Composition for weakening skin barrier function containing anionic long-chain lipid, and dispersion liquid in which complex containing cationic long-chain lipid, anionic long-chain lipid, and drug is dispersed

Abstract

The present invention provides a composition for weakening the skin barrier containing anionic long-chain lipids, in particular long-chain fatty acids. Provided is a dispersion in which a complex (e.g., a solid complex) containing a cationic long-chain lipid, an anionic long-chain lipid, and a drug is dispersed.

Description

Technical Field

[0001] The present invention relates to a composition for weakening the skin barrier containing anionic long-chain lipids (especially long-chain fatty acids). The present invention relates to a dispersion containing a solid complex containing cationic long-chain lipids, anionic long-chain lipids and a drug. Background Art

[0002] The skin has extremely low permeability to hydrophilic molecules exceeding 800Da due to the barrier function of the stratum corneum. The barrier function of the stratum corneum limits the development of preparations for transdermal administration (transdermal absorbents). In contrast, attempts are being made to develop dispersed preparations in which a solid complex formed by coating a hydrophilic drug exceeding 800Da with a surfactant is dispersed in an oil phase (non-patent documents 1 to 4). In this way, various molecules such as low molecular weight and proteins can be administered to an individual transdermally, and even hydrophilic molecules exceeding 800Da can be transferred to the blood through transdermal administration (non-patent documents 1 to 4).

[0003] A technique for forming a solid composite using an ionic liquid is disclosed (Patent Document 5). In Non-Patent Document 5, a new cationic lipid is proposed in order to adapt the ionic liquid to a living body.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 4426749

[0007] Patent Document 2: Japanese Patent No. 4843494

[0008] Patent Document 3: Japanese Patent No. 5752343

[0009] Patent Document 4: WO2021 / 085491A

[0010] Patent document 5: WO2021 / 192861A Summary of the invention

[0011] The present disclosure provides a composition comprising a complex (particle) containing a cation (preferably a cationic surfactant, more preferably a cationic lipid), an anion (preferably anionic surfactant, more preferably a fatty acid, and even more preferably a long-chain fatty acid) and a nucleic acid.

[0012] The present inventors have found that anionic long-chain lipids can weaken the skin barrier function. The present inventors have thus found that anionic long-chain lipids can be used to increase the permeability of drugs to the skin. The present inventors have also found a composition comprising a solid complex containing cations and anions, in particular cationic lipids, anionic lipids and a pharmaceutical agent (e.g., nucleic acid), and an oil phase, wherein the solid complex is dispersed in the oil phase.

[0013] According to the present invention, the following inventions are provided.

[0014] (1) A composition for forming a complex with a pharmaceutical ingredient, comprising a mixture containing cations and anions (preferably, the composition consists of cations and anions or consists of cations, anions and a solvent).

[0015] (2) The composition for percutaneous administration according to (1) above, wherein the cation is a cationic lipid (e.g., EDMPC) and the anion is a fatty acid (e.g., an unsaturated fatty acid, preferably a long-chain unsaturated fatty acid, more preferably linoleic acid and oleic acid).

[0016] (3) The composition according to (1) or (2) above, which does not contain a pharmaceutically active ingredient.

[0017] (4) The composition according to (1) or (2) above, further comprising a transdermally administrable pharmaceutical active ingredient.

[0018] (5) A composition (preferably a composition for transdermal administration) comprising a solid complex containing cations, anions and nucleic acids and an oil phase,

[0019] The solid composite groups are dispersed in the oil phase.

[0020] (6) The composition according to (5) above, wherein the solid composite group contains solid composites having a size of about 5 nm to about 50 nm.

[0021] (7) The composition according to (6) above, wherein the group of solid composites comprises a structure including a plurality of aggregates of solid composites having a size of about 5 nm to about 50 nm.

[0022] (8) The composition according to any one of (5) to (7) above, which is used for transdermal administration of a nucleic acid.

[0023] (9) The composition according to any one of (5) to (8) above, wherein the cation is a cationic lipid and the anion is an anionic lipid.

[0024] (10) The composition according to any one of (5) to (9) above, wherein the cation and the anion are contained in an amount with a charge ratio of 1:0.8 to 1:1.2.

[0025] (11) The composition according to any one of (5) to (10) above, wherein the cations and anions are capable of forming an ionic liquid when mixed at least at a charge ratio of 1:1 in a composition consisting only of the cations and anions.

[0026] (12) A composition comprising an aqueous composition containing a complex containing a cation, an anion and a nucleic acid, which is capable of transfecting the nucleic acid from outside a cell into a cell, thereby producing a viable cell containing the nucleic acid inside the cell.

[0027] (13) A method for producing a composition described in any one of (5) to (11) above, comprising the steps of mixing an aqueous solution containing nucleic acid with a low-grade alcohol solution containing a mixture of cations and anions to obtain a complex containing nucleic acid and the cations and anions.

[0028] (14) The method according to (13) above, further comprising the step of freeze-drying the obtained complex.

[0029] (15) The method according to (12) or (13) above, comprising the step of dispersing the freeze-dried complex in an oil phase.

[0030] (16) The composition according to (12) above, which is used for transfecting nucleic acid from outside cells into cells.

[0031] (17) A composition for weakening the skin barrier of a subject (or increasing the permeability of the skin), comprising an anionic lipid.

[0032] (18) The composition according to (17) above, wherein the anionic lipid is a fatty acid.

[0033] (19) The composition according to (17) or (18) above, wherein the anionic lipid is a long-chain fatty acid.

[0034] (20) The composition according to any one of (17) to (19) above, wherein the anionic lipid is a long-chain unsaturated fatty acid.

[0035] (21) The composition according to any one of the above items, wherein the cationic lipid and the anionic lipid are a cationic fatty acid and an anionic fatty acid.

[0036] (22) A composition wherein, with respect to the cationic lipid and the anionic lipid, a composition containing only the cationic lipid and the anionic lipid at least at a charge ratio of 1:1 can form an ionic liquid.

[0037] (23) The composition according to (21) above, wherein the cationic fatty acid and the anionic fatty acid are capable of forming an ionic liquid when mixed at least at a charge ratio of 1:1 in a composition consisting only of the cationic fatty acid and the anionic fatty acid.

[0038] (31) A composition comprising a mixture of cations and anions for forming a complex with a pharmaceutical ingredient, wherein the cations are cationic surfactants and the anions are anionic surfactants.

[0039] (32) The composition according to (31) above, wherein the cation is a cationic lipid and the anion is a fatty acid.

[0040] (33) The composition according to (32) above, wherein the anion is a long-chain fatty acid (preferably having 14 to 22 carbon atoms).

[0041] (34) The composition according to (33) above, wherein the anion is a long-chain unsaturated fatty acid (preferably having 14 to 22 carbon atoms).

[0042] (35) The composition according to (34) above, wherein the anion is linoleic acid or oleic acid.

[0043] (36) The composition according to any one of (31) to (35) above, wherein the cation is EDMPC.

[0044] (41) A composition comprising a solid complex comprising cations, anions and nucleic acids, and an oil phase,

[0045] The solid complex groups are dispersed in the oil phase.

[0046] The cation is a cationic lipid and / or the anion is a fatty acid.

[0047] (42) The composition according to (41) above, wherein the anion is a long-chain fatty acid (preferably having 14 to 22 carbon atoms).

[0048] (43) The composition according to (42) above, wherein the anion is a long-chain unsaturated fatty acid (preferably having 14 to 22 carbon atoms).

[0049] (44) The composition according to (43) above, wherein the anion is linoleic acid or oleic acid.

[0050] (45) The composition according to any one of (41) to (44) above, wherein the cation is EDMPC.

[0051] (46) The composition according to (41) above, wherein the solid composite group contains solid composites of about 5 nm to about 50 nm.

[0052] (47) The composition according to (42) above, wherein the solid composite group contains solid composites of about 5 nm to about 50 nm.

[0053] (48) The composition according to (43) above, wherein the solid composite group contains solid composites of about 5 nm to about 50 nm.

[0054] (49) The composition according to (44) above, wherein the solid composite group contains solid composites of about 5 nm to about 50 nm.

[0055] (50) The composition according to (45) above, wherein the solid composite group contains solid composites of about 5 nm to about 50 nm.

[0056] (51) The composition according to (46) above, wherein the group of solid composites comprises a structure containing a plurality of aggregates of solid composites of about 5 nm to about 50 nm (e.g., with a diameter of 100 nm to 500 nm).

[0057] (52) The composition according to (47) above, wherein the group of solid composites comprises a structure containing a plurality of aggregates of solid composites of about 5 nm to about 50 nm (e.g., with a diameter of 100 nm to 500 nm).

[0058] (53) The composition according to (48) above, wherein the group of solid composites comprises a structure containing a plurality of aggregates of solid composites of about 5 nm to about 50 nm (e.g., with a diameter of 100 nm to 500 nm).

[0059] (54) The composition according to (49) above, wherein the group of solid composites comprises a structure containing a plurality of aggregates of solid composites of about 5 nm to about 50 nm (e.g., with a diameter of 100 nm to 500 nm).

[0060] (55) The composition according to (50) above, wherein the group of solid composites comprises a structure containing a plurality of aggregates of solid composites of about 5 nm to about 50 nm (e.g., with a diameter of 100 nm to 500 nm).

[0061] (56) The composition according to any one of the above items, wherein the cations and anions are contained in an amount with a charge ratio of 1:0.8 to 1:1.25.

[0062] (57) The composition according to any of the above items, wherein the cations and anions are capable of forming an ionic liquid when mixed at least at a charge ratio of 1:1 in a composition consisting only of the cations and anions.

[0063] (61) A composition comprising a mixture containing cations and anions for forming a complex with a pharmaceutical ingredient, wherein the composition containing only cations and anions at least in a charge ratio of 1:1 can form an ionic liquid or the composition itself is an ionic liquid, and the above-mentioned composition is composed of cations and anions or is composed of cations, anions and a solvent.

[0064] (62) A composition according to any one of (31) to (36) above, which is a composition for improving skin permeability comprising a mixture of cations and anions, wherein the composition containing only cations and anions at least in a charge ratio of 1:1 can form an ionic liquid or the composition itself is an ionic liquid, and the above composition is composed of cations and anions or is composed of cations, anions and a solvent.

[0065] (63) The composition according to (61) or (62) above, wherein the solvent contains a volatile organic solvent or is a volatile organic solvent.

[0066] (64) The composition according to any of the above items, which is a transdermal composition for improving skin permeability, comprising a mixture of cations and anions and a solvent, wherein the solvent contains a volatile organic solvent or is a volatile organic solvent.

[0067] (65) The composition according to (64) above, wherein the volatile organic solvent is a lower alcohol or contains a lower alcohol.

[0068] (66) The composition according to (65) above, wherein the lower alcohol is an alcohol having 1 to 4 carbon atoms.

[0069] (67) The composition according to (66) above, wherein the lower alcohol is ethanol.

[0070] (68) The composition according to any one of (61) to (67) above, wherein the cation is a cationic lipid (preferably a cationic long-chain lipid).

[0071] (69) The composition according to any one of (61) to (68) above, wherein the anion is a fatty acid.

[0072] (70) The composition according to (68) above, wherein the cationic lipid is EDMPC.

[0073] (71) The composition according to any one of (61) to (670) above, wherein the anion is a long-chain fatty acid (eg, having 14 to 22 carbon atoms).

[0074] (72) The composition according to (71) above, wherein the anion is a long-chain unsaturated fatty acid (e.g., having 14 to 22 carbon atoms).

[0075] (73) The composition according to (72) above, wherein the anion is linoleic acid or oleic acid.

[0076] (81) A composition comprising a solid complex containing a surfactant (e.g., a nonionic surfactant (e.g., ER-290), a cationic surfactant, anionic surfactant) and a drug, and an oil phase containing anionic long-chain lipids, wherein the solid complex is dispersed in the oil phase.

[0077] (82) The composition according to (81) above, wherein the anionic long-chain lipid is a long-chain fatty acid.

[0078] (83) The composition according to (81) or (82) above, wherein the anionic long-chain lipid is a long-chain unsaturated fatty acid.

[0079] (84) The composition according to any one of (81) to (83) above, wherein the anionic long-chain lipid is linoleic acid.

[0080] (85) The composition according to any one of (81) to (84) above, which is for transdermal administration (i.e., used for transdermal administration).

[0081] (91) The composition according to any of the above items, wherein the cation is a surfactant having an HLB value of 10 or less, preferably about 1 to about 4.

[0082] (92) The composition according to any of the above items, wherein the anion is a surfactant having an HLB value of 10 or less, preferably about 1 to about 4.

[0083] (93) A composition according to any of the above items, wherein the cation and the anion are surfactants each having an HLB value of 10 or less, preferably about 1 to about 4.

[0084] (101) The composition according to any one of the above items, wherein the oil phase further contains an anionic surfactant.

[0085] (102) The composition according to any one of the above items, wherein the oil phase further contains an anionic surfactant, thereby improving the skin permeability of a drug such as a nucleic acid compared to a composition in which the oil phase does not further contain an anionic surfactant.

[0086] (103) The composition according to (101) or (102) above, wherein the anionic surfactant further contains a fatty acid (preferably a long-chain fatty acid, more preferably a long-chain unsaturated fatty acid). BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The composition composed of a cationic surfactant and an anionic surfactant (forming an ionic liquid) synthesized is shown 1 H-NMR spectrum.

[0088] Figure 2 The method shows the steps of forming a cation-anion-nucleic acid complex through an emulsion formation step, and dispersing the complex in isopropyl myristate (IPM) after freeze-drying to obtain a dispersion liquid in which a solid complex is dispersed in oil.

[0089] Figure 3A The appearance of dispersions of solid complexes of nucleic acids and mixtures of various cationic surfactants and anionic surfactants are shown.

[0090] Figure 3B The particle size distribution of the solid composite in the obtained dispersion liquid measured by dynamic light scattering (DLS) is shown.

[0091] Figure 3C The particle size and polydispersity index (PDI) of the solid composite in the obtained dispersion liquid are shown over time.

[0092] Figure 4 The effect of the concentrations of nucleic acid and surfactant on the particle size distribution of the obtained solid composite is shown.

[0093] Figure 5 The results of evaluation of the skin permeability of the obtained complex are shown.

[0094] Figure 6 The solubility of each lipid or lipid mixture in cyclohexane and the appearance of the dispersion are shown.

[0095] Figure 7 The results of the evaluation of the permeability of the obtained complexes to the skin of hairless mice are shown.

[0096] Figure 8 The effect of the addition of an ionic surfactant on the skin permeability of a dispersion containing an ER-290-nucleic acid solid complex is shown.

[0097] Fig. 9 The effect of ionic surfactants on stratum corneum lipids is shown.

[0098] Fig.10 The results of molecular simulation of stratum corneum lipids coated with an ionic surfactant are shown.

[0099] Fig.11 A scheme showing a method for preparing a surfactant-drug complex using an ethanol dilution method.

[0100] Fig.12 The effect of the ionicity of the surfactant on the particle size distribution of the solid composite obtained by the ethanol dilution method is shown.

[0101] Fig.13 The particle size distributions of the solid composites formed by the emulsion formation method (Pre-method) and the ethanol dilution method (New-method) are shown.

[0102] Fig.14 The permeability and permeability into the skin of the nucleic acid-containing solid complex formed by the emulsion formation method (previous method) and the ethanol dilution method (new method) are shown.

[0103] Fig.15A The stability of the appearance of the dispersion of the solid composite formed by the ethanol dilution method is shown.

[0104] Fig. 15B The stability of the particle size distribution of the dispersion of the solid composite formed by the ethanol dilution method is shown.

[0105] Fig.16 The effect of the mixing ratio of nucleic acid and ionic lipid on the particle size distribution of the solid complex formed by the ethanol dilution method is shown.

[0106] Fig.17 The effects of the mixing ratio of nucleic acid to ionic lipid on the permeation and penetration of a solid complex formed by an ethanol dilution method into the skin are shown.

[0107] Fig.18 The effect of the mixing ratio of nucleic acid and ionic lipid on the particle size distribution of the solid complex formed by the ethanol dilution method is shown.

[0108] Fig.19 The effect of the mixing ratio of nucleic acid and ionic lipid on the zeta potential of a solid complex formed by the ethanol dilution method is shown.

[0109] Fig. 20The relationship between the stirring time after mixing a drug solution and an ionic lipid by the ethanol dilution method and particle formation is shown.

[0110] Fig.21 The relationship between the temperature conditions and particle formation in the step of mixing a drug solution and an ionic lipid by the ethanol dilution method is shown.

[0111] Fig. 22 The relationship between the ratio of the aqueous solution and the ethanol solution mixed in the ethanol dilution method and the particle size distribution is shown.

[0112] Fig.23 The effect of the lipid used on the particle size distribution of the solid composite formed by the ethanol dilution method is shown.

[0113] Fig.24 The cation is shown as (CH 3 ) 3 N ＋ (CH 2 ) 2 Particle size distribution and zeta potential of the solid composite formed by the ethanol dilution method when OH (choline) and the anion is oleic acid.

[0114] Fig.25 The permeability and permeability to the skin of various solid composites formed by the ethanol dilution method are shown.

[0115] Fig.26 The particle size distribution and stability of the solid complex containing peptide nucleic acid formed by the ethanol dilution method are shown.

[0116] Fig. 27 The particle size distribution and stability of the protein-containing solid complex formed by the ethanol dilution method are shown.

[0117] Fig.28 The permeability of a solid complex containing an ionic surfactant and a protein formed by an ethanol dilution method into the skin is shown.

[0118] Fig.29 Transmission electron microscope images of ionic lipid-nucleic acid solid complexes formed by the emulsion method (cyclohexane method) and the ethanol dilution method are shown.

[0119] Fig.30 The results of experiments in which an ionic surfactant-nucleic acid complex formed by the ethanol dilution method was transfected into cultured cells are shown.

[0120] Fig.31 Shown are fluorescence microscopy images of skin sections demonstrating penetration of ionic surfactant-nucleic acid complexes formed by the ethanol dilution method.

[0121] Fig.32The results show that the introduced mRNA is expressed in cells after transfection into cultured cells using an aqueous solution containing an ionic surfactant-mRNA complex formed by the ethanol dilution method.

[0122] Fig.33 The results of evaluating the cytotoxicity of various ionic lipid-nucleic acid solid complexes formed by the ethanol dilution method are shown.

[0123] Fig.34 The knockdown effects of hybrid particles containing ASO in various tissues are shown. DETAILED DESCRIPTION

[0124] The embodiments of the present invention are described with reference to the accompanying drawings. It should be noted that the present invention is not limited to the following embodiments and the accompanying drawings. It should be noted that in the following embodiments, expressions such as "having", "including" or "containing" also include the meaning of "consisting of" or "composed of".

[0125] In this specification, "cation" refers to a molecule having a positive charge under pH 7. In this specification, "cationicity" refers to the property of having a positive charge under pH 7. In this specification, "anion" refers to a molecule having a negative charge under pH 7. In this specification, "anionicity" refers to the property of having a negative charge under pH 7.

[0126] In this specification, "surfactant" is a molecule having a relatively hydrophilic part and a relatively hydrophobic part in the molecule. Surfactants are roughly divided into ionic surfactants and nonionic surfactants. Examples of ionic surfactants include cationic surfactants and anionic surfactants. Typically, an ionic surfactant may have either a cation or an anion and a hydrophobic part. Cationic surfactants may contain a cationic part and a fatty chain (e.g., a long-chain fatty chain) such as an alkyl, alkenyl or alkynyl group. Anionic surfactants may contain an anionic part and a fatty acid such as a long-chain fatty acid, an unsaturated fatty acid (e.g., a monovalent or divalent) or a long-chain unsaturated fatty acid (e.g., a monovalent or divalent). "Long chain" is a term given to compounds having 14 to 22 carbon atoms. The fatty chain may be a fatty chain having 8 to 22 carbon atoms, i.e., a C 22 fatty acid. 8 ~C 22 Alkyl, C 8 ~C 22 Alkenyl or C 8 ~C 22 Alkynyl etc. 8 ~C 22Fatty chain. In addition, the fatty chain may be a long-chain fatty chain. Saturated means that the fatty chain does not have double bonds and triple bonds, and unsaturated means that the fatty chain has at least one of a double bond or a triple bond. Examples of fatty chains having double bonds include cis-type and trans-type, and from the viewpoint of biocompatibility, cis-type is preferred.

[0127] Lipids are molecules from organisms that are soluble in non-polar solvents. Lipids can be divided into glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, pregnenolone lipids, glycolipids and polyketides. Glycerolipids are a general term for molecules in which fatty acids form ester bonds on the hydroxyl groups of glycerol. Glycerol has 3 hydroxyl groups and can be connected to up to 3 fatty acids through ester bonds. In glycerophospholipids, 2 fatty acids are ester-bonded to 2 hydroxyl groups of glycerol and a phosphate group is connected to 1 hydroxyl group. Sphingolipids can be ceramides, sphingomyelins, and glycosphingolipids. Sterol lipids are a subclass of terpenoid compounds. Sterol lipids can be sterols (such as cholesterol). Pregnenolone lipids are terpenoid compounds other than sterol lipids.

[0128] 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. WO96 / 10390, and these disclosures are incorporated herein by reference in their entirety for all purposes. The cationic lipids include 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleoyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane ( DLin-K-C3-DMA), 2,2-dilinoleoyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleoyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpyrazine (methylpepiazino)-[1,3]-dioxolane (DLin-K-M PZ), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2 -Dilinoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linolenoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane hydrochloride (DLin-TMA.Cl), 1,2-dilinolenoyl-3-trimethylaminopropane hydrochloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazine)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (propanedio) (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl Oxy-N,N-dimethylaminopropane (DODMA), 1,2-distearoyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearoyl-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-dimyristoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propane trifluoroacetate ammonium (DOSPA), dioctadecadienylglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2- [5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',1-2'-octadecadienyloxy) propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-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. As the cationic lipid, 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) can be preferably cited. .

[0129] Preferred anionic lipids include those represented by the following general formula (1).

[0130] R-COO - X ＋ …(1)

[0131] In the general formula (1), R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, and at least one vinyl group constituting the alkenyl group may be substituted with a vinylene group.

[0132] The alkyl group in R may be any of linear, branched and cyclic. The preferred carbon number of the alkyl group in R is 8 to 22, more preferably 12 to 22, and further preferably 14 to 22. For example, as the alkyl group in R, linear alkyl groups such as dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl and docosyl, branched alkyl groups thereof, and cyclic alkyl groups thereof may be exemplified. As substituents that may be substituted on the alkyl group, amino groups, benzyl groups and halogen atoms (e.g., fluorine atoms) may be cited. These substituents may be further substituted by substituents. When the alkyl group is substituted by a substituent, the total number of carbon atoms of the alkyl group and the number of carbon atoms of the substituent is preferably 8 to 22, more preferably 12 to 22, and further preferably 14 to 22.

[0133] The alkenyl group in R can be any one of straight chain and branched chain. The preferred carbon number of the alkenyl group in R is 8 to 22, more preferably 12 to 22, and further preferably 14 to 22. For example, as the alkenyl group in R, linear alkenyl groups such as dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosene, hexadecenyl and docosene, and branched alkenyl groups thereof, etc. can be exemplified. As substituents that can be substituted on the alkenyl group, amino groups, benzyl groups and halogen atoms (such as fluorine atoms) can be cited. These substituents can be further substituted by substituents. When the alkenyl group is substituted by a substituent, the total number of carbon atoms of the alkenyl group and the number of carbon atoms of the substituent is preferably 8 to 22, more preferably 12 to 22, and further preferably 14 to 22.

[0134] In addition, at least one vinyl group of the alkenyl group in R may be substituted with a vinylidene 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 bond is not particularly limited, and it is preferred that the double bonds are arranged with at least two single bonds between them. In addition, the double bond may preferably be a cis-type. In addition, R may contain preferably one or more, more preferably two or more, and further preferably three or more double bonds. Among these, R is preferably a group having an unsaturated bond, more preferably a substituted or unsubstituted alkenyl group, a substituted or unsubstituted group having a polyene structure.

[0135] In addition, the alkyl group, alkenyl group and group having a polyene structure in R may be substituted by a substituent, and in this case, R is preferably composed of only carbon atoms and hydrogen atoms. That is, when the alkyl group, alkenyl group and polyene structure are substituted by a substituent, the substituent is also preferably composed of only carbon atoms and hydrogen atoms.

[0136] As R-COO - , for example, carboxylate ions or derivatives thereof formed by the dissociation of hydrogen ions from the carboxyl group of a fatty acid can be used. The fatty acid that produces the carboxylate ion can be a saturated fatty acid or an unsaturated fatty acid, and can be preferably an unsaturated fatty acid, more preferably a cis-type unsaturated fatty acid, and further preferably a cis-type 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), and 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 values ​​in parentheses are the number of carbon atoms and double bonds of each fatty acid. For example, linoleic acid (C18:2) means that the number of carbon atoms is 18 and the number of double bonds is 2. In the general formula (1), X ＋ Represents a phospholipid having a cationic group. Here, "phospholipid" refers to a lipid containing a phosphate structure, and "cationic group" refers to a substituent with a positive charge. ＋ Preferably, it is a glycerophospholipid having a cationic group, and more preferably a derivative of phosphatidylcholine. Here, the derivative of phosphatidylcholine refers to a glycerol backbone, a substituted or unsubstituted alkanoyl group bonded to the 1st and 2nd positions of the glycerol backbone, a phosphate group (-P(=O)(OH)O) bonded to the 3rd position of the glycerol backbone, and a phosphatidylcholine group (-P(=O)(OH)O) bonded to the 3rd position of the glycerol backbone. - ) and a choline residue bonded to the phosphate group. In the phosphatidylcholine derivative, at least one vinyl group of the alkanoyl group may be substituted with a vinylene group, and the hydrogen atom of the hydroxyl group of the phosphate group may be substituted with a substituted or unsubstituted alkyl group.

[0137] In addition, X ＋ The phospholipid having a structure represented by the following general formula (2) is preferred, and the glycerophospholipid having a structure represented by the following general formula (2) is more preferred.

[0138] CH(-CH 2 -OC(=O)-R 10 )(-OC(=O)-R 11 )-CH 2 -OP(OR 1 )(OR 2 )(=O) …(2)

[0139] In the general formula (2), R 1 represents an alkyl group substituted with a cationic group, R 2represents a hydrogen atom or a substituted or unsubstituted alkyl group. The phospholipid having the structure represented by the general formula (2) has a positive charge as a whole, and preferably has no negatively charged part.

[0140] R 1 The alkyl group in R may be any of linear, branched, and cyclic. 1 The preferred carbon number of the alkyl group in is 1 to 20, more preferably 1 to 10, and even more preferably 1 to 6. 1 The alkyl group in the group may be methyl, ethyl, n-propyl, isopropyl, etc., and R 1 The alkyl group in is preferably ethyl.

[0141] R 1 The cationic group in is preferably an ammonium group represented by the following general formula (3).

[0142] -NR 4 R 5 R 6＋ …(3)

[0143] In the general formula (3), R 4 , R 5 and R 6 Each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, Indicates the position of bonding to the alkyl group. 4 , R 5 and R 6 They may be the same or different from each other. 4 , R 5 and R 6 There is no particular limitation on the number of substituted or unsubstituted alkyl groups. 4 , R 5 and R 6 All of them may be hydrogen atoms or substituted or unsubstituted alkyl groups, or one or two of them may be hydrogen atoms and the rest may be substituted or unsubstituted alkyl groups.

[0144] R 4 , R 5 and R 6 The alkyl group in R may be any of linear, branched, and cyclic. 4 , R 5 and R 6 The preferred carbon number of the alkyl group in is 1 to 20, more preferably 1 to 10, and even more preferably 1 to 6. 4 , R 5 and R 6 The alkyl group in the group may be methyl, ethyl, n-propyl, isopropyl, etc., and R 4 , R 5 and R 6The alkyl group in is preferably a methyl group. As substituents that can be substituted on the alkyl group, benzyl and halogen atoms (such as fluorine atoms) can be listed. These substituents can be further substituted by substituents. In a preferred embodiment, R 4 , R 5 and R 6 It is methyl.

[0145] R 1 The cationic group in is preferably a quaternary ammonium group, more preferably a quaternary ammonium group represented by the general formula (5) (R 4 (all of which are substituted or unsubstituted alkyl ammonium groups). In addition, the substitution position of the cationic group in the alkyl group is not particularly limited, but it is preferred that the hydrogen atom bonded to the carbon atom at the terminal of the alkyl group is substituted with a cationic group.

[0146] In the general formula (2), R 2 represents a hydrogen atom or a substituted or unsubstituted alkyl group. 2 Preferably, it is a substituted or unsubstituted alkyl group. 2 The alkyl group in R may be any of linear, branched, and cyclic. 2 The preferred carbon number of the alkyl group in is 1 to 20, more preferably 1 to 10, and even more preferably 1 to 6. 2 The alkyl group in the group may be methyl, ethyl, n-propyl, isopropyl, etc., and R 2 The alkyl group in is preferably an ethyl group. Examples of the substituent group that may be substituted on the alkyl group include a benzyl group and a halogen atom (eg, a fluorine atom). These substituent groups may be further substituted by a substituent group.

[0147] In the general formula (2), R 10 and R 11 Each independently represents an aliphatic chain having 8 to 22 carbon atoms, preferably a long-chain aliphatic chain, and may be a long-chain saturated aliphatic chain or a long-chain unsaturated aliphatic chain. 10 and R 11 are each independently a long-chain saturated aliphatic chain having 12 to 22 carbon atoms. 10 and R 11 It may be a long saturated fatty chain having 12 to 14 carbon atoms (eg, 13). In one embodiment, the glycerophospholipid represented by the general formula (2) may be EDMPC.

[0148] The number of substitutions of the alkylcarbonyloxy group in the alkyl group is preferably 2 to 8, more preferably 2 to 4, and most preferably 2. The substitution position of the alkylcarbonyloxy group in the alkyl group is not particularly limited. 3 When the alkyl group of the main chain is an n-propyl group, the hydrogen atom bonded to the terminal carbon atom and the hydrogen atom bonded to the adjacent carbon atom thereof are substituted by an alkylcarbonyloxy group, which corresponds to a glycerophospholipid and is a particularly preferred phospholipid.

[0149] The description, preferred range and specific examples of the alkyl group in the alkylcarbonyloxy group may refer to the description, preferred range and specific examples of the alkyl group in R described above.

[0150] Preferred examples of compounds having the structure shown in general formula (1) are listed below. The ionic surfactant of this embodiment should not be interpreted restrictively based on this specific example. 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 shown in general formula (2), R1 is -NR 4 R 5 R 6 , R 4 , R 5 and R 6 are independently methyl or ethyl, preferably both are methyl, R 10 and R 11 Each of them is independently a long chain fatty chain, preferably a long chain fatty chain having 12 to 22 carbon atoms, and more preferably a long chain fatty chain having 12 to 14 carbon atoms. As a more specific example, for example, the carboxylate ion is a long chain unsaturated fatty acid, such as linoleic acid or oleic acid, and in the glycerophospholipid represented by the general formula (2), R1 is -NR 4 R 5 R 6 , R 4 , R 5 and R 6 are independently methyl or ethyl, preferably both are methyl, R 10 and R 11 Each independently represents a long chain fatty chain having 12 to 22 carbon atoms, more preferably a long chain fatty chain having 12 to 14 carbon atoms. Furthermore, as a more specific example, for example, the carboxylate ion is a long chain unsaturated fatty acid, such as linoleic acid or oleic acid, and in the glycerophospholipid represented by the general formula (2), R1 is -NR 4 R 5 R 6 , R 4 , R 5 and R 6 is methyl, R 10 and R 11 Each of them is 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. Furthermore, as a more specific example, in the following formula, R—COO - With R-COO in the general formula (1) - The meanings are the same, and specific examples thereof include carboxylate ions of linoleic acid, oleic acid, acetic acid, or stearic acid.

[0151]

[0152] The above shows the case where the carboxylate ion forms an ionic bond with a preferred cationic surfactant. When a mixture of these cationic lipids and anionic lipids is mixed at a charge ratio of 1:1, it can be an ionic liquid at room temperature.

[0153] As anionic surfactants, phospholipids and fatty acids can be cited. As anionic surfactants, for example, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups bonded to neutral lipids can be cited.

[0154] Examples of the phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinolenoylphosphatidylcholine.

[0155] Fatty acids are carbon chains with a monovalent carboxylic acid. Fatty acids are preferably not trans-type but cis-type. Fatty acids are represented by the structural formula CH 3 -R-CO 2 H is represented by the carbon chain in this case, based on the number of carbon atoms and double bonds in the fatty acid. For example, R of decanoic acid is -(CH 2 ) 8 -, expressed as 10:0, the R of lauric acid is -(CH 2 ) 10 -, expressed as 12:0, the R of myristic acid is -(CH 2 ) 14 -, represented by 14:0, the R of pentadecanoic acid is -(CH 2 ) 15 -, represented by 15:0, the R of palmitic acid is -(CH 2 ) 16 -, represented by 16:0, the R of palmitoleic acid is -(CH 2 ) 5 CH=CH(CH 2 ) 7 -, expressed as 16:1, the R of stearic acid is -(CH 2 ) 18 -, represented by 18:0, the R of oleic acid is -(CH 2 ) 7 CH=CH(CH 2 )7 -, expressed as 18:1(n-9), the R of vaccenic acid is -(CH 2 ) 5 CH=CH(CH 2 ) 9 -, expressed as 18:1(n-7), the R of linoleic acid is -(CH 2 ) 3 (CH 2 CH=CH) 2 (CH 2 ) 7 -, represented by 18:2(n-6), the R of (9,12,15)-linolenic acid is -(CH 2 CH=CH) 3 (CH 2 ) 7 -, represented by 18:3(n-3), the R of (6,9,12)-linolenic acid is -(CH 2 ) 3 (CH 2 CH=CH) 3 (CH 2 ) 4 -, expressed as 18:3(n-6).

[0156] In this specification, a "solid composite" is a composite composed of solid components. For example, the freeze-dried composite can be dispersed in an oil layer to maintain it. The solid composite may contain water, but is a composite containing solid components. The solid composite may have a low water content, for example, the water content may be less than 1% by mass, less than 0.9% by mass, less than 0.8% by mass, less than 0.7% by mass, less than 0.6% by mass, less than 0.5% by mass, less than 0.4% by mass, less than 0.3% by mass, less than 0.2% by mass, or less than 0.1% by mass when measured by a known method.

[0157] (Implementation Method)

[0158] According to the present invention, a percutaneous absorption preparation capable of delivering a target molecule (e.g., nucleic acid) to a living body is provided. The percutaneous absorption preparation of the present invention can achieve at least about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, or about 4 times more permeation to the skin compared to a target molecule (e.g., nucleic acid) dissolved in a buffer (e.g., Tris EDTA (TE) buffer). TE buffer generally contains 10mM Tris (pH 7.4-8.0) and 1mM EDTA. Alternatively, the percutaneous absorption preparation of the present invention can achieve at least about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, or about 15 times more skin permeation compared to a target molecule (e.g., nucleic acid) dissolved in a buffer (e.g., Tris EDTA buffer (TE buffer)). In one embodiment, the S / O preparation of the present invention increases the amount of target molecules (e.g., nucleic acids) that penetrate the skin based on any one or more of the particle structure, charge state, and added components. In this specification, the target molecule is a molecule that can be delivered using the method of the present invention to obtain benefits.

[0159] According to the present invention, a composition comprising a solid complex containing a target molecule (e.g., nucleic acid) and a surfactant and an oil phase is provided. In a preferred embodiment, the composition does not contain an aqueous phase. In the above composition, the above complex is dispersed in the above oil phase. A W / O emulsion is formed by mixing an aqueous phase containing a target molecule (e.g., nucleic acid) with an organic solvent phase containing a surfactant, and the obtained W / O emulsion is freeze-dried to remove water and organic solvent components, thereby obtaining a solid complex in a dry state. In the W / O emulsion, particles of the aqueous phase containing the target molecule (e.g., nucleic acid) coated with a surfactant are dispersed in the organic phase. Therefore, it is believed that in the solid complex obtained by drying the above particles, the target molecule (e.g., nucleic acid) is coated with a surfactant. Therefore, in one embodiment, the solid complex contains a target molecule (e.g., nucleic acid) and a surfactant, and the target molecule (e.g., nucleic acid) is coated with a surfactant. The coating is usually completed by using a sufficient amount (especially a saturated amount) of a surfactant on the entire surface of the core containing the target molecule (e.g., nucleic acid). In addition, in the composition or preparation of the present invention, the above solid complex is dispersed in an oil phase. It is believed that the surfactant binds to the target molecule (e.g., nucleic acid) with its hydrophilic part, so that its hydrophobic part faces the outside of the complex and contacts the oil phase. In addition, it is believed that the surfactant plays an important role in promoting the dispersion of the above-mentioned solid complex containing the hydrophilic target molecule (e.g., nucleic acid) in the oil phase. The above-mentioned composition is obtained by dispersing the obtained solid complex in the oil phase. It is speculated that the above-mentioned complex forms solid particles or solid complexes in the above-mentioned composition. The above-mentioned composition of the present invention can be used to make the target molecule (e.g., nucleic acid) percutaneously absorbed. Therefore, the above-mentioned composition of the present invention can be formulated as a percutaneous absorbent. In the above-mentioned complex of the present invention, generally, among the components other than the target molecule (e.g., nucleic acid), about 50% by weight or more, about 60% by weight or more, about 70% by weight or more, about 80% by weight or more, about 90% by weight or more, about 95% by weight or more, or about 100% by weight can be a surfactant. In a preferred embodiment, the above-mentioned complex is composed of a target molecule (e.g., nucleic acid) and a surfactant {wherein the above-mentioned complex may have impurities mixed in that do not cause interference in the manufacturing step}. The above composition may be a dermatologically acceptable composition.

[0160] In one embodiment, a composition (aqueous composition) is provided comprising a complex (preferably complex particles or solid complex) containing a target molecule (e.g., nucleic acid) and a surfactant and an aqueous phase. In a preferred embodiment, the composition does not contain an oil phase. In the above composition, the complex is dispersed in the above aqueous phase. By mixing an aqueous phase containing a target molecule (e.g., nucleic acid) and an organic solvent phase (especially a lower alcohol) containing a surfactant, a dispersion (preferably an aqueous dispersion) having a surfactant-target molecule (e.g., nucleic acid) complex and an aqueous phase and dispersing the surfactant-target molecule (e.g., nucleic acid) complex in an aqueous phase is obtained. According to Zeta potential and DLS studies, it is believed that the target molecule (e.g., nucleic acid) in the complex is coated with a surfactant. The aqueous dispersion preferably does not have cytotoxicity. The above aqueous composition can be used to introduce target molecules (e.g., nucleic acids) into cells of cultured cells.

[0161] The size of the above-mentioned complex is not particularly limited as long as it can maintain a dispersed state in the oil phase and the size of the complex does not impair skin permeability. The average particle size (average particle size based on DLS) of the above-mentioned complex (complex particle) is, for example, about 1 nm to about 3000 nm, about 1 nm to about 2000 nm, about 1 nm to about 1500 nm, about 1 nm to about 1400 nm, about 1 nm to about 1300 nm, about 1 nm to about 1200 nm, about 1 nm to about 1100 nm, about 1 nm to about 1000 nm, preferably about 10 nm to about 900 nm, more preferably about 100 nm to about 700 nm, and further preferably about 200 nm to about 500 nm {herein, when the nucleic acid is mRNA, the average particle size can be about 1 μm or more, and when the nucleic acid is an antisense oligonucleotide or siRNA, the average particle size can be about 1 μm or less.}. In one embodiment, the average particle size of the above-mentioned complex is about 300 nm to about 500 nm. The polydispersity index (PDI) is a measure of the heterogeneity of the particle size. The smaller the PDI, the more uniform the particle size. The polydispersity index of the preparation of the present invention is, for example, about 1 or less, and can be about 0.2 to about 0.8, about 0.3 to about 0.7, about 0.2 to 0.4, or about 0.4 to about 0.6.

[0162] Nucleic acids are hydrophilic compounds. Therefore, they usually cannot penetrate the skin or have low skin permeability when they are alone. The composition or percutaneous absorbent of the present embodiment improves the skin permeability of nucleic acids by forming a complex with a surfactant. Hydrophilic compounds and hydrophilic proteins usually cannot penetrate the skin or have low skin permeability when they are alone. The composition or percutaneous absorbent of the present embodiment improves the skin permeability of hydrophilic physiologically active substances such as hydrophilic compounds and hydrophilic proteins by replacing nucleic acids and forming a complex with a surfactant.

[0163] Nucleic acid can be DNA or RNA. Nucleic acid can be single-stranded or double-stranded. As nucleic acid, mRNA, antisense nucleic acid (such as antisense oligonucleotide) targeting RNA, shRNA, microRNA and siRNA, decoy nucleic acid that binds to transcription factors and inhibits the transcription stage, aptamer that binds to proteins and inhibits function, and CpG oligodeoxynucleotides that act on Toll-like receptor 9 and activate the immune system can be listed. Nucleic acid can be mRNA (mRNA vaccine) that expresses a specified protein in cells. The base length of the nucleic acid is not particularly limited, for example, about 10 to about 80 bases, about 15 to about 50 bases, or about 20 to about 30 bases. The length of siRNA can be, for example, about 20 to about 30 bases, about 20 to about 24 bases, or about 21 to about 23 bases. Nucleic acid can contain modified nucleic acid in its base sequence. That is, the nucleic acid can be a nucleic acid such as DNA, RNA, a hybrid of DNA and RNA, and a nucleic acid containing a modified nucleic acid (gapmer and mixer). Gapmer is a nucleic acid containing a single-stranded nucleic acid, wherein the single-stranded nucleic acid has a DNA portion and an RNA or modified nucleic acid portion and the RNA or modified nucleic acid portion is located at both ends of the DNA portion. Gapmer can be, for example, a double-stranded nucleic acid. Mixmer is an antisense-containing nucleic acid containing different types of nucleic acids. Antisense oligonucleotides, microRNAs, siRNAs, shRNAs, gapmers, mixers, etc. can be designed in a sequence and length in a manner that exerts a knockdown effect on the target RNA.

[0164] As modified nucleic acids, for example, fluorescent dye-modified nucleic acids, biotinylated nucleic acids, and nucleic acids into which cholesterol groups are introduced can be listed. In order to improve stability, the bases of RNA are sometimes modified with 2'-O-methyl, 2'-fluorine, or 2'-methoxyethyl (MOE). In addition, sometimes the phosphodiester bond of the nucleic acid backbone is replaced with a thiophosphate bond, as in thiophosphate-type nucleic acids. As artificial nucleic acids, nucleic acids in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged can be listed. As such artificial nucleic acids, for example, a bridged DNA, i.e., a locked nucleic acid (LNA), in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged by a methylene group, an ENA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged by an ethylene group, and an oxygen atom at the 2' position and the carbon atom at the 4' position are bridged by a -CH 2 OCH 2 - The bridging BNACOC, the oxygen atom at the 2' position and the carbon atom at the 4' position are connected by -NR-CH 2 -{herein, R is a methyl group or a hydrogen atom} and a bridged nucleic acid (BNA) such as BNANC, the oxygen atom at the 2' position and the carbon atom at the 4' position are linked by -CH 2 (OCH 3)- and the bridging cMOE, the oxygen atom at the 2' position and the carbon atom at the 4' position are connected by -CH 2 (CH 3 )- and bridged cEt, AmNA in which the carbon atoms at the 2' and 4' positions are bridged by amide, scpBNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged by methylene and form cyclopropane at the 6' position, and a peptide nucleic acid (PNA) in which a polymer formed by amide bond connection of N-(2-aminoethyl)glycine instead of deoxyribose or ribose is used as the main chain. As RNA, artificial RNAs such as siRNA and shRNA for gene silencing, microRNA (miRNA), non-coding RNAs such as aptamers, and natural RNAs such as mRNA can be listed. These RNAs can be modified to stabilize in vivo.

[0165] As the protein encoded by mRNA, xenobiotics, such as pathogen-specific components or a part thereof and cancer cell-specific components or a part thereof can be enumerated. As pathogen, pathogenic viruses, pathogenic microorganisms and pathogenic parasites can be enumerated. As pathogen, pathogenic viruses, adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses (for example, severe acute respiratory syndrome-related coronaviruses (SARS-CoV), Middle East respiratory syndrome-related coronaviruses (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 virus, poliovirus, rabies virus, John-Cunningham (JC) virus and the virus in the group consisting of arbovirus encephalitis virus can be enumerated. Examples of pathogenic microorganisms include pathogenic bacteria, and examples of pathogenic bacteria include bacteria selected from the group consisting of Chlamydia, Rickettsiales, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, Bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease bacteria. As pathogenic microorganisms, pathogenic fungi can be listed, and as pathogenic fungi, pathogenic fungi selected from the group consisting of Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides braziliensis, Coccidioides immitis, and Histoplasma capsulatum can be listed. As pathogenic parasites, pathogenic parasites selected from the group consisting of Protozoa dysenteriae, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium malariae, Babesia, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma, and Hookworm can be listed. Therefore, in one embodiment of the present invention, mRNA can encode components (preferably surface antigens) of these pathogens or a portion thereof. In particular, it is preferred that the mRNA of the present invention encodes antigens on the surface of pathogens, such as the S protein of the beta coronavirus, for causing the pathogen to infect cells.As cancer, it is possible to list cancer selected from the group consisting of 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, digestive system cancer, Hodgkin or non-Hodgkin tumors, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, renal cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer and head and neck cancer. In addition, in one embodiment of the present invention, mRNA can be these cancer-specific antigens (cancer-specific antigens, preferably cancer-specific surface antigens) or a portion thereof. Cancer-specific antigens, for example, can be a characteristic portion of a neoantigen or a neoantigen. Cancer-specific antigens include, for example, cancer testis antigens (e.g., proteins of the MAGE family, NY-ESO-1), differentiation antigens (e.g., tyrosine kinase of melanoma, Melan-A / MART-1, PSA of prostate cancer), overexpressed cancer antigens (e.g., Her-2 / Neu, survivin, telomerase, and WT-1), cancer-inducing mutants of β-catenin, cancer-inducing mutants of CDK4, MUC-1, especially MUC-1 with a changed glycosylation pattern, and E6 or E7 of human papillomavirus. Regarding these, a person skilled in the art can appropriately select antigens and prepare mRNA suitable for the present invention according to the type of infection and cancer suffered by the subject, and the type of antigen expressed by the cancer. mRNAs against natural mutant proteins can also be appropriately designed.

[0166] The surfactant is not particularly limited as long as it is acceptable in medicine. As the surfactant, for example, ionic surfactants such as anionic surfactants and cationic surfactants are preferably used. From the aspect of excellent complex formation ability with target molecules (such as nucleic acids), anionic surfactants and cationic surfactants are preferred. In particular, a mixture of a cationic surfactant and an anionic surfactant is used as a surfactant in the present disclosure.

[0167] As a surfactant, from the viewpoint of being able to easily disperse the complex in the oil phase, a lipophilic ionic surfactant having an HLB (Hydrophile-Lipophile Balance) value of 10 or less is preferred. The HLB of the ionic surfactant is preferably 8 or less, more preferably 5 or less, and particularly preferably 3 or less. The HLB of the ionic surfactant may be, for example, 1 to 3, 3 to 5, or 5 to 10. In one embodiment, the HLB of the ionic surfactant may be 5 to 10. In one embodiment, the HLB of the cationic surfactant may be 1 to 10, for example, 1 to 3, 3 to 5, or 5 to 10. In one embodiment, the HLB of the cationic surfactant may be 5 to 10.

[0168] The ratio of the mass of the surfactant in the complex to the mass of the target molecule (e.g., nucleic acid) (mass parts of the surfactant relative to 1 mass part of the target molecule (e.g., nucleic acid)) can be, for example, 1 to 200, preferably 20 to 180, and more preferably 20 to 100. When the nucleic acid is an antisense oligonucleotide, the mass ratio can be preferably 30 to 50, and when the nucleic acid is mRNA, the mass ratio can be preferably 50 to 100.

[0169] The content of the complex in the composition or transdermal absorbent of the present embodiment is not particularly limited as long as it is a dispersible concentration. The content of the complex in the composition or transdermal absorbent is, for example, 0.6 w / v% to 60 w / v%. The content of the target molecule (e.g., nucleic acid) in the transdermal absorbent is not particularly limited, for example, it can be 0.1 to 10 mg / mL, 0.2 to 8 mg / mL, 0.3 to 7 mg / mL, or preferably 1 to 5 mg / mL.

[0170] The complex may be composed of only the target molecule (e.g., nucleic acid) 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 can improve the stability of the complex by being coated with the surfactant together with the target molecule (e.g., nucleic acid), prevent the target molecule (e.g., nucleic acid) from leaking out of the complex in the composition or percutaneous absorbent, etc.

[0171] Examples of the protein include serum albumin, ovalbumin, casein, lysozyme, and lipase, etc. These proteins may be used alone or in combination of two or more.

[0172] Examples of the polysaccharide include LM pectin, HM pectin, hydroxypropylmethylcellulose phthalate, heparin, alginic acid, and carboxymethylcellulose, etc. These polysaccharides may be used alone or in combination of two or more.

[0173] The ratio of the mass of the stabilizer in the complex to the mass of the target molecule (eg, nucleic acid) is preferably 0.01 to 100, more preferably 0.1 to 10, and even more preferably 0.5 to 5.

[0174] The oil phase can be liquid or solid with fluidity. The base material of the oil phase is not particularly limited as long as it is an oily base material allowed to be used in pharmaceutical preparations, preferably an oily base material allowed to be used in pharmaceutical preparations for transdermal delivery. Any of the oils that are liquid at room temperature (25°C) or solid at room temperature can be used. There is no restriction on the source of the raw materials, for example, any of natural and synthetic products can be used. In addition, the oil phase can be vegetable oils such as soybean oil, cottonseed oil, rapeseed oil, sesame oil, corn oil, peanut oil, safflower oil, sunflower oil, olive oil, rapeseed oil, perilla oil, anise oil, cocoa butter, cinnamon oil, peppermint oil, bergamot oil, etc., or animal oils such as tallow, lard, and fish oil. In addition, the oil phase can be neutral lipids or synthetic lipids such as glyceride, triolein, trilinolein, tripalmitin, tristearin, trimyristin, and triarachidonic acid glyceride.

[0175] The oil phase can be sterol derivatives such as cholesterol oleate, cholesterol linoleate, cholesterol myristate, cholesterol palmitate and cholesterol arachidate, and can also be long-chain fatty acid esters such as isopropyl myristate (IPM), octyldodecyl myristate, cetyl myristate, ethyl oleate, ethyl linoleate, isopropyl linoleate, isopropyl palmitate and butyl stearate. In addition, the oil phase can be carboxylic acid esters such as ethyl lactate, cetyl lactate, triethyl citrate, diisopropyl adipate, diethyl sebacate, diisopropyl sebacate and 2-ethylhexanoic acid cetyl ester, and can also be hydrocarbons such as vaseline, paraffin, squalane and vegetable squalane, and can also be silicone. One kind of oily base material can be used alone, or two or more kinds can be mixed and used.

[0176] As the oil phase, triglyceride or an edible oil containing it as the main component can be used. In practical terms, soybean oil is preferred, and soybean oil purified to high purity is more preferred. In addition, neutral lipids or long-chain fatty acid esters can also be preferably used, and long-chain fatty acid esters are more preferred, and IPM is further preferred.

[0177] The content of the oil phase in the composition or percutaneous absorption preparation of the present embodiment varies depending on the type of the oil component, other components, etc., but is preferably 50 w / v% to 99.5 w / v%, and more preferably 60 w / v% to 90 w / v%.

[0178] In this specification, ultrapure water refers to water that meets the ASTM D 5196-06 (2018) standard. In the ASTM D 5196-06 (2018) standard, the total amount of inorganic ions (especially aluminum, ammonia, arsenic, cadmium, chromium, fluoride ion, cobalt, copper, iron, lead, magnesium, nickel, potassium, sodium, titanium and zinc, as well as chloride ion, nitrate ion, phosphate ion and sulfate ion) is 1μg / L or the resistivity is 18.2MΩcm at 25°C, the total organic carbon content is less than 20ppb, the colony formation number is less than 100cfu / 100mL and the endotoxin content is less than 0.01EU / mL. For ultrapure water used in solutions containing nucleic acids, nuclease is required to be below the detection limit. For ultrapure water used in solutions containing proteins, protease is required to be below the detection limit.

[0179] As the cation, various cations can be used, for example, cationic lipids can be used. As the anion, various anions can be used, for example, anionic lipids can be used. Cationic lipids and anionic lipids are as described above. Specifically, cationic lipids, for example, contain a cationic moiety and a fatty chain (e.g., a long fatty chain) such as an alkyl, alkenyl, or alkynyl group.

[0180] Ionic liquids are composed of cations and anions, and are salts that exist in liquid form over a wide temperature range. In the present invention, the ionic liquid is liquid at ambient temperature (e.g., room temperature, e.g., 25° C.). As anions, for example, preferably carboxylate ions or derivatives thereof are formed by the dissociation of hydrogen ions from the carboxyl groups of fatty acids. The fatty acid that produces the carboxylate ion may be a saturated fatty acid or an unsaturated fatty acid. Examples of saturated fatty acids include myristic acid, palmitic acid, stearic acid, and lauric acid, and examples of unsaturated fatty acids include oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and erucic acid.

[0181] As cations that can form ionic liquids, cationic lipids, preferably phospholipids with cationic groups. Here, "phospholipid" refers to lipids containing a phosphate structure, and "cationic group" refers to a positively charged substituent. The two hydroxyl groups of the phosphate group are replaced by lower alkyl groups (such as ethyl) and the charge disappears, and then the lower alkyl groups are replaced by amines (primary amines, secondary amines or tertiary amines) to form cationic lipids. Furthermore, the cations that can form ionic liquids are preferably glycerophospholipids with cationic groups, and more preferably derivatives of phosphatidylcholine. Preferably, the anions that can form ionic liquids are linoleic acid, and the cations that can form ionic liquids are 1,2-dimyristoyl-sn-glycero-3-ethylphosphorylcholine. Among lipids, unsaturated lipids are more likely to form ionic liquids than saturated lipids.

[0182] The content of the skin penetration enhancer in the composition or percutaneous absorbent of this embodiment can be determined from the viewpoint of improving the skin permeability of the target molecule (e.g., nucleic acid), and is, for example, 1 to 20 mass %, 2 to 18 mass %, 3 to 16 mass %, 4 to 15 mass % or 5 to 10 mass %.

[0183] (Preparation of emulsion-based formulations)

[0184] The composition or percutaneous absorbent of the present embodiment can be manufactured, for example, by a method comprising an emulsion preparation step, a complex preparation step and a dispersion step. In the emulsion preparation step, a target molecule (e.g., nucleic acid), a surfactant and an organic solvent solution are mixed to prepare a W / O type emulsion. The emulsion preparation step is carried out under conditions suitable for forming a W / O emulsion. Preferably, in the emulsion preparation step, a W / O type emulsion is prepared by mixing an aqueous solution (the above aqueous solution may also contain cations) containing a target molecule (e.g., nucleic acid) and an organic solvent solution containing a surfactant and emulsifying and dispersing the mixture. From the viewpoint of being easily removed in the subsequent drying step, the organic solvent solution is preferably a volatile organic solvent solution.

[0185] Regarding the concentration of the target molecule (e.g., nucleic acid) in the aqueous solution of the target molecule (e.g., nucleic acid), there is no particular restriction as long as these can be substantially completely dissolved, for example, about 0.1 mg / mL to about 30 mg / mL, about 0.4 mg / mL to about 30 mg / mL, or about 1 mg / mL to about 30 mg / mL. A stabilizer can be added to the aqueous solution as needed. There is no particular restriction on the organic solvent as long as it can dissolve the surfactant and can be removed in subsequent steps. Examples of organic solvents include aliphatic hydrocarbons such as hexane and cyclohexane, and aromatic hydrocarbons such as toluene. The concentration of the surfactant in the organic solvent solution is not particularly limited, for example, 1 mg / mL to 100 mg / mL, 20 mg / mL to 90 mg / mL, or 40 mg / mL to 80 mg / mL.

[0186] Emulsification and dispersion can be carried out using a stirrer, a high-speed rotary shearing stirrer, a colloid mill, a homogenizer, a fluid jet mixer, an ultrasonic emulsifier, a vacuum emulsifier, etc. The particle size of the dispersed droplets can be controlled by adjusting the stirring intensity.

[0187] In the complex preparation step, the W / O type emulsion obtained in the emulsion preparation step is dried to prepare a target molecule (e.g., nucleic acid)-surfactant complex (solid complex or solid particles). The drying method is not particularly limited, and freeze drying and reduced pressure drying can be listed, preferably freeze drying. Water may cause leakage of the target molecule (e.g., nucleic acid) in the preparation. Organic solvents may have adverse effects on organisms. Therefore, it is preferred to substantially completely remove water and organic solvents in the drying step. For example, when measured by a known method, the water content reaches 1% by mass or less, 0.9% by mass or less, 0.8% by mass or less, 0.7% by mass or less, 0.6% by mass or less, 0.5% by mass or less, 0.4% by mass or less, 0.3% by mass or less, 0.2% by mass or less, or 0.1% by mass or less. The complex containing the target molecule (e.g., nucleic acid) and the surfactant thus obtained can be a solid complex or solid particle with a low water content. When the organic solvent is volatile, the organic solvent will naturally be removed in the drying step.

[0188] In the dispersion step, the target molecule (e.g., nucleic acid)-surfactant complex (dispersoid) obtained in the complex preparation step is dispersed in the oil phase (dispersion medium). As a method for dispersing the target molecule (e.g., nucleic acid)-surfactant complex, specifically, the same method as the method exemplified as the method of emulsification and dispersion in the emulsion preparation step can be used. The amount of oil phase used in the dispersion step also depends on the compatibility of the type of surfactant and the oil phase, for example, 1 mL to 200 mL relative to 1 g of the target molecule (e.g., nucleic acid)-surfactant complex. As needed, other ingredients such as a skin penetration enhancer can also be dispersed in the oil phase. In the dispersion step, the composition or percutaneous absorbent of the present embodiment is obtained in the form of a dispersed preparation or a suspension.

[0189] (Preparation of preparations based on ethanol dilution method)

[0190] The preparation of the present invention can also be obtained by ethanol dilution method. In the ethanol dilution method, a composition containing cations and anions or a composition consisting of cations and anions can be used. The cations and anions can be cationic surfactants and anionic surfactants, respectively. The principle of the ethanol dilution method is based on that when ethanol (which can be a lower alcohol) containing water-soluble molecules with ethanol solubility is diluted with water, the molecules that can no longer dissolve will form a complex. Therefore, in the ethanol dilution method, a complex containing cations, anions and target molecules (such as nucleic acids) is formed by mixing a lower alcohol solution containing cations and anions with an aqueous solution containing a target molecule (such as a nucleic acid) and other agents. The obtained complex (dispersed in a low concentration ethanol solution) can be used directly, or it can be freeze-dried and dispersed in an oil phase for use. The obtained complex (dispersed in a low concentration ethanol solution) can be used, for example, to transfect the target molecule (such as a nucleic acid) into the cell of the cell by contacting the cell. The complex that is freeze-dried and dispersed in the oil phase can be used for transdermal administration.

[0191] The lower alcohol is an alcohol having a carbon number of 6 or less, preferably 5 or less, more preferably 4 or less, 3 or less, or 2 or less. Examples of the lower alcohol include methanol, ethanol, 1-propanol, isopropanol, 1-butanol, isobutanol, 2-butanol, and tert-butanol. As the lower alcohol, ethanol can be preferably used.

[0192] Therefore, the present invention provides a composition for forming a complex with a pharmaceutical ingredient, which contains cations and anions. In one embodiment, the composition can be an ionic liquid. Alternatively, the composition can contain a lower alcohol in addition to cations and anions. In one embodiment, the lower alcohol can be ethanol. The cations and anions can use the cations and anions used in the formation of the above-mentioned solid complex, respectively. The cations can preferably be cationic lipids. The anions can preferably be anionic lipids. The cations and anions can be cationic lipids and anionic lipids, respectively. Cationic lipids and anionic lipids act as surfactants, and are therefore used in the same meaning as cationic surfactants and anionic surfactants, respectively. In one embodiment, the composition contains a cationic surfactant, an anionic surfactant and a lower alcohol (preferably ethanol) or is composed of these.

[0193] When a composition containing a cationic surfactant, an anionic surfactant and a lower alcohol (preferably ethanol) or consisting of these is mixed with an aqueous solution containing a target molecule (e.g., a nucleic acid), a lower alcohol aqueous solution containing a complex containing a cationic surfactant, an anionic surfactant and a target molecule (e.g., a nucleic acid) can be instantly obtained. The obtained complex contains particles with a particle size of about 5nm to about 20nm. The obtained complex contains agglomerates containing a plurality of particles with a particle size of about 5nm to about 20nm. The agglomerates can have a particle size of, for example, about 100nm to about 500nm or about 100 to about 300nm. When the lower alcohol is an alcohol with low cytotoxicity such as ethanol or propanol, the obtained lower alcohol solution can directly contact the cells and introduce or transfect the target molecule (e.g., a nucleic acid) into the cells of the cells. The alcohol can also be removed from the obtained solution by treatment such as dialysis. By setting the dialyzed external fluid to an aqueous solution (e.g., physiological saline), an aqueous solution containing the above-mentioned complex (e.g., substantially free of alcohol) can be obtained.

[0194] The obtained lower alcohol solution can be freeze-dried without going through the emulsion formation step, and then the obtained freeze-dried product can be dispersed in the oil phase. Thus, a composition containing a solid complex containing a cationic surfactant, an anionic surfactant and a target molecule (e.g., nucleic acid), containing an oil phase, and the complex is dispersed in the oil phase is obtained. Alternatively, by dispersing the obtained freeze-dried product in an aqueous phase, a composition containing a complex or solid complex containing a cationic surfactant, anionic surfactant and a target molecule (e.g., nucleic acid), containing an aqueous phase, and the complex is dispersed in the aqueous phase is obtained.

[0195] In the above, an organic solvent such as acetone or acetonitrile may be used instead of a lower alcohol. In one embodiment, a lower alcohol is preferably used in the ethanol dilution method, and ethanol is preferably used. In one embodiment, acetone may be used. In one embodiment, acetonitrile may be used.

[0196] The obtained composition can be used as a percutaneous absorbent. The obtained composition can also be used as a composition for percutaneous administration. The obtained composition may also contain anionic surfactants. As described later, anionic surfactants (especially fatty acids) can weaken the skin barrier function. Therefore, further adding anionic surfactants can improve the properties of the composition as a percutaneous absorbent. The obtained composition is applied to the skin, so that the target molecule (such as nucleic acid) can penetrate into the skin. The obtained composition containing the complex and the aqueous phase can be used for the introduction or transfection of the target molecule (such as nucleic acid) into the cell.

[0197] In the above, the nucleic acid may be replaced with a physiologically active substance such as a protein or a low molecular compound. Thus, the physiologically active substance such as a protein or a low molecular compound that replaces the nucleic acid can be effectively administered transdermally.

[0198] In a complex containing a cationic surfactant, an anionic surfactant and a target molecule (e.g., a nucleic acid), the cationic surfactant and the anionic surfactant are contained in an amount of 50 mol% or more, 60 mol% or more, 70 mol% or more, 80 mol% or more, 90 mol% or more, 95 mol% or more, 98 mol% or more, 99 mol% or more, or 100 mol% relative to the surfactant contained. The cationic surfactant and the anionic surfactant are preferably contained in the complex in a ratio of 1:0.5 to 1:2, 1:0.6 to 1:1.5, 1:0.8 to 1:1.25, 1:0.9 to 1:1.11, or about 1:about 1. In one embodiment, the cationic surfactant and the anionic surfactant are in an electrically neutral state in the complex.

[0199] (Composition or percutaneous absorption preparation)

[0200] The composition or percutaneous absorbent of the present embodiment can be formulated by adding other additives. As other additives, for example, excipients, colorants, lubricants, binders, emulsifiers, thickeners, wetting agents, stabilizers, preservatives, solvents, solubilizers, suspending agents, buffers, pH adjusting agents and bases can be listed, and these can be matched with the usual matching amount.

[0201] As excipients, sugars such as white sugar, starch derivatives such as dextrin, cellulose derivatives such as sodium carboxymethylcellulose, and water-soluble polymers such as xanthan gum can be cited. As lubricants, stearic acid metal salts such as calcium stearate and magnesium stearate, lauryl sulfate, and starch derivatives in the above excipients can be exemplified. As binders, for example, the above excipients and macrogol can be cited. Wetting agents are, for example, glycerol. Stabilizers are, for example, alcohols such as methyl paraben, parabens, chlorobutanol, benzyl alcohol, and phenylethyl alcohol, phenols such as benzalkonium chloride, phenol, cresol, thimerosal, anhydrous acetic acid, and sorbic acid. As solvents, water, ethanol, glycerol, etc. can be cited. Suspending agents are, for example, sodium carboxymethylcellulose. As bases, polyethylene glycol, crotamiton, diethyl sebacate, and vaseline can be cited.

[0202] The dispersion or suspension obtained by the above-mentioned manufacturing method can be used directly, or can be mixed with other additives as needed and processed into external preparations such as patches, ointments, lotions, aerosols, plasters, aqueous papules, creams, gels, plasters, reservoir-type patches, skeleton-type patches and tapes by known methods. Thus, a percutaneous absorption preparation can be obtained.

[0203] The dosage of the composition or percutaneous absorption agent of this embodiment can be adjusted according to the patient's age, symptoms, type of disease, etc. For example, generally, the dosage of the composition or percutaneous absorption agent can be adjusted for several weeks to several months in such a manner that the amount of the target molecule (e.g., nucleic acid) reaches 1 μg to 30 mg, preferably 10 μg to 10 mg, and more preferably 30 μg to 3 mg per administration per adult.

[0204] As the applicable object of the composition or percutaneous absorption agent of this embodiment, vertebrates are preferred, and mammals are more preferred. Examples of mammals include domestic animals such as humans, chimpanzees and other primates, dogs, cats, rabbits, horses, sheep, goats, cattle, pigs, rats, mice, and guinea pigs, ornamental animals, and experimental animals. As mammals, humans are particularly preferred. The composition or percutaneous absorption agent of this embodiment can be administered to the subject percutaneously or can be applied to the skin of the subject.

[0205] The composition or percutaneous absorbent of this embodiment disperses a solid target molecule (eg nucleic acid)-surfactant complex in an oil phase, thereby enabling target molecules (eg nucleic acid) that are almost impermeable to the skin to penetrate into the body from the skin as shown in the following examples.

[0206] The composition or percutaneous absorbent of the present embodiment can be used for diseases that can be treated with nucleic acids or nucleic acid drugs. The disease is particularly a disease that can be treated or prevented by targeting RNA, or the progression of the disease can be stopped or suppressed. As the disease, for example, skin diseases such as skin cancer, rheumatoid arthritis and atopic dermatitis can be listed. In addition, the composition or percutaneous absorbent of the present embodiment is also suitable for the percutaneous DDS of RNA vaccines.

[0207] In another embodiment, a method for treating the above-mentioned disease is provided, which includes the step of using the above-mentioned composition or percutaneous absorption agent containing a therapeutically effective amount of a target molecule (e.g., a nucleic acid) on a subject in need of treatment. It should be noted that the therapeutically effective amount of a target molecule (e.g., a nucleic acid) refers to the amount shown as the dosage of the target molecule (e.g., a nucleic acid) in the above-mentioned composition or percutaneous absorption agent, which is a dosage set according to the disease. In addition, in another embodiment, the above-mentioned composition or percutaneous absorption agent for treating the above-mentioned disease is provided.

[0208] In another embodiment, a method for improving the transdermal absorbability of a target molecule (e.g., nucleic acid) is provided. The method includes the step of preparing a complex into an external preparation, wherein the complex is prepared by coating the target molecule (e.g., nucleic acid) with a surfactant to prepare a solid target molecule (e.g., nucleic acid)-surfactant complex, and dispersing the complex in an oil phase. According to this method, the transdermal absorbability of a target molecule (e.g., nucleic acid) that is difficult to use as an external preparation due to low transdermal absorbability and cannot fully exert its effect as an external preparation can be improved.

[0209] In this specification, drugs other than nucleic acids can be used in place of nucleic acids in the above invention. As drugs other than nucleic acids, for example, proteins and compounds (preferably hydrophilic proteins and hydrophilic compounds) can be listed. The protein is not particularly limited, and for example, it can be a protein with a molecular weight of 5kDa to 100kDa (preferably a hydrophilic protein, more preferably a cationic hydrophilic protein). The compound is not particularly limited, and for example, it can be a compound with a molecular weight of 800 to 10000 (preferably a hydrophilic compound, more preferably a cationic hydrophilic compound).

[0210] 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). As proteins, for example, agalsidase β, laronidase, alteplase, papinofop α, urokinase, idurase, cerebella lipase α, monteplasmase, asforase α, imiglucerase, velaglucerase, agalsidase α, alglucosidase α, efglucosidase α, idurase, galactosidase, elosulfatase α, rasburicase, dornase α, asforase α, collagenase, cerebella lipase, condoliase, elapegademase, serripase α, glutarcapase, vedronic acidase α, orlipidase α, coagulation factors (e.g., I, II, III, IV, V, VI, VII, VIII, IX, X, XI, and XII), insulin, somatotropin, erythropoietin, and chemokines can be mentioned. Examples of proteins include 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 factor (FGFs), insulin-like growth factor (IGFs), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor (TGFs), colony stimulating factor (CSFs), erythropoietin (EPO), granulocyte growth factor (GFR), IL-67 (IL-7), IL-83 (IL-8), IL-11 (IL-12), IL-13 (IL-13), IL-17 (IL-18), IL-20 (IL-21), IL-22 (IL-23), IL-24 (IL-25), IL-28 (IL-29), IL-34 (IL-35), IL-36 (IL-37), IL-38 (IL-39), IL-40 (IL-50), IL-61 (IL-71), IL-8 The present invention relates to proteins such as cell colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), interleukin (some interleukins have growth factor activity), bone morphogenetic protein (BMPs), growth differentiation factor (GDF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophic factor (NTs), keratinocyte growth factor (KGF), thrombopoietic factor (TPO)). In addition, serum albumin, ovalbumin, casein, lysozyme and lipase can be listed. These proteins can be used alone or in combination of two or more. Proteins include protein complexes.

[0211] As nucleic acid, DNA or RNA (such as mRNA) can be listed. As the protein encoded by nucleic acid, xenobiotics, such as pathogen-specific components or a part thereof and cancer cell-specific components or a part thereof can be listed. As pathogen, pathogenic viruses, pathogenic microorganisms and pathogenic parasites can be listed. As pathogenic viruses, viruses selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus (for example, severe acute respiratory syndrome-related coronavirus (SARS-CoV), Middle East respiratory syndrome-related 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 virus, poliovirus, rabies virus, John-Cunningham (JC) virus and arbovirus encephalitis virus can be listed. Examples of pathogenic microorganisms include pathogenic bacteria, and examples of pathogenic bacteria include bacteria selected from the group consisting of Chlamydia, Rickettsiales, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, Bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease bacteria. As pathogenic microorganisms, pathogenic fungi can be listed, and as pathogenic fungi, pathogenic fungi selected from the group consisting of Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides braziliensis, Coccidioides immitis, and Histoplasma capsulatum can be listed. As pathogenic parasites, pathogenic parasites selected from the group consisting of Protozoa dysenteriae, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium malariae, Babesia, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma, and Hookworm can be listed. Therefore, in one embodiment of the present invention, mRNA can encode components (preferably surface antigens) of these pathogens or a portion thereof. In particular, it is preferred that the mRNA of the present invention encodes antigens on the surface of pathogens, such as the S protein of the beta coronavirus, for causing the pathogen to infect cells.As cancer, it is possible to list cancer selected from the group consisting of 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, digestive system cancer, Hodgkin or non-Hodgkin tumors, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, renal cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer and head and neck cancer. In addition, in one embodiment of the present invention, mRNA can be these cancer-specific antigens (cancer-specific antigens, preferably cancer-specific surface antigens) or a portion thereof. Cancer-specific antigens, for example, can be a characteristic portion of a neoantigen or a neoantigen. Cancer-specific antigens include, for example, cancer testis antigens (e.g., proteins of the MAGE family, NY-ESO-1), differentiation antigens (e.g., tyrosine kinase of melanoma, Melan-A / MART-1, PSA of prostate cancer), overexpressed cancer antigens (e.g., Her-2 / Neu, survivin, telomerase, and WT-1), cancer-inducing mutants of β-catenin, cancer-inducing mutants of CDK4, MUC-1, especially MUC-1 with a changed glycosylation pattern, and E6 or E7 of human papillomavirus. Regarding these, a person skilled in the art can appropriately select antigens and prepare mRNA suitable for the present invention according to the type of infection and cancer suffered by the subject, and the type of antigen expressed by the cancer. The nucleic acid may also be a nucleic acid encoding the protein exemplified above. mRNA for natural mutant proteins can also be appropriately designed.

[0212] As nucleic acid, other examples include fomivirsen, pegaptanib, mipomersen, edrexin, nusinersen, CpG1018, inotsen, patisilan, voranersen, givosilan, golodisen, vitolasen, inksilan, lumasilan, casimersen, wutrisilan, defibrotide, trabedersen (trabedersen) and tofusen. As nucleic acid, various nucleic acids such as other antisense oligonucleotides, siRNA, shRNA, miRNA, etc. can be used.

[0213] As target molecules, for example, low molecular weight compounds can be cited. As low molecular weight compounds, for example, antioxidants (such as ascorbic acid, isoascorbic acid, vitamin E, catechins, butylated hydroxytoluene, tocopherol, butylated hydroxyanisole, polyphenols, resveratrol (resveratrol 3,5,4'-trihydroxy trans-stilbene), coenzyme Q10, N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyl 1-oxyl (TEMPO), superoxide dismutase (SOD), nicotinamide mononucleotide (NMN), astaxanthin, β-carotene and glutathione can be cited. As low molecular weight compounds, for example, antitumor agents can be cited. As antitumor agents, for example, alkylating agents, anthracyclines, antihormonal agents, aromatic The invention relates to a kind of antitumor agent, for example, erlotinib, bortezomib, disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant, sunitinib, letrozole, mesylate, etc. Alkyl sulfonates such as imatinib, fenasone, oxaliplatin, 5-FU (5-fluorouracil), folinic acid, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carboquinone, metodepa and uredepa; ethyleneimine and methylmelamine (including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide); polyacetyl groups (especially bratacin and bratacinone); camptothecins (including topotecan and irinotecan); bryostatin; caritastatin; CC-1065 (including its a- dolesin, carzeresin and biszeresin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocortical steroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, moxitinot, dolastatin; aldesleukin, duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); cypermethrin; cypermethrin; spongestatin; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II and calicheamicin omega 1 I (Angew Chem. Intl. Edition Engl. 1994 33: 183-186); danimicin (danimicin A); bisphosphonates such as clodronate; esperamicins;and new carcinostatisin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carbicin, carminomycin, carmomycin, chromomycinis, actinomycin D, daunorubicin, detoxorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin, doxorubicin and doxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin C and other mitomycins, mycophenolic acid, noramycin, olive mycin, peplomycin, porfiromycin, puromycin, tri-iron doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, netastatin, daunorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethoate, methotrexate, pteropterin, trimetrexate; fludarabine, 6-mercaptopurine, thiopurine , thioguanine and other purine analogs; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine and imatinib (2-phenylaminopyrimidine derivative) and other pyrimidine analogs; androgens such as calutosterone, drostanolone propionate, cyclothiodine, melastosane, and testolactone; antiadrenergic drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronosyl lactone; aldehyde phosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; amustine; bisantrene; edatrexate; diphosphamide; dimethoate Mecocin; oxyspermone; ilonicet; elliptonium acetate; epothilone; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine and ansamitocin and other maytansinoids; mitoxantrone; mitoxantrone; mopidarol; diamine nitracrine; pentostatin; methamine mustard; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; razoxane; rhizomycin; sizolan; spirogermanium; tenuisporic acid; triazoquinone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A (verracurin A), bacitracin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; piperobroman; gacitocin; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes such as padocetaxel, docetaxel, and docetaxel; levofloxacin; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; norsulfuron; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine; ibandronate; CPT-11;Topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above. As low molecular weight compounds, low molecular weight compounds with physiological activity can be listed. ;

[0214] According to the present invention, a composition for weakening the skin barrier or barrier function containing an anionic surfactant is provided. According to the present invention, an anionic surfactant for weakening the skin barrier or barrier function or its use is provided. According to the present invention, the use of anionic surfactants in the manufacture of a composition for weakening the skin barrier or barrier function is provided. Among the above, the anionic surfactant may be preferably an anionic long-chain lipid. The anionic long-chain lipid may be preferably a fatty acid. The fatty acid may be preferably a long-chain fatty acid, and may more preferably be a long-chain unsaturated fatty acid. As long-chain unsaturated fatty acids, long-chain unsaturated fatty acids containing one or more double bonds can be listed. Long-chain unsaturated fatty acids containing one or more double bonds are preferably cis-type. Long-chain unsaturated fatty acids containing one or more double bonds may be, for example, the fatty acids exemplified above, for example, linoleic acid or oleic acid. In one embodiment, a composition for weakening the skin barrier or barrier function containing an anionic surfactant may contain cations (e.g., cationic lipids) in addition to fatty acids. The composition for weakening the skin barrier or barrier function containing anionic surfactants may contain cations (eg, cationic lipids) and target molecules (eg, nucleic acids) in addition to fatty acids.

[0215] According to the present invention, a composition is provided, which comprises a solid complex containing a surfactant and a drug and an oil phase, wherein the oil phase contains anionic long-chain lipids, and the solid complex is dispersed in the oil phase. The surfactant can preferably use a surfactant having an HLB value of 10 or less.

[0216] The above-mentioned surfactant may preferably be a nonionic surfactant. As the nonionic surfactant, there is no particular limitation, for example, as the nonionic surfactant, polyglycerol condensed ricinoleic acid ester, decaglycerol, glycerol fatty acid ester, polyglycerol fatty acid ester, polyoxyethylene glycerol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene castor oil-hydrogenated castor oil, sucrose fatty acid ester (sucrose stearate, sucrose palmitate, sucrose myristate, sucrose oleate, sucrose laurate, sucrose erucate, sucrose mixed fatty acid ester) etc. can be listed. One can be selected from these to use or a mixture of two or more can be used. As the nonionic surfactant, it is preferably an ester compound with unsaturated fatty acids such as acid and oleic acid as raw materials, and sucrose erucate, sucrose oleate, and sucrose mixed fatty acid ester are more preferably listed. Alternatively, one or more selected from the group consisting of glycerol fatty acid esters, polyglycerol fatty acid esters, polyoxyethylene glycerol fatty acid esters, sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oil, and hydrogenated castor oil may be used.

[0217] Among the above, the surfactant may preferably be a cationic surfactant. As the cationic surfactant, for example, the cationic surfactants exemplified above can be used.

[0218] The present invention will be further specifically described by the following examples, but the present invention is not limited to the examples.

[0219] Example

[0220] Example 1: Ionized surfactant composed of cationic surfactant, anionic surfactant and physiologically active substance Preparation of ionic surfactant-nucleic acid complexes

[0221] In this example, a nucleic acid-surfactant solid complex composed of a cationic surfactant, an anionic surfactant and a nucleic acid was prepared. Hereinafter, unless otherwise specified, a mixture of EDMPC as a cationic surfactant and linoleic acid as an anionic surfactant ([EDMPC][Lin]) was used as the surfactant mixture. This mixture exhibits properties as an ionic liquid at room temperature.

[0222] As a cationic surfactant, 1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine (EDMPC) is used (WO2021 / 192861, which is incorporated herein by reference in its entirety). As an anionic surfactant, linoleic acid (Lin), oleic acid (Ole) or stearic acid (Ste) is used. These cationic and anionic surfactants are both biocompatible.

[0223]

[0224] As the nucleic acid, any of mRNA, antisense oligonucleotide, model protein and model low molecular compound was used. As the mRNA, mRNA containing an open reading frame (884mer) encoding green fluorescent protein in a translatable manner and having a 5' cap and poly A was used.

[0225] Reagents

[0226] 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), linoleic acid (Lin), oleic acid (Ole) or stearic acid (Ste) were purchased from Tokyo Chemical Industry Co., Ltd., 5 mol / L hydrochloric acid, chloroform (super dehydrated), and ammonium formate were purchased from Kishida Chemical Co., Ltd., cyclohexane and acetonitrile were purchased from Fujifilm Wako Pure Chemical Industries, Ltd., ER-290 and L-195 were purchased from Mitsubishi Chemical Products, 4% paraformaldehyde was purchased from Muto Chemical, and Trabedersen was purchased from Eurofins Genomics Corporation.

[0227] Experimental animals

[0228] Mouse skin (Hos: HR-1) used in the in vitro skin permeation test was purchased from Hoshino Laboratory Animal Care Co., Ltd.

[0229] Apparatus and equipment

[0230] Slides and cover slips were purchased from Matsunami Glass Industries, Ltd., and molds were purchased from Fisher Scientific. Freeze dryer (FDU-1110, EYELA), NMR (ECZ400S), Polytron homogenizer (Kinematica), dynamic light scattering measurement device (Zetasizer Nano ZS, Malvern), fluorescence microplate reader (LS-55KG, Perkin Elmer), stirrer (RCH-1000, EYELA), heating stirrer (RSH-6DN, REXIM), confocal laser fluorescence microscope (LSM700, CarlZeiss), Franz diffusion cell (Franz Cell) (Asahi Manufacturing Co., Ltd.), reversed-phase high-performance liquid chromatography device (1260InfinityII), and cryo-microtome (CM1510, Leica) were used.

[0231] First, cationic and anionic surfactants were synthesized according to reported protocols.

[0232]

[0233] (1)EDMPC-SO 3 CF3 Synthesis: 677.94 mg (1 mmol) of DMPC powder and 129 μL (1 mmol) of ethyl trifluoromethanesulfonate were stirred in chloroform solvent, and the reaction was carried out under nitrogen flow at 50° C. for 12 hours.

[0234] (2) Synthesis of EDMPC-Cl: Prepare the obtained EDMPC-SO 3 CF 3 After the salt was completely dissolved in chloroform, 5 mL (1 mmol) of 0.2 N hydrochloric acid was added and stirred, and then the transparent aqueous phase (containing excess hydrochloric acid) was removed by washing with Milli-Q water, followed by freeze drying for 24 hours.

[0235] (3) Synthesis of ionic surfactant mixture: 100 mg of the obtained EDMPC-Cl salt was dispensed into three vials, dissolved in chloroform, and 42 μL of linoleic acid, 42 μL of oleic acid, and 38 mg of stearic acid were added respectively, and stirred (molar ratio 1:1). The reaction was carried out under light-shielding conditions at 50°C for 12 hours. The solution was then freeze-dried for 24 hours to obtain three ionic surfactant mixtures [EDMPC][Lin], [EDMPC][Ole], and [EDMPC][Ste], respectively.

[0236] (4) Identification of ionic surfactant mixtures: For the synthesized ionic liquids, chloroform-d was used as the solvent. 1 H-NMR determination.

[0237] A mixture of three ionic surfactants: [EDMPC][Lin], [EDMPC][Ole] and [EDMPC][Ste] were prepared. 1 H-NMR spectrum is shown in Figure 1 The integrated values ​​were analyzed based on the proton of b, and the results were consistent with the theoretical values, so it was confirmed that a mixture of three ionic surfactants was synthesized at a ratio of cation:anion = 1:1.

[0238] The ionic surfactant mixture containing the cationic surfactant and the anionic surfactant obtained above is mixed with nucleic acid to form an emulsion, which is then frozen and stored and then dispersed in an oil phase to obtain a dispersion solution containing a solid complex of nucleic acid and the ionic surfactant mixture as a dispersoid and an oil phase as a dispersion medium. Specifically, as described below.

[0239] (1) Formation of emulsion: 1 mg of Trabedersen (ssDNA) was dissolved in 1 mL of Milli-Q water. In addition, 50 mg of a mixture of three ionic surfactants was dissolved in 2 mL of cyclohexane. These aqueous solutions and cyclohexane solutions were stirred at 26,000 rpm for 2 minutes using a Polytron homogenizer to form an emulsion.

[0240] (2) Removal of the inner aqueous phase and the oil phase: The emulsion was immediately immersed in liquid nitrogen for 20 minutes, frozen, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ionic surfactant-nucleic acid solid complex.

[0241] (3) Dispersion in oil: The above-mentioned ionic surfactant-nucleic acid solid complex is dispersed in 1 mL of an oily base IPM having a high transdermal permeation promoting effect to obtain a dispersion solution containing the ionic surfactant-nucleic acid solid complex at a final concentration of 1 mg / mL.

[0242] The physical properties of the prepared ionic surfactant-nucleic acid solid complex were evaluated by visual observation and dynamic light scattering (DLS) measurement. In the DLS measurement, the sample was diluted 100 times with IPM and the concentration was 0.01 mg / mL. The obtained dispersion solutions were all transparent and had uniform optical properties (refer to Figure 3A ). According to DLS measurement, all of them contain nanoparticles with a single peak and a hydrodynamic particle size of about 100nm to 200nm (refer to Figure 3B The solid composite was stable in the dispersion for 28 days ( Figure 3C ).

[0243] The mass ratio of nucleic acid to surfactant was changed to 1:10, 1:20, 1:30, 1:40, 1:50 and 1:100, and the particle size was measured and the long-term stability was evaluated. It should be noted that this study was conducted using only [EDMPC][Lin] from the three surfactant mixtures. The results of the DLS measurement showed that a single peak was shown at any mixing ratio, which is suitable for the preparation of ionic surfactant-nucleic acid solid complexes (refer to Figure 4 ).

[0244] A dispersion containing a conventional surfactant-nucleic acid solid complex was prepared in the following manner.

[0245] (1) Emulsion formation: 1 mg of Trabedersen (ssDNA) was dissolved in 1 mL of Milli-Q water. 50 mg of ER-290 was dissolved in 2 mL of cyclohexane. The aqueous solution and cyclohexane solution were stirred at 26,000 rpm for 2 minutes using a Polytron homogenizer to form an emulsion.

[0246] (2) Removal of the inner aqueous phase and the oil phase: The emulsion was immediately immersed in liquid nitrogen for 20 minutes, frozen, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ER-290-nucleic acid solid complex.

[0247] (3) Dispersion in oil: The above-mentioned ER-290-nucleic acid solid complex was dispersed in 1 mL of IPM, an oily base having a high transdermal permeation promoting effect, to obtain a dispersion having a final concentration of 1 mg / mL (existing type).

[0248] A percutaneous permeation experiment was performed.

[0249] (1) Preparation of skin slices: The hairless mouse skin was thawed and cut into pieces of approximately 2 cm × 2 cm in size.

[0250] (2) Preparation of Franz diffusion cell: Vertical Franz diffusion cell (effective area 0.785cm 2 ) was added to the receptor chamber of the cell, 4 mL of PBS was added, and after the mouse skin was placed with the SC side facing the donor chamber, 1 mL of PBS was further added to expel the air in the Franz diffusion cell.

[0251] (3) Preparation addition: 200 μL of each sample was added to each supply chamber while maintaining the temperature at 32.5° C. and stirring with a stirrer.

[0252] (4) Washing: After 6 hours, the mouse skin was removed and washed four times on the stratum corneum side and once on the opposite side with 500 μL of a washing solution (1×TE buffer:acetonitrile=1:1).

[0253] (5) Extraction: The washed skin tissue was cut into 16 equal parts, placed in a tube, immersed in 500 μL of extraction solution (PBS), and shaken for 24 hours.

[0254] (6) Quantitative analysis: The extract was filtered and then quantitatively analyzed by HPLC (see Table 1).

[0255] [Table 1]

[0256]

[0257] The skin permeation of various samples is shown in Figure 5. This result shows that the dispersion of the ionic surfactant-nucleic acid solid complex obtained this time has improved the transdermal permeability of nucleic acid compared with the existing dispersion. As a reason for the improved permeability, it can be cited that the liquid crystal structure of the stratum corneum is destroyed due to the presence of cationic surfactants and anionic surfactants. It is believed that the particle size of the obtained solid complex is smaller than that of the existing solid complex, which may also contribute to the improvement of skin permeability.

[0258] In addition, among the three dispersions, the [EDMPC][Lin] solid composite had the highest skin permeation rate. This is believed to be because linoleic acid, an anionic fatty acid, has two cis-type C=C bonds and is bulky, and thus has a higher ability to disrupt stratum corneum lipids than oleic acid and stearic acid.

[0259] In the above, a mixture containing anionic surfactants and cationic surfactants is used as the ionic surfactant mixture. - As an ionic surfactant mixture containing anions and cations, a dispersion preparation containing an ionic surfactant-nucleic acid solid complex was prepared using [EDMPC][Cl] as an ionic liquid, [Cho][Ole] as another hydrophilic ionic liquid, and DES [DMPC][Lin] which is a simple mixture of DMPC and linoleic acid. The results showed that a stable dispersion preparation containing an ionic surfactant-nucleic acid solid complex could be prepared in any case (refer to Figure 6 ). It was found that the stability of the composite of the dispersion preparation containing the ionic surfactant-nucleic acid solid composite prepared using [Cho][Ole] was relatively low due to the low solubility of the ionic surfactant mixture in cyclohexane. The permeability of the obtained dispersion preparation to the skin of hairless mice was evaluated in vitro using a Franz diffusion cell as described above. As a result, in any case, the permeability of nucleic acid to the skin was good (refer to Figure 7). In particular, in the case of a dispersed preparation ([EDMPC][Lin]) containing an ionic surfactant-nucleic acid solid complex containing a cationic surfactant and an anionic surfactant, excellent permeability of nucleic acid to the skin was confirmed. In the case of [DMPC][Lin], although the effect of improving the permeability of nucleic acid to the skin was confirmed, the improvement effect was limited. DMPC is charge neutralized within the molecule, indicating that removing the negative charge in DMPC and becoming cationic is important for improving permeability to the skin. In addition, in the case of choline and Ole, the effect of improving permeability to the skin was also confirmed, but the improvement effect was limited, indicating that it is preferred that the cation has a long-chain fatty chain. It should be noted that when the fatty acid is changed to propionic acid, the effect of improving permeability to the skin is also weak, indicating that it is preferred that the anionic lipid also has a long-chain fatty acid. Lipid mixtures such as [EDMPC][Lin] and [EDMPC][Ole] show the properties of ionic liquids. It should be noted that when the same test was performed on pig skin, [EDMPC][Lin], [EDMPC][Ole], and [EDMPC][Ste] all showed high permeability of nucleic acid to the skin compared to the solid complex prepared with ER-290 or PBS.

[0260] The permeation state of the dispersed preparation containing the ionic surfactant-nucleic acid solid complex containing anionic surfactant and cationic surfactant into the skin was analyzed under a fluorescence microscope. For this purpose, the dispersed preparation containing the ionic surfactant-nucleic acid solid complex was prepared from a fluorescent labeled ionic surfactant and a fluorescent labeled nucleic acid.

[0261] Ionic surfactant mixture containing cationic surfactant and anionic surfactant Skin permeability enhancement effect

[0262] The skin permeability-enhancing effect of an ionic surfactant mixture containing a cationic surfactant and an anionic surfactant was confirmed.

[0263] (1) Emulsion formation: 1 mg of Trabedersen (ssDNA) was dissolved in 1 mL of Milli-Q water. 50 mg of ER-290 was dissolved in 2 mL of cyclohexane. These aqueous solutions and cyclohexane solutions were stirred at 26,000 rpm for 2 minutes using a Polytron homogenizer to form an emulsion.

[0264] (2) Removal of the inner aqueous phase and the oil phase: The emulsion was immediately immersed in liquid nitrogen for 20 minutes, frozen, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ER-290-nucleic acid complex.

[0265] (3) Dispersion in oil: The above-mentioned ER-290-nucleic acid complex was redispersed in IPM without an ionic surfactant mixture and in IPM containing 10 mg of an ionic surfactant mixture, respectively, to obtain a dispersion containing a solid complex with a final nucleic acid concentration of 1 mg / mL.

[0266] The results showed that the addition of an ionic surfactant mixture containing a cationic surfactant and an anionic surfactant to the preparation increased the permeability of the preparation ingredients to the skin (refer to Figure 8 ).

[0267] Disruption of stratum corneum lipid structure caused by ionic surfactant mixtures

[0268] Thaw the frozen YMP skin (back) at room temperature. Heat to 60°C in an aluminum incubator for 2 minutes and peel off the epidermis with tweezers. With the stratum corneum side facing up, float the epidermis sheet in a 0.25% trypsin solution and let it stand at room temperature for 24 hours. Remove the living epidermis with a cotton swab and dry for more than 24 hours to obtain a stratum corneum sheet. Cut the stratum corneum sheet into 0.5 cm squares and wrap it in plastic wrap until use. Add a mixture of three ionic surfactants [EDMPC][Lin], [EDMPC][Ole] and [EDMPC][Ste] directly to the stratum corneum sheet, respectively. After standing at 32.5°C for 1 hour, wash the stratum corneum sheet thoroughly with 20% ethanol and dry it for 24 hours. Perform FT-IR measurement on the stratum corneum sheet and observe the 2920cm -1 and 2850cm -1 CH appears nearby 2 Symmetric stretching vibration (symCH 2 ) and CH 2 Asymmetric stretching vibration 2 ) peak shift.

[0269] As a result, the peak shift was the largest when [EDMPC][Lin] was added, indicating that the liquid crystal structure of the stratum corneum lipids was the most disordered (refer to Fig. 9 ). The reason why the liquid crystal structure is most disordered when linoleic acid is used as an anion is believed to be because the lipid molecule itself has the largest volume due to its two cis-type C=C bonds. Akinshina et al. evaluated the interaction between linoleic acid and intercellular lipids through molecular simulation and disclosed that linoleic acid with cis-type C=C bonds is actually embedded in the lamellar structure, and the regular structure is destroyed (refer to Fig.10)(Anna Akinshina et al., Phys. Chem. Chem. Phys., 18, 17446-17460(2016)). This result is also related to the fact that [EDMPC][Lin]-S / O has the highest permeation amount among the three ionic surfactant mixtures. Therefore, it is believed that the ionic surfactant in the ionic surfactant-nucleic acid solid complex is also beneficial to improve the permeability of nucleic acid to the skin.

[0270] Example 2: Ethanol dilution method

[0271] The ionic surfactant-nucleic acid solid complex is prepared by a process different from that described above.

[0272] Reagents

[0273] 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), linoleic acid, oleic acid, stearic acid, ethyl trifluoromethanesulfonate, isopropyl myristate, and Span-20 were purchased from Tokyo Chemical Industry Co., Ltd. 5 mol / L hydrochloric acid, chloroform (super dehydrated), ammonium formate, and ethanol (99.5) were purchased from Kishida Chemical Co., Ltd. Cyclohexane and acetonitrile were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Co., Ltd., ER-290, L-195 were purchased from Mitsubishi Chemical Corporation, 4% paraformaldehyde was purchased from Muto Chemical, Trabedersen and FAM labeled Trabederse n was purchased from Furusu Co., Ltd., PNA was purchased from Furusu Co., Ltd., 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole MTT medium was purchased from Wako Pure Chemical Industries, Ltd.

[0274] Experimental animals and cells

[0275] Mouse skin (Hos: HR-1) used in the in vitro skin permeation test was purchased from Hoshino Laboratory Animal Care Co., Ltd., B16 melanoma cells were purchased from RIKEN, and human three-dimensional cultured epidermis LabCyte EPI-MODEL was purchased from Diapan Technological Engineering Co., Ltd.

[0276] Apparatus and equipment

[0277] Slides and cover slips were purchased from Matsunami Glass Industries Co., Ltd., and molds were purchased from Fisher Scientific. Freeze dryer (FDU-1110, EYELA), Polytron homogenizer (Kinematica), dynamic light scattering measurement device (Zetasizer Nano ZS, Malvern), fluorescence microplate reader (LS-55KG, Perkin Elmer), heating stirrer (RSH-6DN, REXIM), confocal laser microscope (LSM700, Carl Zeiss), Franz diffusion cell (Asahi Manufacturing Co., Ltd.), reversed-phase high-performance liquid chromatography device (1260 InfinityII), cryostat (CM1510, Leica), transmission electron microscope (JEM-2010, JEOL), small-angle X-ray scattering device (Nanoviewer, Rigaku) ​​were used.

[0278] In Example 1, the dispersion was obtained by mixing an ionic surfactant dissolved in a cyclohexane solution with an aqueous solution containing nucleic acid to form an emulsion, followed by freeze drying, and finally dispersing the obtained solid complex in an oil phase. In this example, an ionic surfactant-nucleic acid complex was formed by an ethanol dilution method, followed by freeze drying, and finally dispersing the obtained solid complex in an oil phase ( Fig.11 ). The ethanol dilution method does not require an emulsion formation step, and can minimize damage to the sample. In this example, it was verified whether solid composite particles can be obtained using such a method.

[0279] (1) Formation of ionic surfactant-nucleic acid solid complex: 1 mg of Trabedersen (ssDNA) was dissolved in 5 mL of Milli-Q water. Separately, 50 mg of the ionic surfactant mixture was dissolved in 1 mL of 99.5% ethanol. These aqueous solutions and ethanol solutions were mixed and gently stirred at room temperature for 6 hours to spontaneously form an ionic surfactant-nucleic acid solid complex.

[0280] (2) Removal of the inner aqueous phase and the oil phase: The water / ethanol solution was immersed in liquid nitrogen for 20 minutes, frozen, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ionic surfactant-nucleic acid complex.

[0281] (3) Dispersion in oil: The above-mentioned ionic surfactant-nucleic acid solid complex is dispersed in 1 mL of IPM, an oily base having a high transdermal permeation promoting effect, so that the final concentration of nucleic acid is 1 mg / mL to obtain a dispersion of the solid complex.

[0282] The above steps yielded an ionic surfactant-nucleic acid solid complex. As surfactants, ER-290 (nonionic surfactant) and a mixture of a cationic surfactant and an anionic surfactant (a mixture of EDMPC and linoleic acid) were used. When the obtained dispersion was analyzed by DLS measurement, the particle size distribution was as follows: Fig.12 When ER-290 having no charge was used as a surfactant, the particle size distribution varied in 10 independent experiments, but when a mixture of EDMPC and linoleic acid was used, the solid composite in the obtained dispersion showed a constant particle size distribution.

[0283] A dispersion containing an ionic surfactant-nucleic acid solid complex prepared as shown in Example 1 (here, the ionic surfactant is composed of a mixture of EDMPC and linoleic acid) (the previous method) was prepared, and the skin permeability was compared with the dispersion containing an ionic surfactant-nucleic acid solid complex prepared by the above-mentioned ethanol method (here, the ionic surfactant is composed of a mixture of EDMPC and linoleic acid) (the new method). First, the particle size distribution of the obtained solid particles was as follows: Fig.13 The shown are equivalent.

[0284] Each dispersion was applied to the skin to investigate the amount of absorption into the skin and the amount of permeation into the skin. Fig.14 As shown, the previous method and the new method showed equivalent skin absorption and skin penetration.

[0285] The dispersion obtained by the new method was evaluated for stability over 4 weeks. The particle size and PDI were measured by DLS immediately after preparation and after 4 weeks. Fig.15A As shown in Figure 2, no change was observed even after 4 weeks. Fig. 15B As shown, even after 4 weeks, no changes were shown.

[0286] The composition ratio of the nucleic acid and ionic surfactant mixture when forming the solid complex was changed. Specifically, the ionic surfactant-nucleic acid complex was re-formed with three composition ratios of mass ratio (N:P ratio when the anions in the lipids were ignored) of 1:3 (1:1), 1:15 (1:5), and 1:30 (1:10), and the particle size / PDI and skin permeability, and the particle size / zeta potential in water were compared.

[0287] The hydrodynamic particle size and PDI obtained by DLS are Fig.16 As shown. Fig.16 As shown in Figure 2, when the amount of surfactant relative to nucleic acid increases, the particle size tends to decrease. Then, the skin permeability and skin permeability of the obtained ionic surfactant-nucleic acid complex were investigated. The results are shown in Figure 2. Fig.17 As shown, as the amount of surfactant relative to nucleic acid increases, the permeability and permeability to the skin increase, but when the amount of surfactant exceeds a certain value, the permeability and permeability to the skin decrease.

[0288] The dispersion prepared by the ethanol method was further analyzed by DLS ( Fig.18 ) and Zeta potential analysis using a Zeta potential analyzer ( Fig.19 ). The zeta potentials were all positive, indicating that the nucleic acid may be enclosed in a lipid double membrane.

[0289] Using Trabedersen as a nucleic acid, EDMPC as a cationic surfactant, and linoleic acid as an anionic surfactant, a dispersion containing an ionic surfactant-nucleic acid solid complex was prepared by an ethanol dilution method. The aqueous solution containing nucleic acids and the ethanol solution containing ionic surfactants were mixed, frozen in liquid nitrogen (0 seconds), stirred with a vortex mixer for 10 seconds, frozen in liquid nitrogen (10 seconds), stirred with a stirrer for 10 minutes or 6 hours, frozen in liquid nitrogen, and then freeze-dried and dispersed in 1 mL of an oily substrate IPM.

[0290] According to the analysis of particle size distribution (refer to Fig. 20 ) showed that ionic surfactants instantly formed ionic surfactant-nucleic acid solid complexes when mixed with nucleic acids.

[0291] The relationship between the mixing temperature of an aqueous solution containing nucleic acid and an ethanol solution containing an ionic surfactant in an ethanol dilution method and the particle size distribution of the obtained ionic surfactant-nucleic acid solid complex was investigated. Fig.21 As shown, the particle size distribution of the obtained ionic surfactant-nucleic acid solid complex does not change with temperature.

[0292] In the ethanol dilution method, the ratio of the mixed aqueous solution to the ethanol solution was set to 1:5 or 1:10, and the relationship with the particle size distribution of the obtained ionic surfactant-nucleic acid solid complex was investigated. Fig. 22 As shown, the particle size distribution does not change depending on the mixing ratio of the aqueous solution and ethanol.

[0293] In the ethanol dilution method, the cationic surfactant was set to EDMPC, and the anionic surfactant was set to linoleic acid, oleic acid, or stearic acid, and the relationship with the particle size distribution of the obtained ionic surfactant-nucleic acid solid complex was investigated. Fig.23 As shown, when any anionic surfactant was used, an almost unimodal particle size distribution was exhibited.

[0294] In the ethanol dilution method, the mixed cation was set to choline (choline does not have a long fatty chain) and the anionic surfactant was set to oleic acid, and the particle size distribution and zeta potential of the obtained ionic surfactant-nucleic acid solid complex were investigated. Fig.24 As shown, although the particle size distribution showed a single peak, the zeta potential of the particles was negative. This suggests that the nucleic acid may not be included in the particles if a cation with a small carbon number such as choline is used.

[0295] Various ionic surfactant-nucleic acid solid complexes were prepared by ethanol dilution method and the skin permeability and permeability were evaluated. Fig.25 As shown. Fig.25 In addition, when a cationic surfactant is used as a cation, skin permeability is improved (refer to Fig.25 ).

[0296] Example 3: Peptide Nucleic Acid

[0297] A dispersion containing an ionic surfactant-peptide nucleic acid solid complex was obtained in the above-described manner by the ethanol dilution method.

[0298] (1) PNA aqueous solution (1 mg nucleic acid / 5 mL Milli-Q water) was added to an ethanol solution (5 mg / 1 mL ethanol) in which [EDMPC] and [Lin] were dissolved.

[0299] (2) The mixture was gently stirred with a stirrer at room temperature for 6 hours to spontaneously form an ionic surfactant-nucleic acid complex.

[0300] (3) After freeze-drying for 24 hours to remove the internal aqueous phase and oil phase, the mixture was dispersed in isopropyl myristate (IPM) to prepare an ionic surfactant-PNA complex preparation (final concentration 1 mg / mL).

[0301] The particle size distribution of the composite in the obtained dispersion was measured by DLS, which showed a unimodal particle size distribution (see Fig.26 ). In addition, it is stable even for 14 days of storage (refer to Fig.26 ).

[0302] Example 4: Ionic Surfactant-Protein Complex

[0303] As protein, insulin is used.

[0304] (1) An aqueous solution of insulin (1 mg of insulin / 5 mL of water) was added to an ethanol solution (50 mg / 1 mL of ethanol) in which [EDMPC] [Lin] was dissolved.

[0305] (2) The mixture was stirred gently with a stirrer at room temperature for 6 hours to spontaneously form an ionic surfactant-protein complex.

[0306] (3) After freeze-drying for 24 hours to remove the internal aqueous phase and oil phase, the mixture was dispersed in isopropyl myristate (IPM) to prepare a dispersion containing an ionic surfactant-protein solid complex (final concentration 1 mg / mL).

[0307] The particle size distribution was measured by DLS measurement, and the obtained composite showed a unimodal particle size distribution (refer to Fig. 27 The obtained complex was also stable for 14 days of storage (ref. Fig. 27 ).

[0308] As protein, ovalbumin was used.

[0309] (1) An OVA aqueous solution (OVA 1 mg / Milli-Q water 5 mL) was added to an ethanol solution (5 mg, 10 mg, 25 mg, 50 mg / ethanol 1 mL) in which [EDMPC] and [Lin] were dissolved.

[0310] (2) The mixture was stirred gently with a stirrer at room temperature for 6 hours to spontaneously form an ionic surfactant-protein complex.

[0311] (3) The mixture was freeze-dried for 24 hours to remove the inner aqueous phase and the oil phase, and then redispersed in isopropyl myristate (IPM) (with 0, 5, 10, and 25 mg of Span 20 added as an auxiliary surfactant) to prepare a dispersion containing an ionic surfactant-protein solid complex (final concentration 1 mg / mL).

[0312] As a result, the obtained complex did not show a unimodal particle size distribution. Here, when an ionic surfactant mixture containing Span 20 as an auxiliary surfactant was used, a complex with a unimodal particle size distribution was obtained by setting the mass ratio of ovalbumin:ionic surfactant mixture:Span 20 to 1:25:25. Ovalbumin is a protein of about 45 kDa, which is much larger than insulin. The use of an auxiliary surfactant is beneficial for forming a complex with large proteins. The permeability of the obtained complex to the skin was studied. Fig.28 As shown, the obtained complex significantly improves the permeability of ovalbumin to the skin.

[0313] Example 5: Structure of the Ionic Surfactant-Nucleic Acid Solid Complex Obtained by the Ethanol Dilution Method

[0314] (1) Formation of ionic surfactant-nucleic acid complex: 1 mg of Trabedersen (ssDNA) was dissolved in 5 mL of Milli-Q water. In addition, 50 mg of the ionic surfactant mixture was dissolved in 1 mL of 99.5% ethanol. These aqueous solutions and ethanol solutions were mixed and gently stirred at room temperature for 6 hours, thereby spontaneously forming an ionic surfactant-nucleic acid complex.

[0315] (2) Removal of the inner aqueous phase and the oil phase: The water / ethanol solution was immersed in liquid nitrogen for 20 minutes to freeze, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ionic surfactant-nucleic acid solid complex.

[0316] Example 6: Electron microscopic observation of the composite

[0317] The sample to be observed was prepared in the following manner.

[0318] Sample 1: Composite based on high-speed stirring process (IPM)

[0319] (1) Emulsion formation: Dissolve 1 mg of Trabedersen (ssDNA) in 1 mL of Milli-Q water. Dissolve 50 mg of [EDMPC] [Lin] in 2 mL of cyclohexane. Stir the aqueous solution and cyclohexane solution at 26,000 rpm for 2 minutes using a Polytron homogenizer to form an emulsion.

[0320] (2) Removal of the inner aqueous phase and the oil phase: The emulsion was immediately immersed in liquid nitrogen for 20 minutes to freeze, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ionic surfactant-nucleic acid complex.

[0321] (3) Dispersion in oil: The above-mentioned ionic surfactant-nucleic acid solid complex was dispersed in 1 mL of IPM to obtain a dispersion with a final concentration of 1 mg / mL.

[0322] Sample 2: Composite based on ethanol dilution method (ethanol-water solution)

[0323] Formation of ionic surfactant-nucleic acid complex: 1 mg of Trabedersen (ssDNA) was dissolved in 5 mL of Milli-Q water. In addition, 50 mg of [EDMPC] [Lin] was dissolved in 1 mL of 99.5% ethanol. These aqueous solutions and ethanol solutions were mixed and gently stirred at room temperature for 6 hours to spontaneously form an ionic surfactant-nucleic acid complex.

[0324] Sample 3: Ethanol dilution based composite (IPM)

[0325] (1) Formation of ionic surfactant-nucleic acid complex: 1 mg of Trabedersen (ssDNA) was dissolved in 5 mL of Milli-Q water. In addition, 5 mg of [EDMPC] [Lin] was dissolved in 1 mL of 99.% ethanol. These aqueous solutions and ethanol solutions were mixed and gently stirred at room temperature for 6 hours to spontaneously form an ionic surfactant-nucleic acid complex.

[0326] (2) Removal of the inner aqueous phase and the oil phase: The water / ethanol solution was immersed in liquid nitrogen for 20 minutes to freeze, and then treated with a freeze dryer for one day and night to remove the inner aqueous phase and the oil phase, thereby preparing an ionic surfactant-nucleic acid solid complex.

[0327] (3) Dispersion in oil: The above-mentioned ionic surfactant-nucleic acid solid complex was dispersed in 1 mL of IPM to obtain a dispersion with a final concentration of 1 mg / mL.

[0328] TEM observation

[0329] Each sample was appropriately diluted with cyclohexane and cast on a carbon film grid for a scanning transmission electron microscope (STEM), dried for 3 hours, and then observed at a magnification of 2000× and an accelerating voltage of 120 kV.

[0330] The results are as follows Fig.29 As shown. In the case of the ionic surfactant-nucleic acid solid complex obtained by the high-speed stirring process (cyclohexane method), spherical particles with a particle size of about 10 nm were observed to aggregate to form aggregates with a particle size of about 200 nm. In the case of the ionic surfactant-nucleic acid complex in the ethanol aqueous solution obtained by the ethanol dilution method, disc-shaped structures with a diameter of about 10 nm were observed to aggregate to form a structure of about 600 nm ( Fig.29 In the case of the ionic surfactant-nucleic acid solid complex obtained by the ethanol dilution method, it was observed that the disc-shaped structures with a particle size of about 10 nm aggregated to form aggregates with a particle size of about 200 nm ( Fig.29 ).

[0331] The ionic surfactant-nucleic acid solid complex was prepared in the following manner. Specifically, FAM-labeled Trabedersen was used as the nucleic acid, and a freeze-dried product was obtained by ethanol dilution as described in this example and dispersed in IPM to obtain a dispersion containing the ionic surfactant-nucleic acid solid complex. The dispersion was then further dispersed in Milli-Q water to obtain a dispersion solution with a nucleic acid concentration of 1 mg / mL.

[0332] The results are as follows Fig.30 As shown in the figure, the ionic surfactant-nucleic acid solid complex was efficiently introduced into the cells. Considering the production process, the complex is considered to be an S / O / W emulsion. The complex was applied to the skin and the permeability to the skin was confirmed. The skin was collected, fixed and sliced, and the FAM signal was confirmed under a fluorescence microscope. The results are shown in the figure. Fig.31 As shown, it was confirmed that the ionic surfactant-nucleic acid solid complex entered the basal layer cells and dermal cells.

[0333] Example 8: Introduction of EGFP mRNA into cells and gene expression

[0334] A complex of a cationic surfactant, an anionic surfactant and mRNA is formed, and transfection of mRNA into cells and expression of mRNA are observed. EGFP is used as mRNA, and expression is detected using fluorescence from EGFP as an indicator.

[0335] Using a linear double-stranded DNA containing a T7 promoter sequence and encoding green fluorescent protein (EGFP) as a template, EGFP mRNA was synthesized using the T7 mScriptTM Standard mRNA production system (CellScript) kit. The obtained EGFP mRNA was dissolved in Milli Q water to a concentration of 20 ng / μl.

[0336] [EDMPC][Lin] was dissolved in 99.5% ethanol to a concentration of 6 μg / μl. 10 μl of the EGFP mRNA aqueous solution and 1 μl of the [EDMPC][Lin] ethanol solution were mixed and gently vortexed to form a cationic surfactant-anionic surfactant-mRNA complex.

[0337] B16 melanoma cells were cultured at 1×10 4 The cells were inoculated into a multi-well plate (Matsunami) at a rate of 100 cells / well. The next day, when the cells reached 70% to 80% confluence, the medium was replaced with serum-free medium, and then a cationic surfactant-anionic surfactant-mRNA complex was added to the medium at a rate of 200 ng / well of mRNA. The culture medium was incubated at 5% CO 2 , cultured in an incubator at 37°C, and observed the green fluorescence from EGFP using a confocal fluorescence microscope (200 times) 48 hours later.

[0338] The results are as follows Fig.32 As shown. Fig.32As shown, fluorescence from green fluorescent protein was not observed from NonTreat cells. On the other hand, in the case of a drug delivery system using a cationic surfactant and an anionic surfactant, green fluorescence was observed after 48 hours, indicating that mRNA was introduced into the cell and EGFP was expressed. It can be seen from this that even a long and unstable nucleic acid such as mRNA can be introduced into the cell in a complete state. When utilizing the ethanol dilution method, the emulsion formation step can be omitted, and lipid-nucleic acid complexes can be formed by simply mixing an aqueous solution containing nucleic acid and a low-grade alcohol solution containing a surfactant, and the resulting complex is in a state dispersed in an aqueous solution, so it can be added to a culture medium. The complex in contact with cells in the culture medium is taken into the cell. In the case of this method, a lipid having high solubility to alcohol and low solubility to water can be preferably used.

[0339] Example 9: Skin Irritation

[0340] The human three-dimensional cultured epidermal model (LabCyte EPI-MODEL trademark) was used to conduct an in vitro skin irritation test. LabCyte EPI-MODEL (LabCyte EPI model) refers to a human three-dimensional cultured epidermal model formed by multilayer culture of normal human epidermal cells, which was developed as an alternative material for skin irritation tests using experimental animals. Since human cells are used, there is no species difference compared to animal experiments, and there is less fluctuation between wells and batches, so test results with high reproducibility can be obtained. Here, the 24-well LabCyte EPI-MODEL24 is used to evaluate the skin irritation in 15 minutes of exposure for PBS (negative control), IPM (negative control), SDS (positive control), a nucleic acid preparation using a mixture of 3 ionic surfactants and [Cho][Ole] as a surfactant (ethanol dilution method), and an ionic surfactant-nucleic acid solid complex based on [EDMPC][Lin].

[0341] (1) Pre-culture

[0342] After heating the test medium in a thermostatic bath (37°C), add 500 μL / well to a 24-well plate. Use sterile tweezers to take out the culture cup containing the human epidermis model and transfer it to the well containing the test medium. At this time, since the culture cup is initially on the agar medium, it is slowly taken out in a way that allows air to enter between the culture cup and the agar medium. When agar medium is attached to the outside of the culture cup, it is carefully removed with tweezers. In addition, transfer to a new well in a way that does not touch the surface of the epidermis and avoids bubbles from entering the bottom of the culture cup. After that, pre-culture in an incubator for one night (15-30 hours).

[0343] (2) Sample administration and cleaning

[0344] 100 μL of each sample was carefully added to the center of the human epidermis model and exposed for 15 minutes (n=3). When the sample did not spread throughout the epidermis, the test plate was covered with a lid and the side of the plate was gently tapped. Then, the sample on the epidermis model was removed, and the culture cup was washed with 100 μL of PBS at 15 times / well (after the last wash, only the outside of the culture cup was wiped with a sterile cotton swab to remove the PBS).

[0345] (3) Post-cultivation

[0346] The test medium heated in a thermostatic chamber (37° C.) was added to the new wells at 1 mL / well, and the washed culture cup was transferred to the new wells. The culture was then cultured in an incubator for 24 hours to perform post-culture.

[0347] Next, the MTT assay was performed.

[0348] Preparation of 5.0 mg / ml 3-(4,5-dimethylthiazol-2-thiazole)-2,5-diphenyl-2H-tetrazolyl Bromide (MTT) / DMEM medium (MTT medium) was filter sterilized. 500 μL / well of this MTT medium was added to new wells, and the culture cup was transferred thereto. Then, the reaction was allowed to proceed in an incubator for 3 hours.

[0349] Extraction of MTT reaction product (formazan)

[0350] After the MTT reaction, the human epidermal tissue in the culture cup was taken out with forceps and immersed in 300 μL of isopropanol in a cool and dark place overnight (15 hours or more) to extract the pigment.

[0351] Absorbance measurement and viable cell count

[0352] The absorbance of each extract at 570 and 650 nm was measured using an ELISA instrument. Then, the viable cell rate was calculated using the following formula, with the cell viability of the negative control (PBS) set to 100%:

[0353] Measured value = [absorbance of sample (570nm) - absorbance of sample (650nm)] - [absorbance of blank (570nm) - absorbance of blank (650nm)]

[0354] Viable cell rate (%) = (average of the measured value of the test substance) / (average of the measured value of the negative control) × 100

[0355] like Fig.33 As shown, none of the ingredients had a significant effect on cell viability and was considered non-irritating to the skin.

[0356] Example 10: Transdermal Nucleic Acid Delivery to Tumors, Skin, and Liver

[0357] Preparation of a SNORA23-targeted preparation. 3×10 ES2 ovarian cancer cells were cultured in vitro. 6 The cells were transplanted subcutaneously into mice, and the formation of subcutaneous tumors was confirmed after 1 week. 50 μL of hybrid particles containing 250 μg of ASO targeting SNORA23 were applied or injected subcutaneously on the upper part of the tumor (1 cm × 1 cm) or around the tumor. In the transdermal administration group, a protective tape was wrapped around the trunk including the administration site.

[0358] ASO has the following sequence.

[0359] 5'-GAAacctatgmCAmCa-3' (SEQ ID NO: 2)

[0360] {Here, lowercase letters represent DNA bases, uppercase letters represent LNA-modified bases, and mC represents LNA-modified methylcytosine. In addition, all phosphate groups are phosphorothioate-modified. )

[0361] Two days after administration, the skin, subcutaneous tumor, and liver were removed and total RNA was extracted. The amount of the target RNA (SNORA23) was quantified and the expression was confirmed by RT-PCR to see whether it was suppressed. Fig.34 As shown. Fig.34 As shown, the expression of the target RNA was decreased in both the transdermal and subcutaneous administration groups. The knockdown effect in the liver indicates that ASO is transferred to the blood regardless of the administration route.

Claims

1. A composition for forming a complex with a pharmaceutical ingredient, comprising a mixture containing cations and anions.

2. The composition according to claim 1, wherein The cations are cationic lipids and the anions are fatty acids.

3. The composition according to claim 1 or 2, which does not contain any active pharmaceutical ingredient.

4. The composition according to claim 1 or 2, wherein It also contains active ingredients for transdermal administration.

5. A composition comprising a solid complex comprising cations, anions and nucleic acids, and an oil phase, The solid composite groups are dispersed in the oil phase.

6. The composition according to claim 5, wherein The solid composite group contains solid composites of about 5 nm to about 50 nm.

7. The composition according to claim 6, wherein The group of solid composites includes a structure including a plurality of aggregates of solid composites having a size of about 5 nm to about 50 nm.

8. The composition according to any one of claims 5 to 7, which is used for transdermal administration of a nucleic acid.

9. The composition according to any one of claims 5 to 8, wherein Cations are cationic surfactants, and anions are anionic surfactants.

10. The composition according to any one of claims 5 to 9, wherein The cation and the anion are contained in an amount with a charge ratio of 1:0.8 to 1:1.

2.

11. The composition according to any one of claims 5 to 10, wherein The cations and anions can form an ionic liquid when mixed at least in a charge ratio of 1:1 in a composition consisting only of the cations and anions.

12. A composition comprising an aqueous composition containing a complex containing cations, anions and nucleic acids, which is capable of transfecting nucleic acids from outside cells into cells, thereby producing viable cells containing the nucleic acids inside the cells.

13. A method for producing the composition according to any one of claims 5 to 11, comprising the steps of: An aqueous solution containing nucleic acid is mixed with a lower alcohol solution containing a mixture of cations and anions to obtain a complex containing nucleic acid and the cations and anions.

14. The method according to claim 13, wherein: The method also includes the step of freeze-drying the obtained complex.

15. The method according to claim 12 or 13, wherein: The method comprises the step of dispersing the freeze-dried complex in an oil phase. The composition according to claim 12 , which is used for transfecting nucleic acid from outside a cell into a cell.

17. A composition for weakening the skin barrier (or increasing the permeability of the skin) of a subject, comprising anionic lipids.

18. The composition according to claim 17, wherein Anionic lipids are fatty acids.

19. The composition according to claim 17 or 18, wherein Anionic lipids are long-chain fatty acids.

20. The composition according to any one of claims 17 to 19, wherein Anionic lipids are long-chain unsaturated fatty acids.

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