0, and myristic acid is where R is -(CH2) 14 -, represented by 14:0, and pentadecylic acid is a compound in which R is -(CH2) 15 -, represented by 15:0, and palmitic acid is where R is -(CH2) 16 - and is represented by 16:0, palmitoleic acid has R -(CH2)5CH=CH(CH2)7- and is represented by 16:1, and stearic acid has R -(CH2) 18 - and is represented by 18:0, oleic acid has R -(CH2)7CH=CH(CH2)7- and is represented by 18:1(n-9), vaccenic acid has R -(CH2)5CH=CH(CH2)9- and is represented by 18:1(n-7), linoleic acid has R -(CH2)3(CH2CH=CH)2(CH2)7- and is represented by 18:2(n-6), (9,12,15)-linolenic acid has R -(CH2CH=CH)3(CH2)7- and is represented by 18:3(n-3), and (6,9,12)-linolenic acid has R -(CH2)3(CH2CH=CH)3(CH2)4- and is represented by 18:3(n-6).

[0054] As used herein, the term "target molecule" refers to molecules such as polypeptides (proteins), nucleic acids, and low-molecular-weight compounds, as well as complexes thereof, and preferably has physiological activity. The target molecule may be a fusion protein of multiple proteins, a complex of multiple proteins (e.g., an antibody), or a complex of a protein and a nucleic acid (e.g., an RNA-protein complex (RNP)). In a preferred embodiment, the target molecule may be a water-soluble molecule. As used herein, the term "water-soluble molecule" refers to a molecule that is water-soluble and can be dissolved in an effective amount to produce the expected effect. A water-soluble molecule can be sufficiently water-soluble to achieve a concentration of, for example, 100 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less. A water-soluble molecule can be sufficiently water-soluble to achieve a concentration of, for example, 1 pM or more, 10 pM or more, 100 pM or more, or 1 nM or more.

[0055] (Embodiment 1) According to the present disclosure, a molecule (preferably a water-soluble molecule) and 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) +The present invention provides a dispersion formulation comprising particles comprising a solid complex of a lipid mixture (containing one or more lipid surfactants) containing EDMPC or a pharmaceutically acceptable salt thereof, and an oily base, wherein the particles are dispersed in the oily base. In this embodiment, the lipid mixture may or may not contain an anionic lipid. When an anionic lipid is contained, the anionic lipid preferably contains one or more selected from the group consisting of oleic acid (OA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), cardiolipin (CL), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (DOPS), and phosphatidylinositol (PI). Also provided is a dispersion formulation comprising particles comprising a solid complex of a lipid mixture (containing one or more lipid surfactants) containing EDMPC or a pharmaceutically acceptable salt thereof, and an oily base, wherein the particles are dispersed in the oily base. In this embodiment, the lipid mixture may or may not contain an anionic lipid. When an anionic lipid is contained, the anionic lipid preferably contains one or more selected from the group consisting of oleic acid (OA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), cardiolipin (CL), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (DOPS), and phosphatidylinositol (PI). + Pharmaceutically acceptable salts of include EDMPC + A salt in which the charge of + Chloride salt of (EDMPC + Cl - This solid composite can be advantageously used as the first composite described below. In addition, this solid composite can be advantageously produced by the production method for the first composite described below.

[0056] (Embodiment 2) According to the present disclosure, a complex (first complex) comprising a lipid (lipid mixture) and a target molecule is provided. According to the present disclosure, a complex comprising a nonionic lipid and a target molecule is provided. According to the present disclosure, a complex comprising an ionic lipid and a target molecule is provided. In one aspect, the ionic lipid comprises 30 mol% or more, 40 mol% or more, 50 mol% or more, 60 mol% or more, 70 mol% or more, 80 mol% or more, or 90 mol% or more of cationic lipid. In one aspect, the ionic lipid comprises 30 mol% or more, 40 mol% or more, 50 mol% or more, 60 mol% or more, 70 mol% or more, 80 mol% or more, or 90 mol% or more of anionic lipid. In one aspect, the ionic lipid comprises a cationic lipid and an anionic lipid. The cationic lipid / anionic lipid ratio (molar ratio) in the ionic lipid may be, for example, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1 or more, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, for example, 0.1 to 10, 0.2 to 5, 0.25 to 4, 0.33 to 3, 0.4 to 2.5, 0.5 to 2, 0.67 to 1.5, 0.75 to 1.33, 0.8 to 1.25, 0.9 to 1.1, or about 1.0. According to the present disclosure, a complex (target molecule-lipid complex) is provided, which includes a liquid (liquid lipid composition) composed of cationic lipids and anionic lipids (hereinafter sometimes referred to as an ionic surfactant or simply a surfactant) and a target molecule. The nonionic lipids form micellar particles in water, and the target molecule is encapsulated within the micellar particles. It is believed that within the micellar particles, the lipid portion of the nonionic lipid faces toward the core and the hydrophilic portion faces outward. The cationic lipids and anionic lipids form micellar particles in water, and the target molecule is encapsulated within the micellar particles. It is believed that within the micellar particles, the lipid portion of the cationic lipids and anionic lipids faces toward the core and the ionic portion faces outward. Within the micellar particles, the target molecule interacts with the ionic portion and / or the lipid portion. The first complex is preferably used for introducing a target molecule into extracellular vesicles (e.g., exosomes).

[0057] (Embodiment 3) The present disclosure provides a dispersion formulation comprising a complex comprising an antibody and a lipid (lipid mixture) and a base, wherein the complex is dispersed in the base. In a preferred embodiment, the complex is a solid complex, and the base is an oily base. In another preferred embodiment, the base is an aqueous base. In this embodiment, where the base is an aqueous base, the complex may or may not contain water. In a preferred embodiment, the lipid is an ionic lipid. In one embodiment, the ionic lipid comprises 30 mol% or more, 40 mol% or more, 50 mol% or more, 60 mol% or more, 70 mol% or more, 80 mol% or more, or 90 mol% or more of cationic lipid. In one embodiment, the ionic lipid comprises 30 mol% or more, 40 mol% or more, 50 mol% or more, 60 mol% or more, 70 mol% or more, 80 mol% or more, or 90 mol% or more of anionic lipid. In one embodiment, the ionic lipid comprises a cationic lipid and an anionic lipid. The cationic lipid / anionic lipid ratio (molar ratio) in the ionic lipid may be, for example, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1 or more, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, for example, 0.1 to 10, 0.2 to 5, 0.25 to 4, 0.33 to 3, 0.4 to 2.5, 0.5 to 2, 0.67 to 1.5, 0.75 to 1.33, 0.8 to 1.25, 0.9 to 1.1, or about 1.0. The complex of this embodiment may form a further complex with exosomes (or may contain extracellular vesicles such as exosomes), but may not form a further complex with exosomes (or may not contain extracellular vesicles such as exosomes). All components of the complex of this embodiment may be synthetic (artificially synthesized), and preferably isolated. In a preferred embodiment, the antibody may be a recombinant antibody produced from antibody-producing cells, and the lipid may be a synthetic lipid. The antibody contained in the formulation retains its antigen-binding property even after administration.

[0058] In all aspects (including embodiments 1 to 3 and other aspects), in a preferred aspect, the target molecule is a water-soluble molecule, the lipid is a surfactant (lipidic surfactant), and preferably the lipid comprises a cationic lipid. The target molecule may be, for example, a water-soluble protein (e.g., a glycoprotein, an enzyme, an antibody, etc.), a nucleic acid, or a water-soluble small molecule compound.

[0059] The first complex may consist of only the target molecule and a surfactant, or may contain other components such as a stabilizer. The stabilizer is, for example, a hydrophilic protein or polysaccharide, preferably having a molecular weight of 10,000 or more. The stabilizer improves the stability of the complex by being coated with the surfactant together with the target molecule, and can prevent leakage of the target molecule from the complex in the composition.

[0060] Examples of proteins include physiologically active substances such as cytokines (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, α-interferon, β-interferon, γ-interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, and tumor necrosis factor). Proteins also include, for example, agalsidase beta, laronidase, alteplase, havinafsp alfa, urokinase, idursulfase, sebelipase alfa, monteplase, asfotase alfa, imiglucerase, velaglucerase, agalsidase alfa, alglucosidase alfa, avalglucosidase alfa, idursulfase, galsulfase, elosulfase alfa, rasburicase, dornase, alfa, asfotase alfa, collagenase, sebelipase, condoliase, elapeguademase, cerliponase, alfa, glucarpidase, bestronidase alfa, oliprodase alfa, blood clotting factors (e.g., I, II, III, IV, V, VI, VII, VIII, IX, X, XI, and XII), insulin, somatropin, erythropoietin, and chemokines.Further examples of proteins include, for example, Cas9 protein, RNA polymerase, transcription factors, ribosomes, cyclin-dependent kinases, growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factors (TGFs), colony-stimulating factors (CSFs), erythropoietin (EPO), granulocyte colony-stimulating factor (GST), and the like. Examples of proteins include granulocyte-macrophage colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), interleukins (some interleukins have growth factor activity), bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial cell line-derived neurotrophic factor (GDNF), ganglionic cell trophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophins (NTs), keratinocyte growth factor (KGF), and thrombopoietic factor (TPO). Proteins also include serum albumin, ovalbumin, casein, lysozyme, and lipase. These proteins may be used alone or in combination of two or more. Examples of proteins include protein complexes. Examples of protein complexes include antibodies or antigen-binding fragments thereof.

[0061] Nucleic acids include, for example, nucleic acids (DNA or mRNA) encoding any one or more of the above proteins. Nucleic acids also include nucleic acids such as siRNA, miRNA, shRNA, and antisense oligonucleotides.

[0062] Examples of polysaccharides include LM pectin, HM pectin, hydroxypropylmethylcellulose phthalate, heparin, alginic acid, carboxymethylcellulose, etc. These polysaccharides may be used alone or in combination of two or more.

[0063] As the lipid, an amphipathic lipid (e.g., having a hydrophobic portion and a hydrophilic portion and exhibiting surfactant properties) can be preferably used. The lipid is preferably a dermatologically or pharmaceutically acceptable lipid. Examples of such lipids are as described above. In one embodiment, the lipid can be a nonionic lipid having a hydrophilic portion. In one embodiment, the lipid is an ionic lipid. In one embodiment, the lipid is a mixture of a cationic lipid and an anionic lipid. In this embodiment, the mixture can preferably be a 1:1 mixture of a cationic lipid and an anionic lipid. In one preferred embodiment, the mixture is liquid. As the cationic lipid, for example, the cationic lipids described above can be used, and as the anionic lipid, for example, the anionic lipids described above can be used. In one preferred embodiment, the cationic lipid is EDMPC + The anionic lipid may comprise one or more lipids selected from the group consisting of linoleic acid (Lin), oleic acid (Ole), and stearic acid (Ste). A liquid mixture is preferred because the manufacturing process is simple. Specifically, when the mixture is liquid, lipid nanoparticles encapsulating the target molecule can be obtained simply by mixing a solution (particularly an aqueous solution) containing the target molecule with an ethanol solution containing the liquid lipid composition. In this case, the emulsification process and subsequent solvent removal are unnecessary. Due to this manufacturing advantage, the lipid is preferably a liquid lipid composition. By reducing the mixing ratio of ethanol, a complex composition with a reduced ethanol content (e.g., ethanol concentration of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) can be obtained. If necessary, the ethanol concentration may be further reduced by solution exchange using various techniques such as columns or dialysis.

[0064] Examples of target molecules include polypeptides. Polypeptides may be linear or cyclic. The molecular weight of the polypeptide is not particularly limited, but may be, for example, 1 kDa to 500 kDa, 1 kDa to 400 kDa, 1 kDa to 300 kDa, 1 kDa to 200 kDa, e.g., 5 kDa to 160 kDa, or 10 kDa to 100 kDa. The polypeptide may be, for example, a major histocompatibility complex (MHC) class I-restricted or class II-restricted polypeptide. The polypeptide may also be an enzyme (e.g., a metabolic enzyme). The polypeptide may also be a mitochondrial protein. The polypeptide may also be a cytoplasmic protein. The polypeptide may also be a nuclear protein. Cyclic polypeptides (e.g., 600 to 6,000 Da) and intracellular antibodies (e.g., scFv fragments of antibodies, approximately 30 kDa) may exert a desired physiological effect by binding to a target molecule within a cell. For example, cyclic polypeptides and intracellular antibodies are expected to have the effect of inhibiting protein-protein interactions (PPI) of target molecules, and by introducing them into cells, they can inhibit intracellular PPI, thereby providing valuable pharmaceutical value. Cyclic polypeptides and intracellular antibodies can also provide valuable pharmaceutical value by inducing degradation of target molecules by attaching a degradation-inducing tag (e.g., a degron). Therefore, the target molecule can be a polypeptide (preferably a cyclic polypeptide or an intracellular antibody) in the form of a fusion protein with a degradation-inducing tag.

[0065] Examples of target molecules include antibodies. Examples of antibodies include full-length antibodies. The antibodies can be, for example, polyclonal antibodies or monoclonal antibodies. The antibodies are preferably human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies are not particularly limited, but any currently commercially available antibodies can be used. Examples of antibodies include monospecific antibodies and multispecific antibodies. The antibodies are preferably monoclonal antibodies. Examples of antibodies include muromonab-CD3, ibritumomab tiuxetan, iodine 131 tositumomab, catumaxomab, blinatumomab, and moxetumomab. pasudotox: moxetumomab, abciximab: abiximab, rituximab: rituximab, basiliximab: basiliximab, infliximab: infliximab, cetuximab: cetuximab, brentuximab vedotin: brentuximab vedotin, siltuximab: siltuximab, dinutuximab: dinutuximab, obiltoxaximab: obiltoxaximab, isatuximab: isatuximab, margetuximab: margetuximab, cetuximab saratalocan Sodium: cetuximab sarotarocan sodium, daclizumab: daclizumab, palivizumab: palivizumab, trastuzumab: trastuzumab, gemtuzumab ozogamicin: gemtuzumab ozogamicin, alemtuzumab: alemtuzumab, omalizumab: omalizumab, efalizumab: efalizumab, bevacizumab: bevacizumab, natalizumab: natalizumab, tocilizumab: tocilizumab, ranibizumab: ranibizumab, eculizumab: eculizumab, certolizumab pegol: certolizumab pegol, mogamulizumab: mogamulizumab, pertuzumab: pertuzumab,trastuzumab emtansine: trastuzumab emtansine, obinutuzumab: obinutuzumab, vedolizumab: vedolizumab, pembrolizumab: pembrolizumab, idarucizumab: idarucizumab, mepolizumab: mepolizumab, elotuzumab: elotuzumab, ixekizumab: ixekizumab, reslizumab: reslizumab, atezolizumab: atezolizumab, ocrelizumab: ocrelizumab, inotuzumab ozogamicin: inotuzumab ozogamicin, emicizumab: emicizumab, benralizumab: benralizumab, galcanezumab: galcanezumab, fremanezumab: fremanezumab, tildrakizumab: tildrakizumab, caplacizumab: caplacizumab, ibalizumab: ibalizumab, ravulizumab: ravulizumab, romosozumab: romosozumab, risankizumab: risankizumab, polatuzumab vedotin: polatuzumab vedotin, romosozumab: romosozumab, brolucizumab: brolucizumab, crizanlizumab: crizanlizumab, trastuzumab deruxtecan: trastuzumab deruxtecan, eptinezumab: eptinezumab, sacituzumab govitecan: sacituzumab govitecan, inebilizumab: inebilizumab, tafasitamab: tafasitamab, belantamab mafodotin: belantamab mafodotin, satralizumab: satralizumab, naxitamab: naxitamab, loncastuximab tesirine: loncastuximab tesirine, dostarlimab: dostarlimumab, bimekizumab: bimekizumab, sutimlimab: sutimlimab, adalimumab: adalimumab, panitumumab: panitumumab, golimumab: golimumab, ustekinumab: ustekinumab, canakinumab: canakinumab, ofatumumab: ofatumumab, denosumab: denosumab,ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, necitumumab, daratumumab, brodalumab, olaratumumab Laratumab, bezlotoxumab, avelumab, durvalumab, dupilumab, bezlotoxumab, guselkumab, sarilumab, burosumab, erenumab, lanadelumab, emapalumab, enfortumab Vedotin: Enfortumab vedotin, Teprotumumab: Teprotumab, Atoltivimab, Maftivimab, Odesivimab: Atoltivimab, Maftivimab, Odesivimab, Ansuvimab: Ansuvimab, Evinacumab: Evinacumab, Amivantamab: Amivantamab, Aducanumab: Aducanumab, Tralokinumab: Tralokinumab, Anifrolumab: Anifrolumab, Tisotumab vedotin: Tisotumab vedotin, Tezepelumab: Tezepelumab, Casirivimab + imdevimab: casirivimab + imdevimab, Regdanvimab: regdanvimab, Sotrovimab: sotrovimab, cemiplimab: cemiplimab, Faricimab: faricimab, and Tebentafusp: an antibody selected from the group consisting of an antibody having three complementarity determining regions (CDRs) in its heavy chain variable region and three complementarity determining regions (CDRs) in its light chain variable region, or an antigen-binding fragment thereof. Antibodies also include antibodies that bind to intracellular factors (e.g., intracellular proteins, cytoplasmic proteins). Antibody fragments include, for example, Fab, Fab',Examples of antibodies include F(ab')2, F(ab'), and scFv. Antibodies can bind to intracellular factors and directly or indirectly inhibit or activate the activity of the intracellular factor, or neutralize the interaction of the intracellular factor with other factors and inhibit or activate the function of the intracellular factor. Therefore, antibodies or antigen-binding fragments thereof are preferably capable of directly or indirectly inhibiting or activating the activity of intracellular factors. Furthermore, antibodies or antigen-binding fragments thereof are preferably capable of neutralizing the interaction of the intracellular factor with other factors.

[0066] Examples of target molecules include nucleic acids. Nucleic acids include DNA and RNA, as well as modified versions thereof (i.e., modified nucleic acids). DNA includes linear or circular single-stranded DNA and linear or circular double-stranded DNA. RNA includes linear single-stranded RNA and linear double-stranded RNA. For example, RNA includes mRNA, microRNA (miRNA), siRNA, shRNA, tRNA, aptamer, and rRNA. Nucleic acids also include antisense oligos. Antisense oligos generally contain DNA and / or RNA and have a sequence complementary to a target nucleic acid. siRNA and shRNA have an appropriate length (e.g., 20-25 mer) and an appropriate sequence (complementarity to the target sequence) for knocking down a target nucleic acid. Nucleic acids may also include modified nucleic acids. The length of siRNA may be, for example, about 20 to about 30 bases, about 20 to about 24 bases, or about 21 to about 23 bases. Nucleic acids also include gapmers and mixmers. Gapmers are single-stranded nucleic acids having a DNA portion and an RNA or modified nucleic acid portion, with the RNA or modified nucleic acid portion located at both ends of the DNA portion. Gapmers can be, for example, double-stranded nucleic acids. Mixmers are antisense nucleic acids containing different types of nucleic acids. Nucleic acids can also be vectors (e.g., expression vectors) encoding any of the above. MicroRNAs are RNA molecules approximately 21 to 25 nucleotides in length. They typically bind to mRNA to inhibit its translation or degrade mRNA, thereby regulating its expression level. Expression vectors have a gene expression cassette containing the above nucleic acid operably linked to a regulatory sequence. Expression vectors can be circular vectors (e.g., plasmid vectors). In some preferred embodiments where the target molecule is a nucleic acid, the lipid can be a nonionic lipid, or more preferably, a cationic lipid or a mixture of cationic lipid and anionic lipid (e.g., a liquid lipid composition that is a 1:1 mixture).In some preferred embodiments where the target molecule is a nucleic acid, the cationic lipid may also be an ionizable lipid. In some preferred embodiments where the target molecule is a nucleic acid, the first complex may be a liposome encapsulating the nucleic acid, rather than a simple complex of the nucleic acid and the cationic lipid.

[0067] Liposomes include unilamellar liposomes, multilamellar liposomes (particularly those having one or more lipid bilayer membranes), stealth liposomes (for example, those whose surfaces are coated with uncharged hydrophilic molecules or polymers such as PEG, which allow them to evade immune defenses or improve circulation compared to uncoated liposomes), immunoliposomes (those with cancer antigen-targeting molecules such as antibodies or peptides attached to the surface), and thermoresponsive liposomes (which release encapsulated components in a thermoresponsive manner), all of which can be advantageously used in the present invention. Coating the surface with an anti-CD71 antibody may also promote permeability through the blood-brain barrier.

[0068] Lipids for forming liposomes are not particularly limited, but examples thereof include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, PEG-modified lipids (e.g., PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol)), cationic lipids (e.g., DOTAP (1,2-dioleoyl-3-trimethylammonium propane)), and sphingomyelin. Other pharmaceutically acceptable lipids may also be used.

[0069] Proteins encoded by mRNA include components or portions thereof specific to heterologous organisms, such as pathogens, and components or portions thereof specific to cancer cells. Pathogens include pathogenic viruses, pathogenic microorganisms, and pathogenic parasites. Pathogenic viruses include viruses selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, herpesvirus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus (e.g., severe acute respiratory syndrome-associated coronavirus (SARS-CoV), Middle East respiratory syndrome-associated coronavirus (MERS), SARS-CoV-2), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-cell leukemia (HTL) virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, John Cunningham (JC) virus, and arboviral encephalitis virus. Pathogenic microorganisms include pathogenic bacteria, including bacteria selected from the group consisting of chlamydia, rickettsiales, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria. Pathogenic microorganisms include pathogenic fungi, such as Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), Mucorales (e.g., Mucor, Abscisicida, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.Pathogenic parasites include those selected from the group consisting of Entamoeba histolytica, Balanchine coli, Naegleria fowleri, Acanthamoeba spp., Giardia lamblia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Oncus brasiliensis. Thus, in one embodiment of the present invention, the mRNA can encode a component (preferably a surface antigen) of these pathogens or a portion thereof. In particular, among the surface antigens of pathogens, antigens used by pathogens to infect cells, such as the S protein of a betacoronavirus, can be preferably encoded by the mRNA of the present invention. Examples of cancer include bladder cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, squamous cell carcinoma, skin cancer, central nervous system neoplasms, lymphoma, leukemia, sarcoma, virus-related cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, and head and neck cancer. In some embodiments of the present invention, the mRNA may be an antigen specific to these cancers (cancer-specific antigen, preferably a cancer-specific surface antigen) or a portion thereof. The cancer-specific antigen may be, for example, a neoantigen or a portion thereof characteristic of a neoantigen. Cancer-specific antigens also include, for example, cancer-testis antigens (e.g., proteins of the MAGE family, NY-ESO-1), differentiation antigens (e.g., tyrosine kinase in melanoma, Melan-A / MART-1, PSA in prostate cancer), overexpressed cancer antigens (e.g., Her-2 / Neu, survivin, telomerase, and WT-1), cancer-induced mutants of β-catenin, cancer-induced mutants of CDK4, MUC-1, particularly MUC-1 with altered glycosylation patterns, and E6 or E7 of human papillomavirus.In regard to these, a person skilled in the art can select an appropriate antigen depending on the type of infectious disease and cancer suffered by the subject and the type of antigen expressed in the cancer, and prepare mRNA suitable for the present invention. mRNA for naturally occurring mutant proteins can also be designed appropriately.

[0070] In one embodiment, the molecule of interest is mRNA, and the mRNA is a cancer antigen. In this embodiment, a complex of the molecule of interest, lipid, and extracellular vesicle can be administered to a subject with cancer. The subject receiving the administration can be administered an immune checkpoint inhibitor. Examples of immune checkpoint inhibitors that can be used include checkpoint inhibitors that block PD-1-mediated signaling or checkpoint inhibitors that block CTLA-4-mediated signaling. Examples of immune checkpoint inhibitors include antibodies that can neutralize the binding of PD-1 to PD-L1 or PD-L2, and antibodies that can neutralize the binding of CTLA-4 to CD80 or CD86. Examples of antibodies that can neutralize the binding of PD-1 to PD-L1 include anti-PD-1 antibodies and anti-PD-L1 antibodies that can neutralize the binding of PD-1 to PD-L1. Examples of antibodies that can neutralize the binding of PD-1 to PD-L2 include anti-PD-1 antibodies and anti-PD-L2 antibodies that can neutralize the binding of PD-1 to PD-L2. Antibodies capable of neutralizing the binding of CTLA-4 to CD80 or CD86 include anti-CTLA-4 antibodies capable of neutralizing the binding of CTLA-4 to CD80 or CD86. Examples of immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies such as nivolumab, pembrolizumab, and pidilizumab; anti-PD-L1 antibodies such as atezolizumab, durvalumab, and avelumab; anti-CTLA-4 antibodies such as ipilimumab; anti-TIM-3 antibodies such as TSR-022 (WO 2016 / 161270) and MBG453 (WO 2015 / 117002); anti-LAG-3 antibodies such as LAG525 (U.S. Publication No. 2015 / 0259420); anti-TIGIT antibodies such as MAB10 (WO 2017 / 059095); Investigation, 2010, Vol. 120, No. 1, pp. 157-167), and anti-VISTA antibodies such as JNJ-61610588 (WO 2016 / 207717).In a preferred embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody, more preferably an anti-PD-1 antibody or an anti-PD-L1 antibody capable of neutralizing the binding of PD-1 to PD-L1, and even more preferably an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab, or one or more anti-PD-L1 antibodies selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0071] In some embodiments, at least one uridine in an mRNA may be changed to a pseudouridine. The pseudouridine may be 1-methylpseudouridine. The mRNA may be transcribed from a cDNA, i.e., it may not have an intron. The mRNA may also have a cap structure at the 5' end (Furuichi Y & Miura K. Nature. 1975;253(5490):374-5). The cap structure can be added to an mRNA by the Anti-Reverse Cap Analogues (ARCA) method using a cap analog (Stepinski J et al. RNA. 2001 Oct;7(10):1486-95). The Cap0 structure of mRNA can be converted to a Cap1 structure by further performing 2'-O methyltransferase treatment. This can be performed by conventional methods, for example, using commercially available kits such as the ScriptCap m7G Capping System and the ScriptCap 2'-O-Methyltransferase Kit, or the T7 mScript Standard mRNA Production System (AR Brown CO., LTD). The mRNA can have a polyA tail. The addition of the polyA tail can be performed by conventional methods, for example, using the A-Plus Poly(A) Polymerase Tailing Kit (AR Brown CO., LTD). Therefore, in one embodiment, the mRNA may be an mRNA having a Cap structure at the 5'-end and a polyA at the 3'-end, and preferably in which at least a portion of the uridines is pseudouridine (preferably 1-methylpseudouridine). The mRNA may be isolated or synthetic.

[0072] In one aspect, the target molecule is an RNP. Furthermore, the RNP is not particularly limited, and can be, for example, a complex of a Cas endonuclease and a guide RNA (gRNA) of the CRISPR / Cas system. The Cas endonuclease has a size of about 130 kDa, and the RNP complex has a size of about 180 kDa, which is a size that can be sufficiently encapsulated in an exosome. The gRNA can include a crRNA and a tracrRNA, and the crRNA and the tracrRNA may be linked by a hairpin to form a single-stranded gRNA (sgRNA).

[0073] Cas endonucleases are broadly classified into Class 1, Class 2, and others. Class I includes Cas nucleases derived from Escherichia coli, such as Cascade (CRISPR-associated complex for antiviral defense), Cas3 nuclease, and Csm / Cmr nuclease. Class 2 includes Cas9 endonuclease, Cas12a, Cas13, Cas14, and CasΦ derived from Staphylococcus aureus, Streptococcus pyogenes, or Neisseria meningitidis. Some nucleases include RNA-protein complexes. The Cas endonuclease may be a nickase. For example, a nickase is one in which either the RuvC domain or the HNH domain of Cas9 has been inactivated, and can introduce a single-strand break into a target sequence.

[0074] The gRNA is an RNA that can form a complex with the Cas protein and guide the Cas protein to the target region. The crRNA contains a spacer region having a sequence complementary to the nucleotide sequence of the target region and a repeat region having a sequence complementary to the tracrRNA, and the tracrRNA has an anti-repeat region having a sequence complementary to the repeat region and a 3' tail region that forms a complex with the Cas nuclease. The 3' flanking region of the cleavage region typically requires a protospacer adjacent motif (PAM). Therefore, the spacer region of the gRNA can be designed toward the sequence adjacent to the 5' side of the PAM. Those skilled in the art can design gRNAs using well-known conventional techniques.

[0075] When exosomes encapsulate a Cas-gRNA complex, the exosomes can be contacted with target cells in vivo or in vitro to target a target region in the genome of the target cell, thereby cleaving the target region or modifying the target region. Modifications include the introduction of indels (insertions or deletions), insertion of a nucleotide sequence of interest, and nucleotide substitution. These can be achieved, for example, by targeting a target region of the genome with a Cas-gRNA complex in the presence of template DNA. The template DNA comprises an upstream homology arm and a downstream homology arm, and there may or may not be a sequence between the upstream and downstream homology arms. The upstream and downstream homology arms have sequences (particularly complementary sequences) capable of homologous recombination with the upstream and downstream of the target region. When the Cas-gRNA complex targets a target region of the genome in the presence of template DNA, the genomic sequence between the upstream and downstream of the target region is replaced with the sequence between the upstream and downstream homology arms of the template DNA. It is practical to encapsulate template DNA in exosomes, and due to the high loading rate, many exosomes can encapsulate both template DNA and RNPs.

[0076] As used herein, a "control sequence" refers to a sequence that drives a gene operably linked to it and has the activity of transcribing RNA from that gene. A control sequence is, for example, a promoter. Examples of promoters include class I promoters (which can be used to transcribe pre-rRNA), class II promoters (which contain a core promoter and upstream promoter elements and can be used to transcribe mRNA), and class III promoters (further classified into types I, II, and III). Control sequences, also known as regulatory sequences, may be any promoter that can transcribe mRNA in cells such as animal cells and plant cells. For example, various Pol II promoters can be used as the first control sequence. Pol II promoters include, but are not limited to, the CMV promoter, the EF1 promoter (EF1α promoter), the SV40 promoter, the MSCV promoter, the hTERT promoter, the β-actin promoter, the CAG promoter, and the CBh promoter. Promoters may also include promoters that drive bacteriophage-derived RNA polymerases, such as the T7 promoter, T3 promoter, and SP6 promoter, as well as Pol III promoters, such as the U6 promoter. The T7 promoter is preferably used for transcription from circular DNA, and the SP6 promoter is preferably used for transcription from linear DNA.

[0077] Examples of target molecules include low molecular weight compounds. Examples of low molecular weight compounds include antioxidants (e.g., ascorbic acid, isoascorbic acid, vitamin E, catechin, dibutylhydroxytoluene, tocopherol, butylhydroxyanisole, polyphenols, resveratrol (resveratrol 3,5,4'-trihydroxy-trans-stilbene), coenzyme Q10, N-acetylcysteine, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), superoxide dismutase (SOD), nicotinamide mononucleotide (NMN), astaxanthin, β-carotene, and glutathione. Examples of low molecular weight compounds include antitumor agents. Examples of antitumor agents include paclitaxel, actinomycin, camptothecin, irinotecan, epirubicin, etoposide, oxaliplatin, L-amycin, benzodiazepine, benzocaine ... Sparginase, alectinib, pemetrexed, ifosfamide, gefitinib, cyclophosphamide, carboplatin, cabazitaxel, imatinib, capecitabine, amrubicin, gemcitabine, crizotinib, cisplatin, afatinib, regorafenib, cytarabine, sunitinib, lapatinib, erlotinib, docetaxel, tegafur / uracil, temozolomide, liposomal doxorubicin Examples of low molecular weight compounds include cyclosporine, dacarbazine, dasatinib, vinorelbine, nimustine, nedaplatin, sorafenib, nogitecan, eribulin, vincristine, bleomycin, vinblastine, fluorouracil, methotrexate, and mitomycin. Examples of low molecular weight compounds include those with physiological activity. Examples of low molecular weight compounds include water-soluble compounds and water-insoluble compounds.

[0078] A complex of a target molecule and a lipid can be obtained by mixing the target molecule with the lipid. Mixing is performed under conditions suitable for complex formation, and usually, complex formation is promoted simply by mixing. The complex usually contains micelles formed by the lipid. The target molecule is preferably encapsulated in the micelle, for example, the target molecule interacts with the hydrophilic and / or hydrophobic portions of the lipid constituting the micelle. In a preferred embodiment, the lipid is an ionic lipid or a mixture thereof, and may be a nonionic lipid or may contain a nonionic lipid. More preferably, the lipid is a cationic lipid or contains a cationic lipid, and even more preferably, it is a mixture of a cationic lipid and an anionic lipid, and even more preferably, it is a 1:1 mixture of a cationic lipid and an anionic lipid. Mixing is performed under suitable conditions.

[0079] The complex of a target molecule and a lipid may be a lipid nanoparticle (LNP). As used herein, "lipid nanoparticle" (LNP) refers to a nanovesicle composed of lipid molecules. Examples of lipid nanoparticles include the nucleic acid-lipid particles described in U.S. Pat. No. 8,058,069B, the entire contents of which are incorporated herein by reference. Amphiphilic lipids can form lipid nanoparticles in aqueous solution. Those skilled in the art can appropriately select lipids and form lipid nanoparticles from the lipids. Lipid nanoparticles are used to deliver nucleic acids, such as single-stranded nucleic acids and double-stranded nucleic acids, into cells, and can also be preferably used to deliver nucleic acids to extracellular vesicles.

[0080] The lipid nanoparticles may comprise one or more, or preferably all, selected from the group consisting of cationic lipids, non-cationic lipids (or neutral lipids), sterols, and PEG lipids. In some embodiments, the cationic lipids are ionizable cationic lipids. In some embodiments, the non-cationic lipids are neutral lipids. In some embodiments, the sterols include cholesterol.

[0081] In some embodiments, the cationic lipids include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)-N,N-dimethyl- 2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530). In some embodiments, the cationic lipid can include or is L608. In some embodiments, the cationic lipid can include or is L530.

[0082] In some embodiments, the cationic lipid is selected from the group consisting of 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 of US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0083] In some embodiments, the ionizable cationic lipid (also referred to as an ionizable lipid) can be 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0084] In some embodiments, neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), PO phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and sphingomyelin (SM). Neutral lipids may be neutral as a whole molecule under neutral pH conditions.

[0085] Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as zymosterol and ergosterol. The lipid nanoparticles preferably contain a sterol, and more preferably contain cholesterol.

[0086] The lipid nanoparticles may contain a lipid modified with polyalkylene glycol. Examples of polyalkylene glycols that can be used include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The weight-average molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.

[0087] Modification of lipids with polyethylene glycol can be achieved by stearylated polyethylene glycol (e.g., PEG 45 stearate (STR-PEG45)). PEG lipids include N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and N- Polyethylene glycol derivatives such as [carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) can also be used.

[0088] The lipid nanoparticles preferably have an average particle size of 400 nm or less, and more preferably an average particle size of 300 nm or less. The average particle size of lipid nanoparticles refers to the number-average particle size measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed by standard methods using a commercially available DLS device. The polydispersity index (PDI) of lipid nanoparticles can be approximately 0.05-0.5, approximately 0.05-0.4, approximately 0.05-0.3, preferably approximately 0.05-0.2, more preferably approximately 0.05-0.15, or approximately 0.1. Lipid nanoparticles can be prepared using a microfluidic device according to standard methods. Furthermore, it is easy to control the PDI of LNPs to approximately 0.1 using standard methods.

[0089] In some embodiments, the lipid nanoparticles may comprise about 20 mol% to about 60 mol% cationic lipids, about 0.5 mol% to about 15% PEG-modified lipids, about 25 mol% to about 55% sterol, and about 20 mol% to about 60 mol% non-cationic lipids. In some preferred embodiments, the lipid nanoparticles comprise about 45 mol% to about 50 mol% cationic lipids, about 9-10 mol% non-cationic lipids (preferably neutral lipids), about 35 mol% to about 45 mol% sterol (preferably cholesterol), and about 1.5 mol% to about 2 mol% PEG-lipids.

[0090] The lipid nanoparticles may have a positive charge in aqueous solution. Alternatively, the lipid nanoparticles may have a negative charge in aqueous solution. Alternatively, the lipid nanoparticles may be uncharged in aqueous solution. In a preferred embodiment, the lipid nanoparticles have a positive charge in aqueous solution.

[0091] The oily base may be, but is not limited to, a sterol derivative such as cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, or cholesteryl arachidate, or a long-chain fatty acid ester such as isopropyl myristate (isopropyl myristate; IPM), octyldodecyl myristate, cetyl myristate, ethyl oleate, ethyl linoleate, isopropyl linoleate, isopropyl palmitate, or butyl stearate. The oil phase may be a carboxylic acid ester such as ethyl lactate, cetyl lactate, triethyl citrate, diisopropyl adipate, diethyl sebacate, diisopropyl sebacate, or cetyl 2-ethylhexanoate, or a hydrocarbon such as petrolatum, paraffin squalane, or vegetable squalane, or a silicone. The oily base may be used alone or in combination of two or more.

[0092] Examples of aqueous bases include, but are not limited to, water, distilled water, Milli-Q water, and physiological saline. The aqueous base may contain pharmaceutically acceptable additives. Examples of pharmaceutically acceptable additives include salts, pH adjusters, thickeners, colorants, surfactants, chelating agents, solubilizers, preservatives, isotonicity agents, and stabilizers.

[0093] The complex of a target molecule and lipid can be mixed with extracellular vesicles. This results in a complex of a target molecule, lipid, and extracellular vesicles (second complex), and a composition containing the complex. Mixing is performed under conditions suitable for complex formation, but typically, complex formation is promoted simply by mixing. The mixing ratio of the complex to the extracellular vesicles can be determined appropriately. The mixing ratio can be, for example, 1:1000-1000:1, 1:100-100:1, 1:50-50:1, 1:30-30:1, 1:20-20:1, 1:10-10:1, 1:5-5:1, or 1:2-2:1 (approximately 1:1). In a preferred embodiment, the lipid is an ionic lipid or a mixture thereof, more preferably a mixture of a cationic lipid and an anionic lipid, and even more preferably a 1:1 mixture of a cationic lipid and an anionic lipid. Typically, in this complex, the extracellular vesicle, the target molecule, and the lipid complex fuse to form a vesicle, which encapsulates the target molecule. For example, the target molecule interacts with the hydrophilic and / or hydrophobic portions of the lipids that make up the vesicle. This makes it possible to introduce the target molecule into the extracellular vesicle. Introduction of the target molecule into the extracellular vesicle can contribute to the enhancement of the functionality of the extracellular vesicle. Mixing is performed under appropriate conditions.

[0094] The complex of a target molecule, lipid, and extracellular vesicles can be brought into contact with cells to introduce the target molecule into the cells. Therefore, it is desirable that the target molecule has an activity that regulates the physiological activity of the cells. The contact is carried out under suitable conditions.

[0095] The first complex effectively delivers a molecule of interest to the brain, lungs, kidneys, muscles (e.g., cardiac muscle), and spleen. Therefore, the first complex can be used to deliver a molecule of interest to any of these tissues. The second complex delivers a molecule of interest to the brain, kidneys, muscles (e.g., cardiac muscle), ovaries, pancreas, and spleen. Therefore, the second complex can be used to deliver a molecule of interest to any of these tissues.

[0096] Therefore, the first complex can be used to treat the disease in each organ or tissue by delivering a target molecule that provides a therapeutic benefit to the disease in the organ or tissue. The second complex can be used to treat the disease in each organ or tissue by delivering a target molecule that provides a therapeutic benefit to the disease in the organ or tissue. The second complex is particularly easy to introduce into cells and may be more beneficial in treating the disease. Therefore, the target molecule may be a molecule that functions intracellularly.

[0097] The pathogenesis of neurodegenerative diseases is thought to be the accumulation of abnormal proteins in brain tissue or neurons. For example, accumulation of amyloid beta or tau is observed in Alzheimer's disease, accumulation of α-synuclein is observed in Parkinson's disease and dementia with Lewy bodies, accumulation of polyglutamine is observed in polyglutamine diseases (e.g., Huntington's disease and spinocerebellar ataxia), accumulation of TDP-43 or SOD1 is observed in amyotrophic lateral sclerosis, and accumulation of tau or TDP-43 is observed in frontotemporal dementia. Antibodies that bind to and remove any of these proteins may be therapeutically beneficial for the above diseases. Target molecules may include antibodies or antigen-binding fragments thereof, particularly intracellular antibodies (e.g., scFv), that bind to the above proteins or abnormal proteins. Degradation of target proteins can be induced by imparting a proteolysis-promoting signal, such as a chaperone-mediated autophagy (CMA) signal, to the intracellular antibody.

[0098] The present disclosure provides a method for treating a disease in a subject having a disease that would benefit from administration, the method comprising administering to the subject an effective amount of a conjugate or composition of the present disclosure. The present disclosure provides a conjugate or composition of the present disclosure for use in the subject. The present disclosure provides use of a conjugate or composition of the present disclosure for use in the subject in the manufacture of a medicament for use in the method. Here, "effective amount" means an amount sufficient to exert physiological significance. The route of administration is not particularly limited, and examples include transdermal administration, intradermal administration, subcutaneous administration, intravenous administration, intraperitoneal administration, ophthalmic administration, intravitreal administration, intramuscular administration, intramedullary administration, intracerebrospinal fluid administration, intrathecal administration, and intracardiac administration.

[0099] According to the present disclosure, the complex can be obtained by the following method: obtaining a complex of a target molecule and a lipid; and contacting the obtained complex with extracellular vesicles to obtain complex particles of the complex and extracellular vesicles, wherein the target molecule is encapsulated in the extracellular vesicles.

[0100] The present disclosure also provides a method for introducing a target molecule into an extracellular vesicle, the method comprising: obtaining a complex of the target molecule and a lipid mixture; and contacting the obtained complex with extracellular vesicles to obtain a complex particle of the complex and the extracellular vesicles, whereby the target molecule is introduced into the complex particle.

[0101] The present disclosure also provides a method for introducing a target molecule into an extracellular vesicle, the method comprising: obtaining a complex of the target molecule and a nonionic lipid; and contacting the obtained complex with an extracellular vesicle to obtain a complex particle of the complex and the extracellular vesicle, whereby the target molecule is introduced into the complex particle.

[0102] The present disclosure also provides a method for introducing a target molecule into an extracellular vesicle, the method comprising: obtaining a complex of a target molecule and a cationic lipid; and contacting the obtained complex with an extracellular vesicle to obtain a complex particle of the complex and the extracellular vesicle, whereby the target molecule is introduced into the complex particle.

[0103] The present disclosure also provides a method for introducing a target molecule into an extracellular vesicle, the method comprising: obtaining a complex of the target molecule and a cationic lipid or an anionic lipid; and contacting the obtained complex with an extracellular vesicle to obtain a complex particle of the complex and the extracellular vesicle, whereby the target molecule is introduced into the complex particle.

[0104] The present disclosure also provides a method for introducing a target molecule into an extracellular vesicle, the method comprising: obtaining a complex of a target molecule, a cationic lipid, and an anionic lipid; and contacting the obtained complex with an extracellular vesicle to obtain a complex particle of the complex and the extracellular vesicle, thereby introducing the target molecule into the complex particle.

[0105] According to the present disclosure, there is provided a method for introducing a target molecule into a cell, the method comprising: obtaining a complex of the target molecule and a lipid mixture; contacting the obtained complex with extracellular vesicles to obtain a complex particle of the complex and the extracellular vesicles, whereby the target molecule is introduced into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

[0106] According to the present disclosure, there is provided a method for introducing a target molecule into a cell, the method comprising: obtaining a complex of the target molecule and a nonionic lipid; contacting the obtained complex with extracellular vesicles to obtain a complex particle of the complex and the extracellular vesicles, whereby the target molecule is introduced into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

[0107] According to the present disclosure, there is provided a method for introducing a target molecule into a cell, the method comprising: obtaining a complex of a target molecule and a cationic lipid; contacting the obtained complex with extracellular vesicles to obtain a complex particle of the complex and the extracellular vesicles, whereby the target molecule is introduced into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

[0108] According to the present disclosure, there is provided a method for introducing a target molecule into a cell, the method comprising: obtaining a complex of the target molecule and a cationic lipid or an anionic lipid; contacting the obtained complex with extracellular vesicles to obtain a complex particle of the complex and the extracellular vesicles, whereby the target molecule is introduced into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

[0109] According to the present disclosure, there is provided a method for introducing a target molecule into a cell, the method comprising: obtaining a complex of a target molecule, a cationic lipid, and an anionic lipid; contacting the obtained complex with extracellular vesicles to obtain a complex particle of the complex and the extracellular vesicles, thereby introducing the target molecule into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

[0110] In one embodiment of the above method, the target molecule is a nucleic acid, and (i) the complex is a complex of the target molecule and a nonionic lipid, and the complex particle is a complex particle of the target molecule, a nonionic lipid, and an extracellular vesicle; preferably, (ii) the complex is a complex of the target molecule and a cationic lipid, and the complex particle is a complex of the target molecule and a cationic lipid; more preferably, (iii) the complex is a complex of the target molecule, a cationic lipid, and a nonionic lipid, and the complex particle is a complex particle of the target molecule, a cationic lipid, a nonionic lipid, and an extracellular vesicle; even more preferably, (iv) the complex is a complex of the target molecule, a cationic lipid, and an anionic lipid, and the complex particle is a complex particle of the target molecule, a cationic lipid, an anionic lipid, and an extracellular vesicle. If the target molecule has a high isoelectric point (e.g., a pI of greater than 7 or 8 or greater), the complex and complex particle of (ii), (iii), or (iv) above can further contain an anionic lipid. Because nucleic acids are polyanions, they are preferably complexed with ionic lipids, particularly cationic lipids and mixtures of cationic and anionic lipids.

[0111] In one embodiment of the above method, the target molecule is a protein (e.g., an antibody), and (i) the complex is a complex of the target molecule and a nonionic lipid, and the complex particle is a complex particle of the target molecule, a nonionic lipid, and an extracellular vesicle; preferably, (ii) the complex is a complex of the target molecule and a cationic lipid, and the complex particle is a complex of the target molecule and a cationic lipid; more preferably, (iii) the complex is a complex of the target molecule, a cationic lipid, and a nonionic lipid, and the complex particle is a complex particle of the target molecule, a cationic lipid, a nonionic lipid, and an extracellular vesicle; even more preferably, (iv) the complex is a complex of the target molecule, a cationic lipid, and an anionic lipid, and the complex particle is a complex particle of the target molecule, a cationic lipid, an anionic lipid, and an extracellular vesicle. If the target molecule has a high isoelectric point (e.g., a pI of greater than 7 or 8 or greater), the complex and complex particle of (ii), (iii), or (iv) above can further contain an anionic lipid.

[0112] In some embodiments, the method for introducing a molecule of interest into a cell is an in vitro method. In some embodiments, the method for introducing a molecule of interest into a cell is an in vivo method. The present disclosure may provide a molecule of interest, a lipid mixture, a nonionic lipid, a cationic lipid, an anionic lipid, a combination of a cationic lipid and an anionic lipid, a first complex, an extracellular vesicle (particularly an exosome), a second complex, or a composition comprising any of these, for use in these methods.

[0113] In all embodiments, the extracellular vesicles preferably comprise or are exosomes. In some embodiments, the extracellular vesicles comprise or are microvesicles. In some embodiments, the extracellular vesicles comprise exosomes and microvesicles. The ratio of exosomes to microvesicles is not particularly limited, but may be, for example, 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 30:70, 20:80, 10:90, or 100:0, or a ratio between any two of the above.

[0114] Exosomes can be obtained by methods well known to those skilled in the art. Exosomes can be obtained from biological samples (e.g., cell culture supernatants) by, for example, ultracentrifugation, size exclusion chromatography, ultrafiltration, immunoprecipitation such as immunomagnetic separation, density gradient centrifugation, or a combination of two or more of these. When exosomes are obtained from cell culture supernatants, the culture supernatant is preferably obtained by culturing the cells in a medium that satisfies one or more or all of the following conditions: serum-free, albumin-free, cytokine-free, and xeno-free. More preferably, the culture supernatant can be obtained by culturing the cells in a chemically defined medium. Ultracentrifugation can be performed on the exosome-containing supernatant after previously sedimenting solids such as cell debris by centrifugation. Ultrafiltration can concentrate exosomes in a sample using a membrane with a specific molecular weight cutoff (MWCO). By passing a sample through a membrane, large particles such as exosomes remain on the membrane and can be recovered. In immunoprecipitation, exosomes can be isolated using antibodies against specific markers on the surface of exosomes (e.g., CD9, CD63, CD81, etc.). For example, exosomes can be easily isolated from a sample using magnetic beads bound to these antibodies. Density gradient centrifugation is a method in which a biological sample is centrifuged in a density gradient medium to separate exosomes in the sample as layers formed depending on their density. Biological samples include the culture supernatant of stem cells such as adipose-derived mesenchymal stem cells and bone marrow-derived stem cells. Biological samples include the culture supernatant of one or more mesenchymal stem cells selected from the group consisting of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and umbilical cord blood-derived mesenchymal stem cells. Biological samples also include, for example, milk, preferably raw milk, particularly raw cow's milk.

[0115] Microvesicles can also be obtained in a similar manner to the above, but it is known that different conditions can be applied to them compared to exosomes. The differences in the separation of microvesicles and exosomes are primarily based on size, density, and surface markers. However, those skilled in the art can utilize these properties to obtain exosomes and microvesicles separately or as a mixture, as appropriate. In ultracentrifugation, microvesicles can generally be precipitated by centrifugation at a centrifugal force of 10,000 to 20,000 × g for 20 minutes to 1 hour, while exosomes can be centrifuged at a centrifugal force of 100,000 × g or higher for several hours. In ultrafiltration, a 100 kDa MWCO filter can generally be used to separate exosomes, and a 1000 kDa MWCO filter can be used to separate microvesicles. Furthermore, exosomes can be obtained using antibodies against one or more or all of the markers selected from the group consisting of CD9, CD63, D81, ALIX, and TSG101, while microvesicles can be obtained using antibodies against one or more or all of the markers selected from the group consisting of CD40, selectin, and integrin. Thus, the obtained extracellular vesicles can express one or more or all of the above markers.

[0116] The present disclosure provides a composition or pharmaceutical composition containing the complex. The composition or pharmaceutical composition containing the complex may further contain a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include solvents such as water, pH adjusters, osmolality adjusters, thickeners, dispersants, or salts. The composition containing the complex may further contain a biologically acceptable additive in addition to the complex. Biologically acceptable additives include solvents such as water, culture media or culture media components, pH adjusters, osmolality adjusters, thickeners, dispersants, or salts. The composition and pharmaceutical composition containing the complex may be used in vitro and in vivo, respectively. The composition containing the complex may be a cosmetic composition or a dermatological composition. The composition and pharmaceutical composition may preferably be a liquid formulation. The components contained in a composition (particularly a pharmaceutical composition) to be administered to the body are of pharmaceutically acceptable types and / or amounts.

[0117] 1A. Fusion of lipid-protein complexes with exosomes. Exosomes were obtained by purifying Suite 2 cell supernatant using the PS affinity method (Fujifilm Wako Pure Chemical Industries, Ltd.). Cells were cultured in 5 ml of serum-free medium in a 10 cm dish for 24 hours, and the cell supernatant was then collected. MagCapture TM Exosomes were purified according to the protocol of Exosome Isolation Kit PS Ver. 2. The purified exosomes were aliquoted and stored at -30°C until use.

[0118] The lipid-protein complex was prepared as follows. An ionic lipid was used here because of its simple manufacturing process. The ionic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) was used. +) was used as the cationic lipid (WO 2021 / 192861, the entire contents of which are incorporated herein by reference), and linoleic acid was used as the anionic lipid. The cation and anion were mixed at a 1:1 molar ratio. The lipid mixture exhibited the properties of an ionic liquid (IL). An ethanolic lipid solution was obtained by diluting with ethanol to the above-mentioned concentration. Antibodies were used as proteins. 10 μL and 1 μL of a Milli-Q aqueous solution (100 ng / μL) of allophycocyanin (APC)-modified anti-hIL-2 antibody (BioLegend, 500310) or an APC-modified hCD45 antibody (BioLegend, 304012) and an ethanolic lipid solution (5 μg / μL) were added to a 1.5 mL tube, respectively, and vortex-mixed for at least 15 seconds. Thereafter, the mixture was allowed to stand at room temperature for 15 minutes or more, thereby forming complex particles of lipid and APC-modified anti-hIL-2 antibody and complex particles of lipid and APC-modified anti-hCD45 antibody (first complexes, respectively), and first complexes containing each antibody were obtained.

[0119] Subsequently, 11 μL of the obtained lipid-protein complex and 10 μL of an aqueous solution of exosomes (1.0 × 10 8 The exosomes were vortex-mixed with the lipid-protein complex (10 μL) and allowed to stand at room temperature for at least 1 hour to obtain lipid-protein-exosome hybrid particles (second complexes). The particle size distributions of the exosomes and complex particles were investigated by dynamic light scattering (Figure 1). As shown in Figure 1, the peak of the particle size distribution shifted to the right. This indicates that the particle size of the resulting particles had increased, suggesting that the exosomes and lipid-protein complexes had further fused to form particles (hereinafter referred to as "hybrid particles").

[0120] 1B. Loading Rate of Active Ingredients in Exosomes Used: Exosomes derived from cultured cells were obtained by concentrating the culture supernatant of SUIT-2 cells, human adipose-derived stem cells, and 293 cells by ultrafiltration and then purifying by size exclusion chromatography. Cells were cultured in 5 mL of serum-free medium in a 10 cm dish for 72 hours, after which the cell supernatant was collected. The resulting supernatant was concentrated 10-fold using an Amicon Ultra-4 100k (Merck Millipore), and exosomes were purified according to the qEV column (IZON) protocol. The purified exosomes were aliquoted and stored at -30°C until use.

[0121] Ginger-derived exosomes were prepared from the juice obtained by grating commercially available ginger (produced in Fukuoka Prefecture) with a hand blender and squeezing it using a nonwoven fabric, while milk-derived exosomes were prepared from whey obtained from commercially available Itomonogatari (Itoshima Milk Plant Co., Ltd.), which was concentrated 10-fold using an Amicon Ultra-4 100k (Merck Millipore) and purified according to the qEV column (IZON) protocol. Use of other purification kits is described separately.

[0122] Measurement of drug loading rate into exosomes using flow cytometry Reagents: N-terminal FITC-labeled peptide (SEQ ID NO: 1: KKKSVYDFFVWL) was custom synthesized by Biologica. siRNA was prepared using BLOCK-iT TM Fluorescent Oligo for electroporation was used. Cas9 Nuclease GFP NLS Protein and TrueGuide TM sgRNA Positive Control and HPRT1 (human) were purchased from Cosmobio and Thermo Fisher Scientific, respectively, and mixed to form RNP (Cas9-gRNA complex). 1,2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) ＋ The cationic lipid was lysine 10, and the anionic lipid was linoleic acid. The cation and anion were mixed in a 1:1 molar ratio in ethanol.

[0123] Experiment: A peptide aqueous solution (containing 200 ng of FITC-labeled peptide) and a lipid ethanol (ILB) solution (containing 0 or 2 μg of lipid) were vortex-mixed and allowed to stand for 15 minutes to form a first complex. Furthermore, SUIT-2 cell-derived exosomes (containing 20 ng of protein) were added, vortex-mixed, and allowed to stand for 1 hour to form a second complex (50 μL). The resulting second complex was measured using a flow cytometer, and the ratio of peptide-loaded exosome particles to total exosomes was calculated.

[0124] As shown in Figure 1B, when the peptide and exosomes were directly mixed, only about 11.5% of the exosomes were loaded with the peptide (see center panel).In contrast, when the peptide and ILB were first mixed to form the first complex, and then the first complex was mixed with the exosomes, 93.3% of the exosomes were loaded with the peptide (see right panel of Figure 1B).

[0125] (2) Nucleic Acid Loading Rate Experiment: An siRNA aqueous solution (containing 200 ng of FITC-labeled siRNA) and a lipid ethanol solution (containing 0 or 2 μg of lipid) were vortex-mixed and allowed to stand for 15 minutes to form a first complex. Furthermore, SUIT-2 cell-derived exosomes (containing 20 ng of protein) were added, vortex-mixed, and allowed to stand for 1 hour to form a second complex (50 μL). The resulting second complex was measured using a flow cytometer, and the percentage of exosome particles loaded with siRNA was calculated.

[0126] As shown in Figure 1C, only when siRNA and ILB were mixed to form a first complex and then the first complex was mixed with exosomes, did nucleic acids load into almost all exosomes, with a loading rate of 98.6%.

[0127] (3) RNA-protein complex (RNP) loading rate. An RNP aqueous solution (containing 200 ng of FITC-labeled RNP) and a lipid ethanol solution (containing 0 or 2 μg of lipid) were vortex-mixed and allowed to stand for 15 minutes to form a first complex. A Cas9-gRNA complex was used as the RNP. GFP was linked to Cas9. Furthermore, exosomes derived from SUIT-2 cells (containing 20 ng of protein) were added, vortex-mixed, and allowed to stand for 1 hour to form a second complex (50 μL). The resulting second complex was measured using a flow cytometer, and the percentage of exosome particles loaded with RNP was calculated.

[0128] As shown in Figure 1D, only when RNP and ILB were mixed to form the first complex and then mixed with exosomes, RNP was loaded onto the majority of exosomes, with a loading rate of 75.2%.

[0129] In this way, regardless of whether it is a protein, peptide, nucleic acid, or RNP, by forming a complex with lipids and then forming a complex with exosomes, it was possible to introduce components into exosomes very efficiently.

[0130] 1C. Verification of complete encapsulation of various components into exosomes (1) Antibody Reagents Anti-mouse IL-17A-InVivo was purchased from Selleck. Triton X-100 was purchased from Thermo Fisher Scientific. Proteinase K solution (for molecular biology) was purchased from Kanto Chemical. Trans-Blot Turbo Transfer Pack PVDF (mini) was purchased from Bio-Rad. Anti-Mouse IgG, HRP-Linked Whole Ab Sheep and ECL TM Prime Western Blotting Detection Reagent was purchased from Cytiva, and Blocking One solution was purchased from Nacalai Tesque.

[0131] Experiment: An antibody aqueous solution (containing 200 ng of antibody) and a lipid ethanol solution (containing 2 μg of lipid) were vortex mixed and allowed to stand for 15 minutes to form a first complex. Various exosomes (containing 20 ng of protein) were then added, vortex mixed, and allowed to stand for 1 hour to form a second complex (50 μL).

[0132] 6 μL of Proteinase K (0 or 0.64 μg / mL) and 6 μL of Triton X-100 (0 or 1%) were added and incubated at 37 ° C for 60 minutes. 21 μL of 4x sample buffer was added to the resulting enzyme reaction solution, and the mixture was incubated at 95 ° C for 5 minutes, followed by SDS-PAGE. NuPAGE 4-12% Bis-Tris Gel was used for the gel, and the gel was electrophoresed at 100 V for 10 minutes, followed by electrophoresis at 200 V for 40 minutes. In this experimental system, the contents of exosomes are not degraded by Proteinase K in the absence of Triton X-100, but are thought to be degraded by Proteinase K in the presence of Triton X-100.

[0133] The membrane was transferred using a Transblot Turbo transfer system (Bio-Rad) according to the device's protocol, and the antibody was then transferred to a PVDF membrane and detected using HRP-labeled anti-mouse IgG. Blocking was performed using Blocking One solution, and washing was performed using PBS-T. The color reaction was performed using ECL. TM Prime Western Blotting Detection Reagent was used, and signal detection was performed using ChemiDoc XRS+ (Bio-Rad).

[0134] The results of Western blotting are shown in Figure 1E. As shown in Figure 1E, it was found that there was a protein near the 150 kDa marker that was not digested in the absence of Triton X-100. This is because the antibody was protected from enzymatic degradation by being encapsulated in exosomes. The results in Figure 1E demonstrate that antibodies can be encapsulated in exosomes of various origins.

[0135] (2) Nucleic acid reagent AccuTargetTM miRNA Housekeeping Positive Control (GAPDH, Human / Mouse) was purchased from Cosmo Bio. Exosomes derived from the culture supernatant of SUIT-2 cells were obtained from MassiveEV. TM EV Purification Column PS / MassiveEV TM Purification was performed using Purification Buffer Set (Fujifilm Wako Pure Chemical Industries, Ltd.). RNase A, DNase, and protease-free (10 mg / mL) were purchased from Thermo Fisher Scientific, and GelRed® Nucleic Acid Gel Stain, 10,000× in Water, was purchased from Cosmobio.

[0136] Experiment: A miRNA aqueous solution (containing 20 ng of miRNA) and a lipid ethanol solution (containing 0.02 μg of lipid) were vortex mixed and allowed to stand for 15 minutes to form a first complex. Various exosomes (containing 0.02 or 0.2 μg of protein) were then added, vortex mixed, and allowed to stand for 1 hour to form a second complex (50 μL).

[0137] RNase A (0, 0.1, 1, or 10 ng / μL) dissolved in nuclease-free water (6 μL) was added and incubated at 37°C for 30 minutes. 5x Loading Buffer was added to the resulting enzyme reaction solution, which was then applied to a 15% polyacrylamide gel and electrophoresed at 20 mA for 40 minutes. The gel was stained with GelRed, and nucleic acid bands were detected using a ChemiDoc XRS+.

[0138] The results shown in Figure 1F indicate that nucleic acids encapsulated in exosomes were digested in a concentration-dependent manner with the digestive enzyme, and that the amount of degradation decreased with increasing amounts of EV. This indicates that nucleic acids were protected from degradation by exosomes, i.e., nucleic acids were encapsulated in exosomes. Furthermore, it was shown that nucleic acids were encapsulated in both SUIT-2 cell-derived exosomes and MSC-derived exosomes.

[0139] Next, exosomes in the hybrid particles were stained with FITC-conjugated CD9 antibody, and colocalization of exosomes and anti-IL-2 antibody was observed by fluorescence microscopy (Figure 2). As shown in Figure 2, the lipid-anti-IL-2 antibody particles and exosomes colocalized. This suggests that contact of the lipid-anti-IL-2 antibody conjugate particles with exosomes resulted in fusion to form hybrid particles, and that the anti-IL-2 antibody was introduced into the exosomes.

[0140] 2A. Introduction of Proteins into Cells Using Hybrid Particles Serum-free medium was added to the dispersion of the first and second complexes prepared in 1 above, mixed gently, and then added dropwise to the cells. After 48 hours of culture, the cells were fixed, stained with CD9 antibody, and observed under a fluorescent microscope. It was demonstrated that exosomes formed hybrid particles with lipid-protein complexes were taken up by cells (Figure 3A). This demonstrated that proteins and protein complexes can be complexed with lipids and then fused with exosomes, thereby passing through the cell membrane and being introduced into cells.

[0141] 2B-1. Introduction of proteins into cells using hybrid particles 10 μL and 1 μL of a MilliQ solution (20 mg / mL) of APC-labeled PD-L1 antibody (manufactured by BioLegend) and an ethanol solution (6 mg / mL) containing the above liquid lipid composition (IL) were added to a 1.5 mL tube, respectively, and vortex mixed for 15 seconds or more. EDMPC was used as the cationic lipid, and linoleic acid was used as the anionic lipid. Specifically, the lipids were: (i) EDMPC + and linoleic acid, (ii) an ionic liquid lipid composition (IL) containing EDMPC. + Chloride salt (EDMPC + Cl - ) (cationic lipid), and (iii) EDMPC + A mixture of EDMPC and linoleic acid (lipid mixture) was used. (i) EDMPC in chloroform + Cl -and linoleic acid, and then freeze-dried to remove chloroform and HCl. (iii) is EDMPC + Cl - and linoleic acid. The mixture was then left to stand at room temperature for 15 minutes or more, allowing the formation of lipid and PD-L1 antibody complex particles. Next, 11 μL of the lipid-antibody complex was added to 10 μL of an aqueous exosome solution (1.0 × 10 8 10 μL) was added, vortex mixed, and allowed to stand at room temperature for at least 1 hour. The hybrid particles were added to Suit2 pancreatic cancer cells and cultured for 48 hours. APC fluorescence was observed under a microscope. Furthermore, the percentage of cells into which the antibody was introduced was measured using a flow cytometer. The results are shown in Figure 3B. As shown in Figure 3B, the second complex, which was prepared by associating the first complex, a type of lipid and protein complex, with exosomes, was also able to effectively introduce antibody molecules into cells. Furthermore, as shown in Figure 4A, regardless of which lipid was used as the lipid, the first complex and the second complex were successfully introduced into cells. EDMPC + Cl - The fact that the first complex (cationic lipid-antibody complex) using EDMPC was also able to effectively transfer the antibody into cells suggests that the first complex does not need to contain components other than the cationic lipid, such as anionic lipids or neutral lipids (especially lipids other than the cationic lipid). + Cl - The first complex (lipid mixture-antibody complex) using linoleic acid was also able to effectively import the antibody into the cells.

[0142] 2B-2. Formation of a First Complex Containing an Antibody and Lipid and Its Delivery into Cells. APC-labeled anti-human CD274 (BD-H1, PD-L1) Clone: ​​29E.2A3 was purchased from BioLegend. The antibody was diluted to 10 ng / μL with water. EDMPC Cl salt was dissolved in ethanol to a concentration of 2.5 mM to obtain lipid solution (i). EDMPC Cl salt and linoleic acid were mixed in ethanol to a concentration of 2.5 mM each to obtain lipid solution (ii). 50 μL of the antibody solution and 5 μL of lipid solution (i) or (ii) were added to a 1.5 mL tube, vortexed for 60 seconds, and then allowed to stand for at least 15 minutes to obtain a dispersion containing the first complex (i) or (ii), respectively.

[0143] To 55 μL of the dispersion containing the obtained first complex (i) or (ii), 5 μL of milk exosome solution (protein concentration: 0.1 mg / mL) was added, and the mixture was stirred with a vortex for 60 seconds and then allowed to stand for at least 1 hour to obtain the second complex (i) or (ii).

[0144] Cell experiment: Suit2 cell suspension (3 × 10 4 A 200μL / well suspension of the first complex (i) or (ii) or the second complex (i) or (ii) was added to each well of a multi-well plate. After incubation at 37°C overnight, the cells were confirmed to cover approximately 80-90% of the bottom surface the next day. The medium in the dish was aspirated, and 200μL of FBS-free medium was added. 55μL / well of a dispersion containing the first complex (i) or (ii) or the second complex (i) or (ii) was added. After incubation at 37°C for 48 hours, the nuclei were stained with Hoechst 33342 and then observed under a fluorescence microscope.

[0145] Results: When cells were simply exposed to IgG (PDL1 Ab aqueous solution), almost no APC-derived fluorescence was detected within the cells. However, APC-derived fluorescence was detected within the cells administered with either the first or second complex (Figure 4B). This suggests that antibody delivery into cells is enhanced by complexing the antibody with anionic lipids or anionic and cationic lipids. Furthermore, fusion of the complex with exosomes further enhanced antibody delivery into cells. This result was also observed when the mixture ratio of anionic lipid to cationic lipid was changed.

[0146] 2C. Introduction of proteins into cells using hybrid particles 10 μL and 1 μL of a MilliQ solution (1 mg / mL) of a peptide labeled with FITC at the N-terminus (SEQ ID NO: 1: KKKSVYDFFVWL) and an ethanol solution (10 mg / mL) containing the liquid lipid composition (IL) were added to a 1.5 mL tube, respectively, and vortex mixed for 15 seconds or more. The mixture was then left to stand at room temperature for 15 minutes or more, thereby forming lipid and peptide complex particles (first complex). Next, 10 μL of an aqueous exosome solution (1.0 × 10 8 The hybrid particles (10 μL each) were added to a culture containing Suit2 pancreatic cancer cells and allowed to stand at room temperature for at least 1 hour to obtain hybrid particles (second complexes). The hybrid particles were added to a culture containing Suit2 pancreatic cancer cells and cultured for 48 hours. FITC fluorescence was observed under a phase-contrast fluorescence microscope, and phase-contrast images of the same field of view were obtained. The results are shown in Figure 4C. As shown in Figure 4C, both the first complex and the second complex were effectively delivered into cells. Thus, the first complex and the second complex were capable of delivering target molecules into cells.

[0147] 2D. Introduction of siRNA into cells using hybrid particles. Complex preparation: 10 μL of HiLyte488-siRNA aqueous solution (0.2 nmol / mL) and 1 μL of lipid ethanol solution (6 mg / mL) were added to a 1.5 mL tube and vortex-mixed for at least 15 seconds. The siRNA used was HiLyte-488 labeled positive control siRNA (human β-actin) purchased from Nippon Gene. The lipid used was EDMPC. + and linoleic acid liquid lipid composition (IL), EDMPC + Chloride salts, and EDMPC + A mixture of lipid and linoleic acid was used. The mixture was then left to stand at room temperature for 15 minutes or more, thereby forming lipid and siRNA complex particles. Next, 11 μL of the lipid-siRNA complex was added to 10 μL of an aqueous exosome solution (1.0 × 10 8 The hybrid particles were added to Suite 2 pancreatic cancer cells and cultured for 48 hours. The fluorescence of HiLyte 488 was observed under a microscope. The percentage of cells into which the antibody was introduced was also measured using a flow cytometer. The results are shown in Figure 5. As shown in Figure 5, regardless of which lipid was used, the first complex and the second complex were successfully introduced into the cells. EDMPC + Cl - The fact that the first complex using the cationic lipids was also able to effectively transfer the antibody into cells suggests that the first complex does not need to contain components other than the cationic lipids, such as anionic lipids or neutral lipids (especially lipids other than cationic lipids). In this experiment, the first complex and the second complex also had the ability to deliver the target molecule into cells.

[0148] 3A. Examination of the Intracellular MMP-7 (Enzyme) Inhibitory Effect of Antibody First, an anti-matrix metalloproteinase-7 antibody (anti-MMP-7 antibody) was delivered into cells using the first complex, and the intracellular MMP-7 (enzyme) inhibitory effect was examined.

[0149] Materials: Anti-MMP-7 antibody was purchased from Proteintech. As a positive control, Ab-DeliverIN, an antibody transfection reagent, was purchased from OZ Biosciences. Suit2 cell extracellular vesicles (sEVs) were obtained by purifying cell culture supernatant using a PS affinity kit. The protein content of the resulting sEV dispersion was quantified using the BCA method, and the particle count was measured using a NanoSight.

[0150] Complex Preparation: The antibody was diluted to 10 ng / μL with water. EDMPC Cl salt and linoleic acid were mixed in ethanol to a concentration of 2.5 mM each to obtain a lipid solution. 50 μL of the antibody solution and 5 μL of the lipid solution were added to a 1.5 mL tube, vortexed for 60 seconds, and then allowed to stand for at least 15 minutes to obtain a dispersion containing the first complex. sEV was diluted to 0.1 μg / mL, and 5 μL of the sEV dispersion was added to 55 μL of the dispersion containing the first complex. After vortexing for 60 seconds, the mixture was allowed to stand for at least 1 hour to obtain a second complex.

[0151] Cell experiment: Matrigel (20 μg / mL) was added to a 96-well plate for cell invasion assay, and the pores (φ8 μm) of the polycarbonate membrane in the chamber were filled with Matrigel. A suspension of 1 × 10 Suit2 cells was added. 5 The cell concentration was adjusted to 2.5 × 10 cells / mL. 4The cells were then dispensed at 1 / well. The dish was incubated overnight at 37°C, and the next day, it was confirmed that cells covered more than 90% of the bottom surface. The entire medium in the dish was aspirated, and 750 μL of FBS-free medium was added to the lower chamber and 195 μL to the upper chamber. Subsequently, a lipid complex dispersion containing 500 ng of antibody was administered to the cells in the upper chamber (antibody dose: 500 ng / well). Ab-DeliverIN-antibody complexes were administered as a positive control, and an antibody solution was administered as a negative control (antibody dose: 500 ng / well). Cells receiving 50 μL of medium without antibody were treated as non-treated cells. After 48 hours of incubation at 37°C, the cell proliferation rate was calculated by counting the number of cells in the upper chamber. Cells remaining on the top of the polycarbonate membrane were then removed with a cotton swab, and cells that invaded the lower membrane were stained with H&E. The cells that invaded the lower polycarbonate membrane were observed under a microscope. The number of cells present per microscope field was counted, and the number of cells was calculated assuming the number of invaded cells in the non-treated state as 100.

[0152] Results: No significant difference in cell proliferation rate was observed among cells treated with any of the formulations, indicating the absence of cytotoxicity. Meanwhile, cells treated with only the anti-MMP-7 antibody showed no inhibitory effect on cell invasion, whereas cells treated with the first and second conjugates exhibited inhibition of cell invasion (see Figures 6A and 6B). These results suggest that lipid conjugation promotes the uptake of the anti-MMP-7 antibody into the cytoplasm of Suit2 cells, thereby inhibiting cell invasion. Furthermore, lipid conjugation also demonstrated that the antibody retains its antigen-binding ability.

[0153] In addition, a preparation containing a solid complex of an antigen and a lipid and an oily base, in which the solid complex is dispersed in the oily base, enhances the skin permeability of the antigen (see Patent No. 5752343). The antibody was mixed with lipid in cyclohexane to emulsify, and then lyophilized to form a solid complex containing an anti-VEGF antibody and a lipid, which could be dispersed in an oily base (isopropyl myristate). A preparation in which the solid complex containing the antibody and lipid was dispersed in the oily base was obtained (hydrodynamic diameter was about 100 nm). By using aflibercept instead of the anti-VEGF antibody, a preparation in which the solid complex containing aflibercept and a lipid was dispersed in the oily base was obtained. Furthermore, this preparation was able to sustainedly release functional antibodies or proteins by intravitreal administration or ophthalmic administration. Furthermore, this preparation exhibited skin permeability, and the penetrated antibody possessed binding ability to its antigen. Thus, the antigen-lipid complex could be formed into a solid complex and dispersed in an oily base.

[0154] 3B. Examination of the Intracellular MMP-7 (Enzyme) Inhibitory Effect of Antibody Next, the anti-MMP-7 antibody was delivered into cells using the second complex, and the intracellular MMP-7 (enzyme) inhibitory effect was examined. Specifically, 5 mg of anti-MMP-7 antibody (mouse, monoclonal) was dissolved in 200 μL of MilliQ. 30 mg of lipid was dissolved in 20 μL of ethanol. The lipid was EDMPC. + and linoleic acid in a 1:1 ratio (IL), EDMPC + Chloride salt (cationic lipid), and EDMPC + A mixture of 20 μL of antibody aqueous solution and 2 μL of lipid ethanol solution was mixed, vortexed for 60 seconds, and then allowed to stand for 15 minutes to prepare a lipid-protein complex sample (first complex) (22 μL). 22 μL of lipid-antibody complex sample was mixed with 10 μL of exosome dispersion (1 × 10 exosomes). 9 The mixture was added with 200 ng of lipid-antibody-exosome hybrid particles (2nd complex) and vortex mixed, and allowed to stand for 1 hour (32 μL).

[0155] Matrigel PBS dispersion was added at 20 μg / well to the inner cup of a 24-well plate for cell migration assay. After 2 hours, the PBS was removed from the wells, and 250 μL / well of serum-free medium was added to swell the gel. After 30 minutes, 2.5 × 10 Suit2 pancreatic cancer cells were plated on the Matrigel. 4 The cells were seeded per well. After 24 hours, the medium was replaced with serum-free MEM medium, and the entire amount of the sample prepared above was added. After 72 hours of culture, the number of cells remaining on the Matrigel was counted using phase contrast images, and the cell proliferation rate was calculated. In addition, cells that had migrated to the wells below the Matrigel were fixed with ethanol and counted after H&E staining.

[0156] The results are shown in Figures 6C and 6D. As shown in Figure 6D, intracellular introduction of MMP-7 had little effect on cell proliferation. Furthermore, as shown in Figure 6C, the first complex inhibited cell migration, while the second complex significantly inhibited cell migration. These results demonstrate that lipid-antibody-exosome hybrid particles can inhibit cell migration by inhibiting cellular functions (particularly the intracellular function of MMP-7) without inhibiting cell proliferation. The second complex was more effective at inhibiting cell migration than the first complex, suggesting that lipid-antibody-exosome hybrid particles have a higher efficiency of introducing functional antibodies into cells. Comparing the activities of the first and second complexes, both effectively inhibited cell migration, but the second complex exhibited a stronger inhibitory effect on cell migration. Extracellular vesicles (especially exosomes) are thought to contribute to intercellular communication and can efficiently transfer internal substances into recipient cells in a functional state, which is thought to be the cause of the increased inhibitory effect. + Cl - The fact that the first complex using the antibody was also able to effectively transfer the antibody into cells suggests that the first complex does not need to contain components other than the cationic lipid, such as anionic lipids or neutral lipids (especially lipids other than cationic lipids). Furthermore, it was demonstrated that the first complex and the second complex have the ability to deliver large protein complexes (approximately 150 kDa) such as antibodies into cells.

[0157] 4. Cellular delivery using nucleic acid-loaded hybrid particles Lipid-exosome hybrid particles loaded with Alexa647-labeled antisense oligonucleotides (ASO) were prepared using the following method. 10 μL and 1 μL of an ASO Milli-Q aqueous solution (100 ng / μL) and an ethanol solution of the liquid lipid composition (5 μg / μL) were added to a 1.5 mL tube, respectively, and vortex-mixed for at least 15 seconds. The mixture was then left to stand at room temperature for at least 15 minutes, allowing the formation of ASO and lipid complex particles.

[0158] The resulting ASO-lipid complex particles were then mixed with an exosome solution to fuse the ASO-lipid complex particles with the exosomes. The mixture was then left at room temperature for at least one hour to yield lipid-ASO-exosome hybrid particles. Serum-free medium was added to the resulting hybrid particle dispersion, followed by gentle mixing and subsequent dropwise addition to cells. After 48 hours of culture, the cells were fixed and the exosomes were stained with Alexa488-conjugated CD-9 antibody and observed under a fluorescent microscope (Figure 7). As shown in Figure 7, the hybrid particle-treated group demonstrated significant intracellular migration of ASO. This suggests that lipid-exosome hybrid particles efficiently promote intracellular ASO delivery.

[0159] 5. Hybrid particle tissue delivery reagents: 14:0 EPC Cl salt (Avanti Polar Lipids) and linoleic acid (Sigma-Aldrich) were purchased from Merck. EPC stands for 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine. Normal human IgG (143-09501) was purchased from Fujifilm Wako Pure Chemical Industries. Exosomes were purified from milk-derived exosomes purchased from Cosmobio using a PS affinity kit (Fujifilm Wako Pure Chemical Industries). Normal human IgG was obtained by purifying total IgG contained in human serum using protein A chromatography. 14:0 EPC (Cl salt) and linoleic acid were dissolved in chloroform at equimolar ratios to obtain a liquid composition. The liquid was then stirred and lyophilized to obtain the lipid base.

[0160] Particle Preparation: The lipid-based antibody complex (first complex) was prepared as follows: 400 μL and 40 μL of an IgG aqueous solution (0.5 mg / mL) and an ethanolic lipid-based solution (50 mg / mL) were added to a 1.5 mL tube, respectively, and vortex-mixed for at least 60 seconds. The mixture was then left to stand at room temperature for at least 15 minutes to form particles (first complex). The resulting first complex (440 μL) was then mixed with the purified exosome solution (protein concentration 333 μg / mL, 60 μL) and left to stand at room temperature for at least 1 hour to obtain the second complex (500 μL).

[0161] The particle size distributions of commercially available extracellular vesicles, the first complex, and the second complex were investigated by dynamic light scattering using a Malvern Zetasizer Pro. The results are shown in Figure 8 and Table 1. As shown in Figure 8, it was clear that exosomes bound to the first complex in the second complex, increasing the particle size.

[0162]

[0163] Tissue Observation: The first and second complexes were prepared using the same method as above using the Cy5-labeled normal human IgG and administered intravenously to mice. 72 hours later, brains were harvested and cryosectioned. To confirm the localization of IgG (Cy5), the relative position of the red particles (IgG) to the nucleus was examined. As shown in Figure 10A, the first complex tended to reside far from the nucleus, whereas as shown in Figure 10B, the second complex tended to reside close to the nucleus. This suggests that the first complex delivered IgG to brain tissue but with low intracellular delivery efficiency, whereas the second complex delivered IgG more efficiently into brain cells. This suggests that further complexation of exosomes with the first complex improved the intracellular delivery efficiency of the complex in brain tissue. Furthermore, it was revealed that although both the first and second complexes are suitable for delivering substances into brain tissue and brain cells, the lipid-antibody complex (first complex) is relatively superior in delivering antibodies to brain tissue, and the lipid-antibody-exosome complex (second complex) is relatively superior in delivering antibodies into brain cells.

[0164] 6. Localization of antibodies administered intravenously into the mouse brain Reagent: NeuroTrace TM 500 / 525 Green Fluorescent Nissl Stain - Solution in DMSO was purchased from Thermo Fisher Scientific. Iba1 / AIF-1 (E4O4W) XP™ Rabbit mAb (Alexa Fluor™ 488 Conjugate) was purchased from Cell Signaling. ProLong TM Glass Antifade Mount with NucBlue TM Stain was purchased from Thermo Fisher Scientific.

[0165] Experiment: 100 μL of Cy5-labeled human IgG aqueous solution (containing 100 μg of antibody) and 10 μL of the above lipid ethanol solution (containing 1 mg of lipid) were vortex mixed and allowed to stand for 15 minutes to prepare a first complex. 100 μL of commercially available milk-derived exosomes (containing 10 μg of protein) were then added, vortex mixed, and allowed to stand for 1 hour to prepare a second complex (210 μL).

[0166] 200 μL of the solution was administered intravenously to the tail vein of mice. 48 hours later, the brain tissue was perfused with PBS followed by 4% paraformaldehyde (PFA). After harvesting, the brain tissue was immersed in 4% PFA and fixed at room temperature for 3 hours. After immersing in PBS for 1 hour at room temperature, the tissue was immersed in 30% sucrose solution and left overnight at 4°C.

[0167] The submerged tissue was removed and embedded in OCT compound, and 10 μm frozen sections were prepared using a cryostat. After drying, the sections were washed with PBS and blocked for 1 hour at room temperature using Blocking One (blocking solution) containing 0.1% Triton X-100. Nissl staining was performed using Nissl staining solution diluted 1,000-fold with blocking solution and incubated overnight at 4°C. Staining with Iba-1 antibody was performed using antibody diluted 500-fold with blocking solution and incubated overnight at 4°C. After washing with PBS, the sections were mounted using mounting medium containing a nuclear staining dye and observed under a fluorescence microscope.

[0168] The results are shown in Figures 11A to 11C. As shown in Figure 11A, IgG was delivered throughout the brain. It was confirmed that IgG was taken up deep in the cerebral cortex, in the central hippocampus, and near Purkinje cells in the cerebellum (Figure 11A). Close observation of the cerebral cortex, hippocampus, and cerebellum revealed fluorescence derived from IgG-Cy5 within the cells of each tissue, as shown in Figure 11B. The fluorescent areas are indicated by arrows in Figure 11B. Furthermore, as shown in Figure 11C, it was confirmed that IgG was also taken up by microglial cells in the cerebral cortex (see arrow).

[0169] 7. Antibody Loading into Lipid Nanoparticles Preparation of Lipid Nanoparticles The LipidLaunch LNP-0315 Exploration Kit was purchased from Cayman Chemical Company, and particles were prepared without RNA according to the kit's protocol. The lipid concentration and lipid mixing rate during particle preparation were determined according to the kit's protocol. Particles were prepared using a microfluidic device, and then dialyzed for 18 hours using a 3.5k MWCO dialysis membrane. The solvent in which the lipid nanoparticles were dispersed was replaced with 1x PBS.

[0170] Antibody Loading. Normal human IgG, whole molecule, purified product was purchased from Fujifilm Wako Pure Chemical Corporation. A 0.1 mg / mL aqueous solution of antibody (100 μL) and a 1 mg / mL ethanol solution of ionic lipid (10 μL) were vortex-mixed and allowed to stand for 15 minutes to prepare the first particles. Next, the lipid nanoparticles prepared above were added to the 10 mg / mL PBS solution (10 μL), vortex-mixed, and allowed to stand for 1 hour to prepare the second particles. The resulting solution was analyzed by size exclusion chromatography, and the amount of unencapsulated IgG antibody was quantified. The amount of antibody loaded into the lipid nanoparticles was calculated using the following formula: antibody loading amount (mg) = antibody added amount (mg) - free antibody amount (mg).

[0171]

[0172] 8. Antibody Loading into Liposome Particles Preparation of Liposome Particles The mixed lipid Presome ACD-1 was purchased from Nippon Fine Chemical Co., Ltd. Presome ACD-1 was uniformly dispersed in 1x PBS at 60°C, and then passed through a 0.1 μm pore size polycarbonate membrane filter 22 times using an extruder to prepare liposome particles.

[0173] Antibody Loading. Normal human IgG, whole molecule, purified product was purchased from Fujifilm Wako Pure Chemical Corporation. A 0.1 mg / mL aqueous antibody solution (100 μL) and a 1 mg / mL ionic lipid ethanol solution (10 μL) were vortex-mixed and allowed to stand for 15 minutes to prepare the first particles. Next, a 10 mg / mL PBS solution (10 μL) of the liposome particles prepared above was added, vortex-mixed, and allowed to stand for 1 hour to prepare the second particles. The resulting solution was analyzed by size exclusion chromatography to quantify the amount of unencapsulated IgG antibody. The amount of antibody loaded into the liposome particles was calculated using the following formula: antibody loading amount (mg) = antibody added amount (mg) - free antibody amount (mg).

[0174]

Claims

1. A composition comprising a complex of a target molecule, a lipid mixture, and lipid vesicles.

2. The composition of claim 1, wherein the lipid mixture and lipid vesicles form a vesicle, and the target molecule is encapsulated within the vesicle.

3. The composition according to claim 1 or 2, wherein the lipid mixture comprises a cationic lipid.

4. The composition of claim 3, comprising a cationic lipid and an anionic lipid.

5. Anionic lipids are R-COO - wherein R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, at least one ethylene group constituting the alkenyl group may be substituted with a vinylene group, and preferably R is a cis-unsaturated fatty acid.

6. Cationic lipids are R-O-P(OR 1 ) (OR 2 ) (=O), where R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, at least one ethylene group constituting the alkenyl group may be substituted with a vinylene group, and preferably R is a cis-unsaturated fatty acid, and R 1 represents an alkyl group substituted with a cationic group; R 2 The composition according to any one of claims 3 to 5, wherein represents a hydrogen atom or a substituted or unsubstituted alkyl group.

7. A composition according to any one of claims 1 to 6, wherein the target molecule is a polypeptide.

8. The composition according to any one of claims 1 to 6, wherein the target molecule is an antibody or an antigen-binding fragment thereof.

9. The composition according to any one of claims 1 to 6, wherein the target molecule is a nucleic acid.

10. A composition according to any one of claims 1 to 9 for delivering a target molecule to a cell.

11. The composition according to any one of claims 1 to 10, wherein the complex is obtained by a method comprising the steps of: obtaining a complex (first complex) between a target molecule and a lipid mixture; and contacting the obtained complex (first complex) with lipid vesicles to obtain a complex particle (second complex) between the complex and lipid vesicles, wherein the target molecule is encapsulated in the lipid vesicles.

12. A method for introducing a target molecule into a lipid vesicle, comprising: obtaining a complex (first complex) between the target molecule and a lipid mixture; and contacting the obtained complex with a lipid vesicle to obtain a complex particle (second complex) between the complex and the lipid vesicle, thereby introducing the target molecule into the complex particle.

13. A method for introducing a target molecule into a cell, comprising: obtaining a complex (first complex) of the target molecule and a lipid mixture; contacting the obtained complex with lipid vesicles to obtain a complex particle (second complex) of the complex and lipid vesicles, thereby introducing the target molecule into the complex particle; and contacting the obtained complex particle with a cell to introduce the target molecule into the cell.

14. Mix the target molecule (preferably a water-soluble molecule) with 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) + 1. A dispersion formulation comprising particles comprising a solid complex of a lipid mixture (containing one or more lipid surfactants) containing a lipid surfactant or a pharma- ceutically acceptable salt thereof, and an oily base, wherein the particles are dispersed in the oily base.

15. The lipid mixture is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC + 15. The dispersion formulation of claim 14, consisting of:

16. A pharma- ceutically acceptable salt thereof, + Chloride salt of EDMPC + C - 16. The dispersion formulation according to claim 14 or 15, 17. The dispersion formulation according to any one of claims 14 to 16, wherein the lipid mixture further comprises an anionic lipid.

18. The dispersion formulation of claim 17, wherein the anionic lipid comprises one or more selected from the group consisting of oleic acid (OA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), cardiolipin (CL), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (DOPS), and phosphatidylinositol (PI).

19. A composition according to any one of claims 1 to 9 for use in delivering a molecule of interest to the brain.

20. A composition according to any one of claims 1 to 9 for use in delivering a target molecule into brain cells.

21. An aqueous dispersion solution comprising (i) a lipid-antibody complex and (ii) an aqueous or oily base, wherein the complex is dispersed in the base.

22. The aqueous dispersion of claim 21, wherein the lipid comprises or consists of a cationic lipid.

23. The lipid is 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC + 23. An aqueous dispersion according to claim 21 or 22, comprising or consisting of: or a pharma- ceutically acceptable salt thereof (preferably the chloride salt).

24. The aqueous dispersion solution of any one of claims 21 to 23, wherein the lipid further comprises an anionic lipid.

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