Lipid nanoparticles and method for producing same, nucleic acid-encapsulated lipid nanoparticles and method for producing same, and method for imparting acquired immunity to living body using nucleic acid-encapsulated lipid nanoparticles

Abstract

Provided are lipid nanoparticles containing (A) an ionic lipid represented by formula (1) (wherein the symbols in formula (1) are as defined in the description), (B) vitamin E and / or a derivative thereof, (C) a phospholipid, (D) cholesterol, and (E) a PEG lipid. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a lipid nanoparticle that can be used particularly as a nucleic acid vaccine and a method for producing the same, a lipid nanoparticle encapsulating a nucleic acid and a method for producing the same, and a method for conferring acquired immunity to an organism using the lipid nanoparticle encapsulating a nucleic acid. Background Art

[0002] In order to use the nucleic acid therapy of oligonucleic acid such as siRNA, the gene therapy adopting mRNA, pDNA etc. is practical, it is necessary to effectively and safely deliver nucleic acid vectors. Viral vectors are nucleic acid delivery vectors with good expression efficiency, but have problems in practicality from the viewpoint of safety. Therefore, the development of non-viral nucleic acid delivery vectors that can be used more safely is being promoted. Among them, lipid nanoparticles as the carrier using ionic lipids are the most commonly used non-viral nucleic acid delivery vectors now.

[0003] Ionic lipids are generally composed of an amine site and a lipid site. For example, ionic lipids are substances that promote the uptake of nucleic acids into cells by electrostatically interacting with nucleic acids, which are polyanions, under acidic conditions to form lipid nanoparticles, thereby delivering nucleic acids into cells.

[0004] As a well-known ionic lipid that is generally widely used, 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP) can be mentioned. It is known that by combining such a well-known ionic lipid with phospholipids, cholesterol and PEG lipids, lipid nanoparticles can be formed to deliver nucleic acids into cells (for example, see Non-Patent Document 1).

[0005] For example, Patent Document 1 shows an ionic lipid having a structure in which a compound consisting of one or two amine sites and one lipid site is connected by a disulfide bond showing biodegradability. Patent Document 1 shows that the ionic lipid improves pharmacokinetics such as blood stability and tumor targeting. In addition, it is shown that by changing the structure around the amine site, in addition to adjusting the pKa of the lipid membrane structure to a value that is conducive to escaping from the endosome in the cell, the disulfide bond is cut off in the cell, and it also has the effect of dissociating nucleic acids from the lipid membrane structure. In fact, compared to DODAP, which is a well-known ionic lipid, it is obvious that the ionic lipid can improve the intracellular dynamics such as the delivery efficiency of nucleic acids to the cytoplasm due to the high nucleic acid delivery efficiency.

[0006] For example, Patent Document 2 discloses a lipid membrane structure that uses an ionic lipid having a tertiary amine site, a disulfide bond, and an aromatic ring introduced near a lipid site to enhance fusion ability with endosomal membranes and further enhance the efficiency of nucleic acid delivery to the cytoplasm.

[0007] By improving the endosomal escape efficiency and membrane fusion ability in this way, ionic lipids that improve intracellular dynamics are developed. On the other hand, in order to improve the effectiveness of lipid nanoparticles containing ionic lipids as vaccines, in addition to improving the efficiency of nucleic acid delivery, improving acquired immunity (cellular immunity, humoral immunity) is also an important factor.

[0008] There are several examples of lipid nanoparticles containing ionic lipids being used as nucleic acid vaccines. For example, Non-Patent Document 2 shows an example of using 1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP) as an ionic lipid.

[0009] Although nucleic acid vaccines using lipid nanoparticles have been developed in this way, the effect of enhancing acquired immunity is not sufficient and there is still room for improvement. Prior art literature Patent Literature

[0010] Patent Document 1: U.S. Patent No. 9708628 Patent Document 2: International Publication No. 2019 / 188867 Non-patent literature

[0011] Non-patent document 1: Molecular Therapy, 25(7): 1467-1475 (2017) Non-patent document 2: Vaccine, 8(3):433(2020) Non-patent document 3: Hum Vaccin Immunother., 14(3): 550-564 (2018) Non-patent literature 4: Immunity, 54(12): 2877-2892(2021) Summary of the invention Problem that the invention aims to solve

[0012] As a method for improving the effect as a vaccine, a method of adding an adjuvant to the structural components of the vaccine is used. For example, AS03, MF59, aluminum salt (alum), etc. in O / W emulsions are used as adjuvants for influenza vaccines (for example, see Non-Patent Document 3).

[0013] However, although these adjuvants improve the efficacy of vaccines, they also have the problem of increasing side effects such as fever and pain at the administration site. Therefore, the selection of adjuvants is also important.

[0014] It is also suggested that lipid nanoparticles may be used as adjuvants (e.g., non-patent document 4). However, lipid nanoparticles using ionic lipids shown in patent document 2 do not show sufficient effects as nucleic acid vaccines. The effects of lipid nanoparticles that are not vaccine components are not fully satisfied and need to be studied.

[0015] In view of the above problems, the present invention aims to provide a lipid nanoparticle and a method for producing the same for particularly efficiently enhancing acquired immunity, a lipid nanoparticle encapsulating nucleic acid and a method for producing the same, and a method for conferring acquired immunity to an organism using lipid nanoparticles encapsulating nucleic acid. Means to solve the problem

[0016] In view of the above problems, the inventors conducted in-depth research and found that lipid nanoparticles made using ionic lipids with pKa suitable for endosomal escape and which improve the efficiency of nucleic acid delivery into cells by specifically degrading under the reducing environment in cells and vitamin E or vitamin E derivatives with adjuvant ability can effectively improve acquired immunity, thereby completing the present invention. The present invention based on this discovery is as follows.

[0017] [1] A lipid nanoparticle comprising: (A) Ionic lipid represented by formula (1):

[0018] [Chemistry 1]

[0019] (In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom, n a and nb Each independently represents 0 or 1, R 3a and R 3b Each independently represents: (a)R 3a -COO- and R 3b -COO- represents a group derived from a residue of a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, or (b) an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or (c) A group represented by formula (2): R 9 -O-CO-(CH2) a -(2) (In formula (2), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. )) (B) vitamin E and / or its derivatives, (C) phospholipids, (D) Cholesterol and (E) PEG lipids. [2] The lipid nanoparticle according to [1], wherein the vitamin E is any one selected from the group consisting of α-tocopherol, β-tocopherol, α-tocotrienol and β-tocotrienol. [3] According to the lipid nanoparticles described in [1] or [2], the vitamin E derivative is any one selected from the group consisting of α-tocopherol succinate, α-tocopherol glutarate, β-tocopherol succinate, β-tocopherol glutarate, α-tocotrienol succinate, α-tocotrienol glutarate, β-tocotrienol succinate and β-tocotrienol glutarate. [4] The lipid nanoparticle according to [1] or [2], wherein the vitamin E derivative is a compound represented by formula (3):

[0020] [Chemistry 2]

[0021] (In formula (3), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X beach independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3b Each independently is R 3a -COO- and R 3b -COO- represents a group derived from a residue of a reaction product of a hydroxyl group of vitamin E and succinic anhydride or glutaric anhydride. ). [5] The lipid nanoparticle according to [1] or [2], wherein the vitamin E derivative is any one of the compounds represented by formula (4) or (5):

[0022] [Chemistry 3]

[0023] (In formula (4), R is RCOO(CH2)2COO-, which represents a residue formed by the reaction of the hydroxyl group of α-tocopherol, β-tocopherol, α-tocotrienol or β-tocotrienol with succinic anhydride.

[0024] [Chemistry 4]

[0025] (In formula (5), R is RCOO(CH2)2COO-, which represents a residue obtained by the reaction of a hydroxyl group of α-tocopherol, β-tocopherol, α-tocotrienol or β-tocotrienol with succinic anhydride.). [6] The lipid nanoparticle according to any one of [1] to [5], wherein the ionic lipid is a compound represented by the following formula:

[0026] [Chemistry 5]

[0027] [7] According to the lipid nanoparticles described in [5], relative to the total of the ionic lipids, the vitamin E and / or its derivatives, the phospholipids and the cholesterol, the ionic lipids are 20-55 mol%, the vitamin E and / or its derivatives are 4.5-35 mol%, the phospholipids are 5-35 mol%, the cholesterol is 10-50 mol%, and the PEG lipids are 0.5-4 mol%. [8] According to the lipid nanoparticles described in [6], relative to the total of the ionic lipids, the vitamin E and / or its derivatives, the phospholipids and the cholesterol, the ionic lipids are 20-55 mol%, the vitamin E and / or its derivatives are 4.5-35 mol%, the phospholipids are 5-35 mol%, the cholesterol is 10-50 mol%, and the PEG lipids are 0.5-4 mol%. [9] The lipid nanoparticle according to [8], wherein the phospholipid is 1,2-diacyl-sn-glycero-3-phosphocholine or 1,2-diacyl-sn-glycero-phosphoethanolamine.

[10] According to the lipid nanoparticles described in [8], the phospholipid is any one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[11] According to the lipid nanoparticle described in [8], the PEG lipid is dimyristoylglycerol PEG represented by formula (6): CH2(OR 6 )-CH(OR 7 )-CH2(OR 8 )(6) (In formula (6), R 6 , R 7 and R 8 Any two of them represent myristoyl groups, and the remaining one represents an alkyl group having 1 to 6 carbon atoms connected via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000. ).

[12] According to the lipid nanoparticle described in

[10] , the PEG lipid is dimyristoylglycerol PEG represented by formula (6): CH2(OR 6 )-CH(OR 7 )-CH2(OR 8 )(6) (In formula (6), R 6 , R 7 and R8 Any two of them represent myristoyl groups, and the remaining one represents an alkyl group having 1 to 6 carbon atoms connected via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000. ).

[13] A nucleic acid-encapsulated lipid nanoparticle, comprising the lipid nanoparticle according to any one of [1] to

[12] in which a nucleic acid is encapsulated.

[14] A method for producing nucleic acid-encapsulated lipid nanoparticles according to

[13] , comprising a vitamin adding step of adding the vitamin E and / or its derivative to the ionic lipid, the phospholipid, the cholesterol and the PEG lipid.

[15] According to the production method described in

[14] , in the vitamin adding step, after adding the vitamin E and / or its derivative to the alcohol solution containing the ionic lipid, the phospholipid, the cholesterol and the PEG lipid, the step further includes mixing with a buffer solution containing nucleic acid.

[16] According to the production method described in

[14] , in the vitamin addition step, after the alcohol solution containing the ionic lipid, the phospholipid, the cholesterol and the PEG lipid is mixed with the buffer solution containing the nucleic acid, the vitamin E and / or its derivative is added.

[17] A method for conferring acquired immunity against an antigen to an organism, comprising the step of administering to the organism a lipid nanoparticle according to any one of [1] to

[12] encapsulating a nucleic acid expressing the antigen protein. Effects of the Invention

[0028] The lipid nanoparticles of the present invention, which are used for nucleic acid vaccines that particularly efficiently enhance acquired immunity, can efficiently enhance acquired immunity compared to lipid nanoparticles of the prior art by combining ionic lipids with at least one of vitamin E having adjuvant capacity and a vitamin E derivative having a vitamin E skeleton. The ionic lipids have a pKa suitable for endosomal escape and are specifically degraded under the reducing environment within the cell to enhance the efficiency of delivering nucleic acids into the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] [ Figure 1 ] Figure 1 The effect of adding α-tocopherol (VE) to lipid nanoparticles on CTL activity in mice administered with the lipid nanoparticles is shown. Regardless of the timing of VE addition, CTL activity was increased compared to the case where VE was not added. [ Figure 2 ] Figure 2 The graph shows the effect of the added concentration of VE on the CTL activity in mice administered with lipid nanoparticles. [ Figure 3] Figure 3 The figure shows the effect of the added concentration of α-tocopheryl succinate (TS) on the CTL activity in mice administered with lipid nanoparticles. [ Figure 4 ] Figure 4 The results of investigating the effect of the addition method of vitamins / vitamin derivatives (VE, TS, SS-EC) on CTL activity in mice administered with lipid nanoparticles are shown. Regardless of the addition method, CTL activity was increased compared to the case where no vitamin / vitamin derivative was added. [ Figure 5 ] Figure 5 The figure shows the effect of the added concentration of SS-EC on the nucleic acid delivery efficiency (transferred gene expression activity) in mice administered with lipid nanoparticles. The nucleic acid delivery efficiency equivalent to that without addition was obtained at any added concentration. [ Figure 6 ] Figure 6 The figure shows the effect of the added concentration of SS-EC on the CTL activity in mice administered with lipid nanoparticles. Compared with the case of no addition, the CTL activity increased at any added concentration. [ Figure 7 ] Figure 7 The figure shows the effect of the added concentration of SS-EC on the production of anti-OVA antibodies in mice administered with lipid nanoparticles. Compared with the case of no addition, the amount of antibody production increased at all the added concentrations. [ Figure 8 ] Figure 8 The effect of adding SS-EC to lipid nanoparticles on the amount of anti-OVA antibody produced in mice to which the lipid nanoparticles were administered is shown. When SS-EC was not added, no antibody was produced, but the addition of SS-EC induced antibody production. [ Fig. 9 ] Fig. 9 The figure shows the effect of the ratio of RNA to total fat content (L / R ratio) on the production of anti-OVA antibodies in mice administered lipid nanoparticles. The same amount of antibody production was observed at any L / R ratio. [ Fig.10 ] Fig.10 The figure shows the effect of the added concentration of SS-EC on the IL-6 production in mice administered with lipid nanoparticles. Compared with the case without addition, the IL-6 production increased at all the added concentrations. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention relates to lipid nanoparticles (hereinafter referred to as "lipid nanoparticles of the present invention") that are particularly useful as a component of a vaccine and a method for producing the same, lipid nanoparticles encapsulating nucleic acids and a method for producing the same, and a method for imparting acquired immunity to an organism against an antigen using lipid nanoparticles encapsulating nucleic acids expressing an antigen protein, wherein the lipid nanoparticles contain: (A) an ionic lipid represented by formula (1) (i.e., an ionic lipid having a tertiary amino group, a lipid moiety, and a disulfide bond that is a biodegradable group), (B) vitamin E and / or its derivatives, (C) phospholipids, (D) Cholesterol and (E)PEG lipids of.

[0031] Lipid Nanoparticles In this specification, "lipid nanoparticle" (LipidNano Particle, sometimes referred to as "LNP" in this specification) refers to a membrane structure with the hydrophilic groups of amphiphilic lipids arranged toward the aqueous phase side of the interface, and the particle size is less than 1 μm; "amphiphilic lipids" refer to lipids that have both hydrophilic groups and hydrophobic groups.

[0032] The particle size of the lipid nanoparticles of the present invention is preferably 10nm to 500nm, more preferably 30nm to 300nm. The determination of particle size can be carried out using a particle size distribution measuring device such as ZetasizerNano (Malvern Company). The particle size of lipid nanoparticles can be appropriately adjusted according to the manufacturing method of lipid nanoparticles. In this specification, "particle size" refers to the average particle size (Z average) measured by dynamic light scattering.

[0033] Examples of amphiphilic lipids include ionic lipids, phospholipids, and PEG lipids. In the present specification, "PEG" refers to polyethylene glycol, "PEG lipid" refers to a lipid modified with PEG, and "X-modified Y" (e.g., X: PEG; Y: lipid) refers to Y bound to X. In other words, "PEG lipid" refers to a lipid bound to PEG.

[0034] Ionic lipids The ionic lipid used in the present invention is an ionic lipid represented by the following formula (1) (sometimes simply referred to as "ionic lipid (1)" in this specification). The ionic lipid (1) may be used alone or in combination of two or more.

[0035] [Chemistry 6]

[0036] (In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3b Each independently represents: (a)R 3a -COO- and R 3b -COO- represents a group derived from a residue of a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, or (b) an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or (c) A group represented by formula (2): R 9 -O-CO-(CH2) a -(2) (In formula (2), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. ))

[0037] R 1a and R 1bEach independently represents an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 4, more preferably 1 to 2. Specific examples of the alkylene group having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. 1a and R 1b Preferably, each is independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each is an ethylene group.

[0038] R 1a Can be used with R 1b The same or different, but preferably R 1a For R 1b Same group.

[0039] X a and X b Each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, and is preferably a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0040] The alkyl group having 1 to 6 carbon atoms in the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group may be linear, branched, or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl, preferably methyl, ethyl, propyl, or isopropyl, and most preferably methyl.

[0041] The preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is as follows: 1 shown.

[0042] [Chemistry 7]

[0043] X 1 R 5The alkyl group represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, cyclohexyl, etc., preferably methyl, ethyl, propyl or isopropyl, and most preferably methyl.

[0044] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is preferably 4 to 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include aziridinyl, aziridinyl, pyrrolidinyl, piperidinyl, imidazolyl, and piperazinyl, preferably pyrrolidinyl, piperidinyl, and piperazinyl, and most preferably piperidinyl.

[0045] The preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group is as follows: 2 shown.

[0046] [Chemistry 8]

[0047] X 2 p is 1 or 2. When p is 1, X 2 is pyrrolidinylene; when p is 2, X 2 Preferably, p is 2.

[0048] The preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is as follows: 3 shown.

[0049] [Chemistry 9]

[0050] X 3 w is 1 or 2. When w is 1, X 3 When w is 2, X 3 It is piperazinylene.

[0051] X a With X b Can be the same or different, preferably X a With X b are the same group.

[0052] R 2a and R 2bEach independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group, and each independently represents an alkylene group having 8 or less carbon atoms.

[0053] The alkylene group having 8 or less carbon atoms may be straight-chain or branched, but is preferably straight-chain. The number of carbon atoms contained in the alkylene group is preferably 6 or less, and most preferably 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include methylene, ethylene, trimethylene, isopropylidene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, etc., preferably methylene, ethylene, trimethylene, tetramethylene, and most preferably ethylene.

[0054] In this specification, "oxydialkylene having less than 8 carbon atoms" refers to an alkylene group (alkylene-O-alkylene group, in other words, "alkyleneoxyalkylene group") separated by an ether bond, and the total number of carbon atoms of the two alkylene groups present is less than 8. Among them, the two alkylene groups present may be the same or different, and are preferably the same. As the oxydialkylene having less than 8 carbon atoms, specifically exemplified are: oxydimethylene, oxydiethylene, oxybis(trimethylene) groups (i.e., trimethyleneoxytrimethylene), oxybis(tetramethylene) groups (i.e., tetramethyleneoxytetramethylene), etc. Preferred are oxydimethylene, oxydiethylene, oxybis(trimethylene) groups, and most preferably oxydiethylene.

[0055] R 2a and R 2b Can be the same or different, preferably R 2a and R 2b are the same group.

[0056] Y a and Y b Each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently represents an ester bond, an amide bond or a carbamate bond, more preferably each independently represents an ester bond or an amide bond, and most preferably each independently represents an ester bond. a and Y b There is no restriction on the bonding orientation of Y a and Y b When it is an ester bond, it is preferably -Z a -CO-OR 2a -and-Z b -CO-OR 2b -structure.

[0057] Y a and Y b Can be the same or different, preferably Y a For Y b Same group.

[0058] Za and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom. The number of carbon atoms contained in the aromatic compound is preferably 6 to 12, and most preferably 6 to 7. In addition, the aromatic compound preferably has one aromatic ring.

[0059] Examples of the types of aromatic rings contained in the aromatic compound having 3 to 16 carbon atoms include, for aromatic hydrocarbon rings, a benzene ring, a naphthalene ring, and an anthracene ring; and for aromatic heterocyclic rings, an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furathiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, and a benzodiazepine ring. It is preferably a benzene ring, a naphthalene ring or an anthracene ring, and most preferably a benzene ring.

[0060] The aromatic ring may have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylthio group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, The ureido group having 1 to 4 carbon atoms, the alkoxy group having 1 to 4 carbon atoms, the aryl group having 6 to 10 carbon atoms, the aryloxy group having 6 to 10 carbon atoms, and the like. Preferred examples include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetylamino group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylthio group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0061] As Z a and Z b The preferred specific structure of Z 1 .

[0062] [Chemistry 10]

[0063] In the formula, s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R 4 Each independently represents a substituent.

[0064] Z 1 The s is preferably an integer of 0 to 1, and more preferably 0. Z 1 The t is preferably an integer of 0 to 2, and more preferably 1. Z 1 The u is preferably an integer of 0-2, and more preferably an integer of 0-1.

[0065] Z 1 R 4 It is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms which does not inhibit the reaction in the synthesis process of the ionic lipid. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylthio group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a 4 urea groups, alkoxy groups having 1 to 4 carbon atoms, aryl groups having 6 to 10 carbon atoms, aryloxy groups having 6 to 10 carbon atoms, etc. Preferred examples include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetylamino, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylthio, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, urea, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, and phenoxy. 4 When each R 4 Can be the same or different.

[0066] Z a With Z b Can be the same or different, preferably Z a For Z b Same group.

[0067] n a and n b Each independently is 0 or 1.

[0068] n a With n b Can be the same or different, preferably n a For n b Same group.

[0069] R 3a and R 3b Each independently represents: (a)R3a -COO- and R 3b -COO- represents a group of a residue derived from a reaction of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or (b) an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or (c) A group represented by formula (2): R 9 -O-CO-(CH2) a -(2) (In formula (2), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer of 2 to 10. ) are preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0070] The residue derived from the reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride means a group having a structure in which the hydroxyl group of the sterol derivative having a hydroxyl group is substituted with *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2-. * indicates a bonding position with the sterol derivative.

[0071] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and cholesterol or cholestanol is preferred.

[0072] The aliphatic hydrocarbon group having 1 to 40 carbon atoms may be straight-chain or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, preferably carbon-carbon double bonds. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes alkyl, alkenyl, alkynyl, etc., but preferably includes alkyl or alkenyl. Specific examples of the aliphatic hydrocarbon group having 1 to 40 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, tetracontyl, dodecenyl, tridecenyl, tetradecenyl, 1-decenyl, 1-pentadecenyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, 1-hexylhexyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, 1-decylundecyl, 1-hexyl ...hexylhexyl, 1-octylundecyl, 1-decylundecyl, 1-decylundecyl, 1-decylundecyl, 1-hexylhexyl, 1-hexylhexyl, 1-octylundecyl, 1-decylundecyl, 1-decylundecyl, 1-decylundecyl, 1-decylundecyl, 1-decylundecyl, 1-decylundecyl, 1-decyl The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably tridecyl, pentadecyl, heptadecyl, nonadecyl, heptadecenyl, heptadecadienyl, or 1-hexylnonyl, and particularly preferably tridecyl, heptadecyl, heptadecenyl, or heptadecadienyl.

[0073] In one embodiment of the present invention, R 3a and R 3b The aliphatic hydrocarbon group having 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) represented by is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). As a specific example of the aliphatic hydrocarbon group, when linoleic acid is used as the fatty acid, it is heptadecadienyl; when oleic acid is used as the fatty acid, it is heptadecenyl.

[0074] R 3a and R 3bThe alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring in the alkyl group refers to an alkyl group having 3 to 40 carbon atoms and having at least one cyclopropane ring in the alkyl chain. The 3 to 40 carbon atoms of the alkyl group do not include the carbon atoms of the cyclopropane ring. The number of cyclopropane rings in the alkyl group is preferably 1. 3a and R 3b The alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring is preferably a group represented by the formula (6).

[0075] [Chemistry 11]

[0076] (In formula (6), b and c are each independently an integer, and the total of b and c is 2 to 39.) Preferably, b is an integer of 1 to 20, and c is an integer of 1 to 19. More preferably, b is an integer of 2 to 18, further preferably an integer of 3 to 17, and further preferably an integer of 4 to 12. More preferably, c is an integer of 3 to 15, further preferably an integer of 3 to 11, and further preferably an integer of 3 to 9. Examples of the group represented by formula (6) include 7-(2-octylcyclopropyl)heptyl and the like.

[0077] In formula (2), R 9The aliphatic hydrocarbon group having 2 to 20 carbon atoms may be straight-chain or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. When it is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, preferably carbon-carbon double bonds. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, further preferably 13 to 19, and most preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl, alkenyl, alkynyl, etc., preferably include alkyl or alkenyl, and more preferably alkyl. Specific examples of the aliphatic hydrocarbon group having 2 to 20 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecyl, nonadecyl, eicosyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably tridecyl, pentadecyl, heptadecyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl, dodecadienyl, tridecadienyl, tetradecadienyl, pentadecadienyl, hexadecadienyl, heptadecadienyl, octadecadienyl, nonadecadienyl, eicosadienyl, eicosatoenyl, eicosatetraenyl, eicosapentaenyl, isostearyl, 1-hexylheptyl, 1-ethylnonyl, 1-butylnonyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 3-octylundecyl, etc. The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably tridecyl, pentadecyl, heptadecyl, nonadecenyl, heptadecenyl, heptadecadienyl, 1-hexylnonyl, and particularly preferably tridecyl, heptadecyl, heptadecenyl, heptadecadienyl, heptadecadienyl.

[0078] In formula (2), a is preferably an integer of 3 to 9, more preferably an integer of 3 to 7, further preferably an integer of 5 to 7, and most preferably 5 or 7.

[0079] R 3a With R 3b Can be the same or different, preferably R 3a For R 3b Same group.

[0080] In one embodiment of the present invention, R 1a With R 1b Same, X a With X b Same, R 2a With R 2b Same, Y a With Y b Same, Za With Z b Same, R 3a With R 3b same.

[0081] Preferred examples of the ionic lipid (1) include the following ionic lipids. [Ionic lipids (1-1)] An ionic lipid (1), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidinylene); R 2a and R 2b Each is independently an alkylene group having 8 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each independently is an ester bond or an amide bond; Z a and Z b Each is independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); n a and n b Each is independently 0 or 1; R 3a and R 3b Each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms (for example, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0082] [Ionic lipids (1-2)] An ionic lipid (1), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g. -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g. piperidinylene); R2a and R 2b Each is independently an alkylene group having 6 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b each independently an ester bond or an amide bond; Z a and Z b Each is independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); n a and n b Each is independently 0 or 1; R 3a and R 3b Each independently represents an aliphatic hydrocarbon group having 13 to 19 carbon atoms (for example, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0083] [Ionic lipids (1-3)] An ionic lipid (1), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 2 carbon atoms (i.e., methylene or ethylene); X a and X b Each independently is X 1 or X 2 :

[0084] [Chemistry 12]

[0085] (Where R 5 An alkyl group having 1 to 3 carbon atoms (e.g., methyl group).

[0086] [Chemistry 13]

[0087] (where p is 1 or 2); R 2a and R 2b Each is independently an alkylene group having 4 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each independently is Z 1 :

[0088] [Chemistry 14]

[0089] (wherein, s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R 4 Each independently represents a substituent. ); n a and n b Each is independently 0 or 1; R 3a and R 3b Each independently represents an aliphatic hydrocarbon group having 13 to 17 carbon atoms (for example, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0090] Specific examples of the ionic lipid (1) include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, and O-P4C2.

[0091] In addition, specific examples of the ionic lipid (1) include Lipid1 to Lipid20 described in WO 2021 / 195529 A2, and particularly include the following Lipid1, Lipid5, and Lipid8. [Table 1-1] [Table 1-2]

[0094] [Table 1-3]

[0095] Among the specific examples of the ionic lipid (1), SS-OP is also preferred. That is, the ionic lipid (1) is preferably an ionic lipid represented by the following formula.

[0096] [Chemistry 15]

[0097] From the viewpoints of nucleic acid internalization efficiency, nucleic acid release efficiency in cells and stability of lipid nanoparticles, when vitamin E and / or its derivatives are used, the amount of ionic lipid (1) in the lipid nanoparticles of the present invention is preferably 20 to 55 mol%, more preferably 22.5 to 50 mol%, relative to the total of ionic lipid (1), vitamin E and / or its derivatives, phospholipids and cholesterol.

[0098] Ionic lipid (1) can be produced by a known method (for example, the method described in WO 2019 / 188867 A1, US 9708628 B2, and WO2021 / 195529A2).

[0099] Vitamin E and vitamin E derivatives The lipid nanoparticles of the present invention contain at least one of vitamin E and vitamin E derivatives as a lipid constituent component. Among these, only one type may be used, or two or more types may be used.

[0100] Vitamin E Vitamin E is a general term for four tocopherols and four tocotrienols, specifically α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol.

[0101] As vitamin E used in the present invention, any one of the above eight types may be used, or a plurality of types may be used in combination. Preferred are α-tocopherol, β-tocopherol, α-tocotrienol, or β-tocotrienol, and most preferred is α-tocopherol.

[0102] Vitamin E derivatives If it is a compound having a vitamin E skeleton, there is no limitation, for example, it can be cited: a compound in which the hydroxyl group of vitamin E is modified, a compound in which the isoprenoid side chain of vitamin E is modified (for example, a compound in which the terminal is carboxylated or a compound in which a hydroxyl group is introduced into the terminal), a compound in which the chroman ring of vitamin E is modified, etc. Among them, a compound in which the hydroxyl group of vitamin E is modified is preferred. As a compound in which the hydroxyl group of vitamin E is modified, a compound in which vitamin E is reacted with an acid such as carboxylic acid, phosphoric acid or its derivative, sulfonic acid, and its hydroxyl group is esterified can be cited. As carboxylic acid, aliphatic carboxylic acids such as acetic acid, propionic acid, hexane acid, myristic acid, stearic acid, oleic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, fumaric acid, maleic acid, various amino acids, etc. can be cited. As a phosphoric acid derivative, monoalkyl phosphate, dialkyl phosphate, etc. can be cited. As a sulfonic acid, alkyl sulfonic acid, etc. can be cited. Specific examples of compounds in which the hydroxyl group of vitamin E is esterified include: α-tocopherol fatty acid esters such as α-tocopherol acetate; α-tocopherol phosphate; α-tocopherol dibasic acid esters (monoesters or diesters) such as α-tocopherol malonate (monoester or diester), α-tocopherol succinate (monoester or diester), and α-tocopherol glutarate (monoester or diester); and compounds in which vitamin E reacts with other compounds via dibasic acids, such as compounds represented by formula (3) described later. In the present invention, compounds represented by formula (3) described later are preferred. The vitamin E derivatives described above may be in the form of salts, and examples thereof include salts with organic bases, salts with inorganic bases, salts with organic acids, and salts with inorganic acids. The vitamin E derivative may be used alone or in combination of two or more.

[0103] In a preferred embodiment, the vitamin E derivative is a compound represented by the following formula (3) (sometimes simply referred to as "compound (3)" in the present specification).

[0104] [Chemistry 16]

[0105] (In formula (3), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3b Each independently is R 3a -COO- and R 3b -COO- represents a group derived from the residue of a reaction product of a hydroxyl group of vitamin E and succinic anhydride or glutaric anhydride. )

[0106] R 1a and R 1bEach independently represents an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 4, more preferably 1 to 2. Specific examples of the alkylene group having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. 1a and R 1b Preferably, each is independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each is an ethylene group.

[0107] R 1a With R 1b Can be the same or different, preferably R 1a For R 1b Same group.

[0108] X a and X b Each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, and each independently represents a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0109] The alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group may be linear, branched, or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl, preferably methyl, ethyl, propyl, or isopropyl, and most preferably methyl.

[0110] The preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is as follows: 1 shown.

[0111] [Chemistry 17]

[0112] X 1 R 5The alkyl group having 1 to 6 carbon atoms may be straight-chain, branched, or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl, preferably methyl, ethyl, propyl, or isopropyl, and most preferably methyl.

[0113] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is preferably 4 to 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include aziridinyl, aziridinyl, pyrrolidinyl, piperidinyl, imidazolyl, and piperazinyl, preferably pyrrolidinyl, piperidinyl, and piperazinyl, and most preferably piperidinyl.

[0114] The preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group is as follows: 2 shown.

[0115] [Chemistry 18]

[0116] X 2 p is 1 or 2. When p is 1, X 2 is pyrrolidinylene; when p is 2, X 2 Preferably, p is 2.

[0117] The preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is as follows: 3 shown.

[0118] [Chemistry 19]

[0119] X 3 w is 1 or 2. When w is 1, X 3 When w is 2, X 3 It is piperazinylene.

[0120] X a With X b Can be the same or different, preferably X a For X b Same group.

[0121] R 2a and R 2bEach independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group, and each independently represents an alkylene group having 8 or less carbon atoms.

[0122] The alkylene group having 8 or less carbon atoms may be straight-chain or branched, preferably straight-chain. The number of carbon atoms contained in the alkylene group is preferably 6 or less, and most preferably 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include methylene, ethylene, trimethylene, isopropylidene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, etc., preferably methylene, ethylene, trimethylene, tetramethylene, and most preferably ethylene.

[0123] In this specification, "oxydialkylene having 8 or less carbon atoms" refers to an alkylene having an ether bond (alkylene-O-alkylene, in other words, "alkyleneoxyalkylene"), wherein the total number of carbon atoms of the two alkylene groups present is 8 or less. The two alkylene groups present may be the same or different, and are preferably the same. Specific examples of oxydialkylene having 8 or less carbon atoms include oxydimethylene, oxydiethylene, oxybis(trimethylene) groups (i.e., trimethyleneoxytrimethylene), oxybis(tetramethylene) groups (i.e., tetramethyleneoxytetramethylene), etc. Preferred are oxydimethylene, oxydiethylene, oxybis(trimethylene) groups, and most preferred are oxydiethylene.

[0124] R 2a With R 2b Can be the same or different, preferably R 2a For R 2b Same group.

[0125] Y a and Y b Each is independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each is independently an ester bond, an amide bond or a carbamate bond, more preferably each is independently an ester bond or an amide bond, and most preferably each is an ester bond. a and Y b There is no restriction on the bonding orientation of Y a and Y b When it is an ester bond, it is preferably -Z a -CO-OR 2a -and-Z b -CO-OR 2b -structure.

[0126] Y a With Y b Can be the same or different, preferably Y a For Y b Same group.

[0127] Za and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. The number of carbon atoms contained in the aromatic compound is preferably 6 to 12, and most preferably 6 to 7. In addition, the aromatic compound preferably has one aromatic ring.

[0128] Examples of the types of aromatic rings contained in the aromatic compound having 3 to 16 carbon atoms include, for aromatic hydrocarbon rings, a benzene ring, a naphthalene ring, and an anthracene ring; and for aromatic heterocyclic rings, an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furathiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, and a benzodiazepine ring. It is preferably a benzene ring, a naphthalene ring or an anthracene ring, and most preferably a benzene ring.

[0129] The aromatic ring may further have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylthio group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, The ureido group having 1 to 4 carbon atoms, the alkoxy group having 1 to 4 carbon atoms, the aryl group having 6 to 10 carbon atoms, the aryloxy group having 6 to 10 carbon atoms, and the like. Preferred examples include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetylamino group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylthio group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0130] As Z a and Z b The preferred specific structure of Z 1 .

[0131] [Chemistry 20]

[0132] In the formula, s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R 4 Each independently represents a substituent.

[0133] Z 1 The s is preferably an integer of 0 to 1, and more preferably 0. Z 1 The t is preferably an integer of 0 to 2, and more preferably 1. Z 1 The u is preferably an integer of 0-2, and more preferably an integer of 0-1.

[0134] Z 1 R 4 It is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms which does not inhibit the reaction in the synthesis process of the ionic lipid. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylthio group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a 4 urea groups, alkoxy groups having 1 to 4 carbon atoms, aryl groups having 6 to 10 carbon atoms, aryloxy groups having 6 to 10 carbon atoms, etc. Preferred examples include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetylamino, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylthio, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, urea, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, and phenoxy. 4 When each R 4 Can be the same or different.

[0135] Z a With Z b Can be the same or different, preferably Z a For Z b Same group.

[0136] n a and n b Each independently is 0 or 1.

[0137] n a With n b Can be the same or different, preferably n a With n b same.

[0138] R 3a and R 3b Each independently is R 3a -COO- and R3b -COO- represents a group of a residue derived from the reaction of a hydroxyl group of vitamin E with succinic anhydride or glutaric anhydride.

[0139] The residue derived from the reaction product of vitamin E and succinic anhydride or glutaric anhydride means a group having a structure in which the hydroxyl group of vitamin E having a hydroxyl group is substituted with *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2-. * indicates a bonding position with vitamin E.

[0140] Specific examples of vitamin E include α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol. α-tocopherol, β-tocopherol, α-tocotrienol and β-tocotrienol are preferred, and α-tocopherol is most preferred.

[0141] In one embodiment of the present invention, R 1a With R 1b Same, X a With X b Same, R 2a With R 2b Same, Y a With Y b Same, Z a With Z b Same, R 3a With R 3b same.

[0142] Preferred examples of the vitamin E derivative represented by formula (3) include the following compounds. [Vitamin E derivatives (3-1)] A compound (3), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidinylene); R 2a and R 2b Each is independently an alkylene group having 8 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is independently an ester bond or an amide bond; Z aand Z b Each is independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); n a and n b Each is independently 0 or 1; R 3a and R 3b Each independently is R 3a -COO- and R 3b -COO- represents a group of a residue derived from the reaction of a hydroxyl group of vitamin E with succinic anhydride or glutaric anhydride.

[0143] [Vitamin E derivatives (3-2)] A compound (3), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g. -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g. piperidinylene); R 2a and R 2b Each is independently an alkylene group having 6 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each is independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and may or may not have a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); n a and n b Each is independently 0 or 1; For R 3a and R 3b For R 3a -COO- and R 3b -COO- each independently represents a residue derived from the reaction of a hydroxyl group of vitamin E with succinic anhydride or glutaric anhydride.

[0144] [Vitamin E derivatives (3-3)] A compound (3), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 2 carbon atoms (i.e., methylene or ethylene); X a and X b Each independently is X 1 or X 2 :

[0145] [Chemistry 21]

[0146] (Where R 5 An alkyl group having 1 to 3 carbon atoms (e.g., methyl group).

[0147] [Chemistry 22]

[0148] (where p is 1 or 2); R 2a and R 2b Each is independently an alkylene group having 4 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each independently is an ester bond or an amide bond; Z a and Z b Each independently is Z 1 :

[0149] [Chemistry 23]

[0150] (wherein, s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R 4 Each independently represents a substituent. ); n a and n b Each is independently 0 or 1; R 3a and R 3b Each independently is R 3a -COO- and R 3b -COO- represents a group of a residue derived from the reaction of a hydroxyl group of vitamin E with succinic anhydride or glutaric anhydride.

[0151] Specific examples of vitamin E derivatives of formula (3) include the following B-2-5, TS-C4E, TS-P3C1, TS-P4C1, TS-P4C2, E-Ph-P4C2, TS-P4C3, TS-P4C4, TG-C3M, and TSamide-C3M. In addition, compounds in which the residue of α-tocopherol in these derivatives is replaced by the residue of β-tocopherol, α-tocotrienol, or β-tocotrienol can also be mentioned.

[0152] [Table 2-1]

[0153] [Table 2-2]

[0154] Among the specific examples of the vitamin E derivative of formula (3), SS-E and SS-EC are also preferred. That is, the vitamin E derivative of formula (3) is preferably an ionic lipid represented by the following formula.

[0155] [Chemistry 24]

[0156] [Chemistry 25]

[0157] As other preferred specific examples of the vitamin E derivative of formula (3), compounds in which the residue of α-tocopherol in SS-E and SS-EC is replaced by the residue of β-tocopherol, α-tocotrienol or β-tocotrienol can be cited. Therefore, in a preferred embodiment, the vitamin E derivative of formula (3) is any one of the compounds represented by formula (4) or formula (5):

[0158] [Chemistry 26]

[0159] (In formula (4), R is RCOO(CH2)2COO-, which represents a residue formed by the reaction of the hydroxyl group of α-tocopherol, β-tocopherol, α-tocotrienol or β-tocotrienol with succinic anhydride.

[0160] [Chemistry 27]

[0161] (In formula (5), R is RCOO(CH2)2COO-, which represents a substance in which the hydroxyl group of α-tocopherol, β-tocopherol, α-tocotrienol or β-tocotrienol reacts with succinic anhydride to form a residue.

[0162] The vitamin E derivative of formula (3) can be produced by a known method (e.g., US 9708628 B2, WO2019 / 188867A1).

[0163] From the viewpoint of the internal sealing (encapsulation) efficiency of nucleic acid, the release efficiency of nucleic acid in cells, and the stability of lipid nanoparticles, the total amount of vitamin E and / or its derivatives in the lipid nanoparticles of the present invention is preferably 4.5 to 35 mol%, more preferably 10 to 30 mol%, relative to the total amount of ionic lipid (1), vitamin E and / or its derivatives, phospholipids, and cholesterol. In addition, the lipid nanoparticles of the present invention may use only any one of vitamin E or vitamin E derivatives, or may use these in combination, and preferably any one of them may be used.

[0164] Phospholipids The lipid nanoparticles of the present invention contain phospholipids. Only one phospholipid can be used, or phospholipids or other phospholipids can be used in combination. In addition, at this time, if the ratio of other phospholipids to phospholipids is within the scope of achieving the effect of the present invention, there is no particular limitation.

[0165] As specific examples of phospholipids, 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), lysates of these substances, etc. In addition, in this specification, there is a case where phospholipids are recorded by their abbreviations. For example, there is a case where 1,2-diacyl-sn-glycero-3-phosphocholine is recorded as PC, and 1,2-didecanoyl-sn-glycero-3-phosphocholine is recorded as DDPC.

[0166] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) include: 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-diazainoyl-sn-glycero-3-phosphocholine (DAPC) 1,2-di(eicosenoyl)-sn-glycero-3-phosphocholine (DEiPC), 1,2-Dibehenyl-sn-glycero-3-phosphocholine (DBPC), 1,2-Dierucyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

[0167] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) include: 1,2-Didecanoyl-sn-glycero-3-phosphoethanolamine (DDPE), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1,2-Dierucyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine (MPPE), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (MSPE), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphoethanolamine (PMPE), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (PSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

[0168] The phospholipid is not particularly limited, but is preferably at least one selected from the group consisting of DOPC, DSPC, POPC, DOPE, and POPE, and more preferably at least one of DOPC and DOPE. Since these phospholipids have similar acyl chain structures, they are considered to exert the same effect.

[0169] From the viewpoints of nucleic acid internalization efficiency, nucleic acid release efficiency in cells and stability of lipid nanoparticles, the amount of phospholipids in the lipid nanoparticles of the present invention is preferably 5 to 35 mol%, more preferably 6 to 30 mol%, relative to the total of ionic lipid (1), vitamin E and / or its derivatives, phospholipids and cholesterol.

[0170] cholesterol The lipid nanoparticles of the present invention contain cholesterol. From the viewpoint of the internalization efficiency of nucleic acids, the release efficiency of nucleic acids in cells and the stability of lipid nanoparticles, the amount of cholesterol in the lipid nanoparticles of the present invention is preferably 10 to 50 mol%, more preferably 30 to 45 mol%, and further preferably 30 to 40 mol%, relative to the total of ionic lipid (1), vitamin E and / or its derivatives, phospholipids and cholesterol.

[0171] PEG lipids The lipid nanoparticles of the present invention contain PEG lipids. The PEG lipids are not particularly limited, but are preferably compounds represented by formula (7).

[0172] It contains dimyristoylglycerol PEG represented by formula (7) (sometimes referred to as "dimyristoylglycerol PEG (7)" in this specification): CH2(OR 6 )-CH(OR 7 )-CH2(OR 8 )(7) (In formula (7), R 6 , R 7 and R 8 Any two of them represent myristoyl groups, and the remaining one represents an alkyl group having 1 to 6 carbon atoms connected via a polyethylene glycol (PEG) chain having a number average molecular weight of 1,000 to 3,000. ).

[0173] The number average molecular weight of the PEG chain in formula (7) is 1,000 to 3,000, preferably 1,500 to 2,500. The number average molecular weight of PEG used to form the PEG chain can be measured by gel permeation chromatography (GPC).

[0174] The alkyl group having 1 to 6 carbon atoms may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, cyclohexyl, etc. Methyl is preferred.

[0175] From the viewpoints of the internalization (encapsulation) efficiency of nucleic acids, the release efficiency of nucleic acids in cells, and the stability of lipid nanoparticles, the amount of PEG lipid in the lipid nanoparticles of the present invention is preferably 0.5 to 4 mol%, more preferably 1 to 3 mol%, and most preferably 1.5 mol%, relative to the total of ionic lipid (1), vitamin E and / or its derivatives, phospholipids and cholesterol.

[0176] Other lipids The lipid nanoparticles of the present invention may also contain lipids other than the ionic lipids represented by formula (1), vitamin E and / or its derivatives, phospholipids, cholesterol and PEG lipids (hereinafter referred to as "other lipids"). Examples of other lipids include sterols other than cholesterol, PEG phospholipids, and the like.

[0177] The amount of other lipids in the lipid nanoparticles of the present invention is preferably 0 to 50 mol%, more preferably 0 to 30 mol%, and further preferably 0 to 10 mol%, relative to the total amount of lipids in the lipid nanoparticles. The term "the total amount of lipids in the lipid nanoparticles" refers to "the total amount of ionic lipids, vitamin E and / or its derivatives, phospholipids, cholesterol, PEG lipids and other lipids represented by formula (1)" when the particles contain the ionic lipids, vitamin E and / or its derivatives, phospholipids, cholesterol, PEG lipids and other lipids as constituent components. In addition, in the present specification, "the amount of B relative to A (mol%)" refers to "100×the amount of B (mol) / the amount of A (mol)". For example, "the amount of other lipids relative to the total amount of lipids (mol%)" refers to "100×the amount of other lipids (mol) / the total amount of lipids (mol)".

[0178] In the present invention, no other lipids are used, that is, the lipids constituting the lipid nanoparticles of the present invention are most preferably composed of an ionic lipid represented by formula (1), vitamin E and / or its derivatives, phospholipids, cholesterol and PEG lipids.

[0179] Lipid composition As the lipid composition in the lipid nanoparticles of the present invention, the molar ratio of ionic lipid (1): vitamin E and / or its derivatives (excluding the vitamin derivatives of formula (3)): phospholipid: cholesterol: PEG lipid is preferably 30-55:4.5-20:5-15:20-50:1-3, more preferably 40-50:10-20:5-10:30-40:1-2, and most preferably 43.8:16.7:6.2:33.3:1.5.

[0180] On the other hand, when using the vitamin derivative of formula (3), the optimal composition in the lipid nanoparticles of one embodiment of the present invention is: the molar ratio of ionic lipid (1): vitamin E derivative (3): phospholipid: cholesterol: PEG lipid is preferably 20-55:4.5-35:5-35:10-50:1-3, more preferably 20-50:10-30:7.5-30:10-45:1-3, further preferably 25-40:10-30:7.5-10:30-45:1-3, and most preferably 32.5:20:7.5:40:1.5.

[0181] When using the vitamin derivative of formula (3), the optimal composition in the lipid nanoparticles of other embodiments of the present invention is: the molar ratio of ionic lipid (1): vitamin E derivative (3): phospholipid: cholesterol: PEG lipid is preferably 20-55:4.5-35:5-35:10-50:1-3, more preferably 20-50:10-30:7.5-30:10-40:1-3, and most preferably 32.5:20:7.5:40:1.5.

[0182] Method for producing lipid nanoparticles The lipid nanoparticles of the present invention are manufactured by a vitamin addition step of adding vitamin E and / or its derivatives to the ionic lipids, phospholipids, cholesterol and PEG lipids represented by formula (1). Specifically, the lipid nanoparticles can be manufactured by adding a solution obtained by dispersing vitamin E and / or its derivatives in a suitable dispersion medium (e.g., an aqueous dispersion medium, an alcoholic dispersion medium) to a solution in which a lipid raw material containing ionic lipids (1), phospholipids, cholesterol and PEG lipids is dispersed in a suitable dispersion medium (e.g., an aqueous dispersion medium, an alcoholic dispersion medium), preparing a lipid solution, and inducing organization as needed (in this specification, there is a case referred to as "pre-addition method").

[0183] Examples of the "organization-inducing procedure" for producing the lipid nanoparticles of the present invention by the pre-addition method include: an ethanol dilution method using microfluidics or vortex, a simple hydration method (pure water method), ultrasonic treatment, heating, vortexing, ether injection method, French Press method, bile acid method, Ca 2+ method, etc. 2+ Self known methods such as fusion method, freeze-thaw method, reverse phase evaporation method, preferably use the ethanol dilution method of microfluid or eddy current, further preferably use the ethanol dilution method of microfluid.In using the ethanol dilution method of microfluid, for example, can be by using NanoAssemblr (registered trademark) (Precision NanoSystems company), the acidic buffer containing purpose encapsulation (preferably nucleic acid) is mixed with the ethanol solution of the lipid prepared as mentioned above, manufacture contains the dispersion liquid of the lipid nano particle that has been enclosed in this encapsulation.The dispersion liquid manufactured according to this method contains this lipid nano particle and dispersion medium (acidic buffer and ethanol), but can carry out the removal of dispersion medium (especially ethanol), the exchange of dispersion medium (especially buffer) etc. by operations such as ultrafiltration, dialysis, dilution.

[0184] As another embodiment for producing the lipid nanoparticles of the present invention enclosing the target encapsulated object (preferably nucleic acid), it is also possible to produce the lipid nanoparticles by using a lipid solution prepared by dispersing a lipid raw material containing ionic lipid (1), phospholipid, cholesterol and PEG lipid in an appropriate dispersion medium (e.g., an aqueous dispersion medium, an alcoholic dispersion medium), and an acidic buffer containing the encapsulated object to prepare a dispersion liquid, and then removing the dispersion medium (especially ethanol) by ultrafiltration, dialysis, dilution and the like, exchanging the dispersion medium (especially buffer), and adding vitamin E or a vitamin E derivative (in this specification, sometimes referred to as "post-addition method") through steps such as removal of the dispersion medium (especially ethanol) and exchange of the dispersion medium (especially buffer).

[0185] Examples of methods for preparing a dispersion containing lipid nanoparticles by post-addition include: ethanol dilution method using microfluidics or vortex flow, simple hydration method, ultrasonic treatment, heating, vortex flow, ether injection method, French press method, bile acid method, Ca 2+ method, etc. 2+The self known methods such as fusion method, freeze-thaw method, reverse phase evaporation method, etc. are preferably the ethanol dilution method using microfluid or eddy current, and more preferably the ethanol dilution method using microfluid.For example, when adding vitamin E, vitamin E derivative to the dispersion prepared according to the ethanol dilution method using microfluid to manufacture lipid nano particles of the present invention, the ethanol solution of vitamin E, vitamin E derivative can also be directly added in any process after the dispersion is prepared, but preferably after the dispersion is just prepared, during the dilution operation of the dispersion medium, or by ultrafiltration, dialysis, at least any one process when exchanging the dispersion medium (especially buffer) is added.

[0186] Methods for obtaining acquired immunity based on nucleic acid delivery The present invention also provides a method for imparting acquired immunity to a subject by delivery of an encapsulated substance, comprising the step of administering to the subject a lipid nanoparticle of the present invention encapsulated with the target encapsulated substance (preferably a nucleic acid). In this method, the lipid nanoparticle is preferably administered subcutaneously or intramuscularly to the subject.

[0187] The encapsulated material enclosed in the lipid nanoparticles of the present invention is not particularly limited as long as it is given the desired acquired immunity by being delivered into the cells of the object, and examples thereof include: proteins or fragments thereof or nucleic acids, etc. As nucleic acids, for example, DNA, RNA, chimeric nucleic acids of RNA, hybrids of DNA / RNA, etc., can be mentioned, but are not limited to these. In addition, nucleic acids can use any one of 1 to 3 chains, but are preferably single-stranded or double-stranded. Nucleic acids can also be, for example, nucleotides having N-glycosides of purine or pyrimidine bases, oligomers having non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNA), etc.), or oligomers having special bonds (wherein, the oligomer contains nucleotides having a configuration that allows base pairing and base adhesion as found in DNA and RNA), etc.

[0188] Furthermore, the nucleic acid may be, for example, a nucleic acid to which a known modification is added, a nucleic acid having a label known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides are substituted with an analog, a nucleic acid in which nucleotides are modified, a nucleic acid having a non-charged bond (e.g., methanesulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid containing a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having side chain groups such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.), sugars (e.g., monosaccharides, etc.), a nucleic acid containing an intercalating compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., a metal, a radioactive metal, boron, an oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-isomer nucleic acid, etc.), and the like.

[0189] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of use. Examples of DNA include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligonucleotides, etc., preferably plasmid DNA, cDNA, antisense DNA, and more preferably plasmid DNA. Circular DNA such as plasmid DNA can also be consumed by appropriate restriction enzymes and used as linear DNA.

[0190] The type of RNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of use. As RNA, for example, messenger RNA (mRNA), mRNA precursor, single-stranded RNA genome, double-stranded RNA genome, siRNA, miRNA, shRNA, antisense RNA, RNA replicon, transfer RNA, ribosomal RNA, etc. can be mentioned, preferably mRNA, mRNA precursor, siRNA, miRNA, shRNA, antisense RNA, RNA replicon.

[0191] The nucleic acid used in the present invention is preferably purified by a method commonly used by those skilled in the art.

[0192] The nucleic acid enclosed in the lipid nanoparticles of the present invention is preferably a nucleic acid (preventive / therapeutic nucleic acid) having preventive and / or therapeutic activity for a specific disease. As such nucleic acids, for example, nucleic acids used for so-called gene therapy and vaccine applications can be cited. As nucleic acids used for vaccine applications, nucleic acids (expression vectors, mRNA (precursor)) encoding antigenic proteins or peptides presented on the surface of pathogenic cells (including pathogens, cancer cells) and viruses of the target disease can be cited. When the lipid nanoparticles of the present invention enclosed in the nucleic acid are delivered to the cell, the nucleic acid is (transcribed and) translated to generate antigenic proteins or peptides, which are recognized by immune cells such as B cells and T cells, and neutralizing antibodies and killer T cells specific to the antigenic protein or peptide are produced, and acquired immunity is given. Depending on the disease as the target, those skilled in the art can appropriately select nucleic acids encoding such antigenic proteins or peptides for use. For example, when used as a vaccine for COVID-19, DNA and mRNA encoding spike proteins or fragments thereof derived from SARS-CoV-2 viruses can be appropriately designed based on the genome sequence information of the virus.

[0193] The particle size of the lipid nanoparticles encapsulating nucleic acid is not particularly limited, but is preferably 10nm to 500nm, more preferably 30nm to 300nm. For the determination of particle size, for example, a particle size distribution measuring device such as ZetasizerNano (Malvern Company) can be used. The particle size of the lipid nanoparticles encapsulating nucleic acid can be appropriately adjusted by its manufacturing method.

[0194] The surface potential (ZETA potential) of lipid nanoparticles of nucleic acid vaccines for effectively improving acquired immunity is preferably -15 to +15mV, and more preferably -10 to 10mV. In previous gene introduction, particles with positive surface charge were mainly used. This is useful as a method for promoting electrostatic interaction with heparan sulfate on the negatively charged cell surface and promoting cell uptake. However, there is a possibility that (a) the release of nucleic acid from the carrier caused by the interaction with the delivered nucleic acid in the cell is inhibited, and (b) the protein synthesis caused by the interaction between mRNA and the delivered nucleic acid is inhibited due to the positive surface potential. This problem can be solved by adjusting the surface potential (ZETA potential) to the above range. For the determination of the surface potential (ZETA potential), for example, a ZETA potential measuring device such as Zetasizer Nano can be used. The surface potential (ZETA potential) of lipid nanoparticles can be adjusted according to the composition of the constituent components of the lipid nanoparticles.

[0195] By administering the lipid nanoparticle of the present invention enclosed in nucleic acid to an organism, the lipid nanoparticle is taken in by cells, and the nucleic acid enclosed in the lipid nanoparticle is delivered to the cell in vivo to perform a function, thereby obtaining acquired immunity. As the object that can administer the lipid nanoparticle, it is not particularly limited, for example, can be enumerated: the cell of mammals (for example people, monkeys, mice, rats, hamsters, cattle, etc.), birds (for example chickens, ostriches, etc.), amphibians (for example frogs, etc.), fish (for example zebrafish, killifish, etc.). The object of importing the lipid nanoparticle is preferably the cell of people or other mammals.

[0196] As a method for administering lipid nanoparticles encapsulating nucleic acids to a subject, there is no particular limitation as long as the lipid nanoparticles can deliver nucleic acids to spleen cells, and a known method of administration (e.g., oral administration, parenteral administration (e.g., nasal administration, intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray administration, etc.)) can be appropriately selected. As a method of administration, intramuscular administration and subcutaneous administration are preferred. The dosage of the lipid nanoparticles can be appropriately selected in consideration of the type of the subject of administration, the method of administration, etc.

[0197] The lipid nanoparticles of the present invention can be directly or mixed with a pharmaceutically acceptable carrier as an oral preparation (e.g., tablets, capsules, etc.) or a non-oral preparation (e.g., nasal preparations, injections, inhalants, etc.), preferably manufactured as a non-oral preparation (more preferably an injection).

[0198] As pharmaceutically acceptable carriers, carriers commonly used as raw materials for preparations are used, for example, in solid preparations, excipients, lubricants, binders and disintegrants are used; in liquid preparations, solvents, solubilizers, suspending agents, penetrants, buffers and pain-relieving agents are used. In addition, preparation additives such as preservatives, antioxidants, colorants, sweeteners, etc. may be used as needed. For example, in the case of a nasal preparation, it can be used in the form of nasal drops or sprays. Example

[0199] The present invention will be described in further detail below using Examples and the like, but the present invention is not limited to the following Examples and the like.

[0200] In the following examples, etc., the ionic lipid (1) and the ionic lipid having a vitamin E skeleton are represented by the names described in the above table. In addition, the abbreviations used in the following examples, etc. have the following meanings. Chol: Cholesterol DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (PEG number average molecular weight (Mn): 2000) VE: D-α-tocopherol TS: D-α-Tocopheryl Succinate MES: 2-morpholineethanesulfonic acid PBS: Phosphate buffered saline OVA-mRNA: mRNA encoding ovalbumin Luc-mRNA: mRNA encoding luciferase CFSE: Carboxyfluorescein Diacetate Succinimidyl Ester The mol% of the lipid composition indicates the mol% relative to the total of the ionic lipid (1), vitamin E and / or its derivative, phospholipid and cholesterol.

[0201] [Production Example 1] Preparation 1 of mRNA-encapsulated LNPs by ethanol dilution method (post-addition method) (1) Preparation of lipid ethanol solution Each lipid was dissolved in ethanol to obtain 10 mM SS-OP, 5 mM SS-EC, 10 mM VE, 10 mM TS, 10 mM DOPC, 10 mM DOPE, 10 mM Chol, and 1 mM DMG-PEG2000. Each lipid selected from these solutions was mixed at an arbitrary ratio so that the total mole amount of SS-OP, DOPC, and Chol was 1000 nmol, and a 1 mM DMG-PEG2000 ethanol solution was added thereto to obtain the target ratio. Finally, ethanol was added to a total amount of 450 μL to prepare an ethanol solution of lipids.

[0202] (2) Preparation of acidic buffer solution for nucleic acid The acidic buffer solution of nucleic acid is prepared by mixing mRNA encoding luciferase or ovalbumin (Luc-mRNA or OVA-mRNA) in 20 mM acid malic acid buffer (pH 3.0) containing 30 mM NaCl to a concentration of 0.0067 μg / μL.

[0203] (3) Preparation of dispersion by ethanol dilution method Using NanoAssemblr (registered trademark) ultra-high-speed nanomedicine production apparatus (manufactured by Precision NanoSystems), 1200 μL of the acidic buffer solution of nucleic acid and 300 μL of the ethanol solution of lipid were mixed at flow rates of 3 mL / min and 1 mL / min, respectively, and 1 mL of the dispersion was recovered.

[0204] (3-1) Adding VE to the dispersion (post-addition method 1) After adding 10mM VE solution to the recovered dispersion in any proportion, dilute with 3mL of 20mM MES buffer (pH6.5) and transfer to Amicon Ultra 4 (Millipore). The transferred LNP solution is ultrafiltered under centrifugal conditions (25°C, 1000g, about 10 minutes) and concentrated to about 500μL. PBS is used to dilute the obtained concentrate to 4mL, ultrafiltered again under centrifugal conditions (25°C, 1000g, about 10 minutes), and concentrated to about 200μL. Finally, PBS is used to be constant to a nucleic acid concentration of 10μg / mL.

[0205] (3-2) Adding VE to the diluted LNP (post-addition method 2) The recovered dispersion was diluted with 3 mL of 20 mM MES buffer (pH 6.5), 10 mM VE solution was added thereto in any proportion, and transferred to Amicon Ultra 4 (Millipore). The transferred LNP solution was ultrafiltered under centrifugal conditions (25 ° C, 1000 g, about 10 minutes) and concentrated to about 500 μL. PBS was used to dilute the resulting concentrate to 4 mL, and ultrafiltered again under centrifugal conditions (25 ° C, 1000 g, about 10 minutes) and concentrated to about 200 μL. Finally, PBS was used to set the volume to a nucleic acid concentration of 10 μg / mL.

[0206] (3-3) Adding VE to LNP during ultrafiltration (post-addition method 3) The recovered dispersion was diluted with 3 mL of 20 mM MES buffer (pH 6.5) and transferred to Amicon Ultra4 (Millipore). The transferred LNP solution was ultrafiltered under centrifugal conditions (25 ° C, 1000 g, about 10 minutes) and concentrated to about 500 μL. PBS was used to dilute the resulting concentrate to 4 mL, 10 mM VE solution was added thereto in any proportion, and ultrafiltered again under centrifugal conditions (25 ° C, 1000 g, about 10 minutes) and concentrated to about 200 μL. Finally, PBS was used to set the volume to a nucleic acid concentration of 10 μg / mL.

[0207] [Production Example 2] Preparation of mRNA-encapsulated LNP Configuration 2 by ethanol dilution method (pre-addition method) (1-1) Preparation of lipid ethanol solution Each lipid was dissolved in ethanol to 10 mM SS-OP, 5 mM SS-EC, 10 mM VE, 10 mM TS, 10 mM DOPC, 10 mM DOPE, 10 mM Chol, and 1 mM DMG-PEG2000. Each lipid selected from these solutions was mixed at an arbitrary ratio so that the total mole amount of SS-OP, DOPC, and Chol was 1000 nmol, and an ethanol solution of 10 mM VE, 10 mM TS or 5 mM SS-EC and 1 mM DMG-PEG2000 was added thereto at a target ratio. Finally, ethanol was added to a total amount of 450 μL to prepare an ethanol solution of lipids.

[0208] (1-2) Preparation of lipid ethanol solution Each lipid was dissolved in ethanol to 10 mM SS-OP, 5 mM SS-EC, 10 mM DOPC, 10 mM DOPE, 10 mM Chol, and 1 mM DMG-PEG2000. Each lipid selected from these solutions was mixed at an arbitrary ratio so that the total mole amount of SS-OP, SS-EC, DOPC or DOPE, and Chol was 1000 nmol, and a 1 mM DMG-PEG2000 ethanol solution was added thereto at a target ratio. Finally, ethanol was added to a total amount of 450 μL to prepare an ethanol solution of lipids.

[0209] (2) Preparation of acidic buffer solution for nucleic acid The acidic buffer solution of nucleic acid is prepared by mixing mRNA encoding luciferase or ovalbumin (Luc-mRNA or OVA-mRNA) in 20 mM acid malic acid buffer (pH 3.0) containing 30 mM NaCl to a concentration of 0.0067 μg / μL.

[0210] (3) Preparation of LNP by ethanol dilution method Using NanoAssemblr (registered trademark) ultra-high-speed nano drug production device (manufactured by PrecisionNanoSystems), 1200 μL of acidic buffer solution of nucleic acid and 300 μL of ethanol solution of lipid were mixed at flow rates of 3 mL / min and 1 mL / min, respectively, and 1 mL of dispersion was recovered. The recovered dispersion was diluted with 3 mL of 20 mM MES buffer (pH 6.5) and transferred to Amicon Ultra 4 (Millipore). The transferred LNP solution was ultrafiltered under centrifugal conditions (25 ° C, 1000g, about 10 minutes) and concentrated to about 500 μL. The resulting concentrate was diluted to 4 mL with PBS, and ultrafiltered again under centrifugal conditions (25 ° C, 1000g, about 10 minutes) and concentrated to about 200 μL. Finally, PBS was used to make the volume to a nucleic acid concentration of 10 μg / mL.

[0211] [Test Example 1] Evaluation of the physical properties of various mRNA-encapsulated LNP particles The particle size and surface potential of the LNPs in Examples 1 to 31 and Comparative Examples 1 to 4, which had different lipid compositions prepared in Production Example 1 and Production Example 2, were measured by dynamic scattering (Zetasizer Nano; Malvern). The results are shown in Tables 3 to 7. All LNPs had excellent particle size and surface charge.

[0212] [Table 3] Evaluation of VE, TS, SS-EC Addition Methods (In the table, the composition ratio is mol%, and the particle size is the Z average particle size)

[0213] [Table 4] Evaluation of OP / EC gene expression, CTL activity, and antibody production SS-EC was added by the first addition method to prepare LNP (In the table, the composition ratio is mol%)

[0214] [Table 5] Evaluation of the influence of L / R ratio SS-EC was added by the first addition method to prepare LNP. For nucleic acid, OVA-mRNA was encapsulated (in the table, the composition ratio is mol%, and the L / R ratio is the weight of mRNA relative to the total lipid mol amount excluding DMG-PEG2000, in nmol / μg)

[0215] [Table 6] Evaluation of IL-6 production SS-EC was added by the first addition method to prepare LNP (In the table, the composition ratio is mol%) [Table 7] Composition Impact Assessment SS-EC was added by the first addition method to prepare LNP (In the table, the composition ratio is mol%)

[0217] [Test Example 2] Evaluation of CTL activity using LNP containing VE (Study of addition method) Subcutaneous administration to mice The OVA-mRNA encapsulated LNP prepared by the method described in Production Example 1 or Production Example 2 was diluted with PBS to an mRNA concentration of 2.0 μg / mL. The diluted mRNA encapsulated LNP was subcutaneously administered to 6-week-old female C57BL / 6J mice at 50 μL per mouse (0.1 μg per mouse in terms of mRNA administration). Evaluation of CTL activity One week after administration, the CTL assay described below was performed to evaluate the activity of OVA-specific cytotoxic T cells (CTL). As a culture medium for spleen cells, the following composition was used: 500 mL RPMI1640, 50 mL FBS, 5 mL 100 mM sodium pyruvate, 5 mL 1 M HEPES, 500 μL 55 mM 2-mercaptoethanol, 5 mL penicillin / streptomycin (hereinafter referred to as culture medium). Untreated mice were euthanized by cervical dislocation, and the spleen was removed. The spleen was cut in the middle, and spleen cells were removed from the cut surface using forceps and suspended in culture medium. The cell suspension culture medium was recovered into a 50 mL tube through a 40 μm cell strainer and centrifuged (4°C, 500g, 5 minutes). The supernatant was discarded, and 1 mL of red blood cell lysis buffer (SIGMA) was added to each mouse and allowed to stand for 5 minutes. After diluting to 5 times with culture medium, centrifugation was performed (4°C, 500g, 5 minutes). Further suspension was performed in 10 mL of culture medium and centrifugation was performed (4°C, 500g, 5 minutes). The cells were suspended in 30 mL of culture medium and the cell number was measured. Afterwards, the cells were passed through a 40 μm cell strainer and divided equally into two 50 mL test tubes and centrifuged (4°C, 500g, 5 minutes). Suspended in culture medium until it became 1.0×10 7 cells / mL. (1) Add 2 mM OVA epitope (composed of the amino acid sequence of positions 257-264 of OVA) solution in an amount of 1 / 400 of the cell suspension to one cell suspension and let it stand in an incubator for 1 hour. Centrifuge the epitope pulse group (4°C, 500g, 5 minutes). Wash with 10 mL of culture medium and then with 10 mL of PBS. After measuring the cell number again, suspend until it becomes 3.0×10 7 cells / mL, and stained with CFSE at a final concentration of 5 μM to obtain "target cells". (2) The other cell suspension was cultured without adding epitopes, and washed with culture medium and PBS. 7 cells / mL, and stained with CFSE at a final concentration of 0.5 μM to obtain “control cells”. Both target cells and control cells were washed twice with culture medium, and then washed twice with PBS, and suspended to a concentration of 5.0×10 7 Cells / mL. 100 μL of "target cells" and 100 μL of "control group cells" were mixed and administered to immunized mice. The two could be distinguished by the fluorescence intensity of CFSE. 20 hours after administration of the cell mixture, spleens were removed from mice and analyzed by flow cytometry. The presence of "target cells" was corrected by "control group cells" to quantify epitope-specific CTL activity.

[0218] Evaluation results such as Figure 1 As shown. When LNPs prepared by adding VE in any addition method, high CTL activity was shown relative to Comparative Example 1. In particular, the LNPs prepared in Example 3 (post-addition method 2) or Example 4 (post-addition method 3) showed about 4 times the CTL activity relative to Comparative Example 1. CTL activity is an indicator for evaluating whether acquired immunity is obtained, and the higher the CTL activity, the more acquired immunity can be obtained. Therefore, the LNPs of Examples 1 to 4 show improved acquired immunity compared to Comparative Example 1 of the prior art.

[0219] [Test Example 3] Evaluation of CTL activity using LNP containing VE (Study of VE addition amount) (1) Subcutaneous administration to mice According to the method described in [Test Example 2], LNPs encapsulating OVA-mRNA were subcutaneously administered to mice (the dosage of mRNA was 0.1 μg per mouse). (2) Evaluation of CTL activity The CTL activity was evaluated according to the method described in [Test Example 2]. Figure 2 When any one of the LNPs of Examples 4 to 6 was used, high CTL activity was shown relative to Comparative Example 1. In particular, the LNP of Example 6 to which 16.7 mol% VE was added showed about 2.3 times the CTL activity relative to the LNP of Comparative Example 1. This result shows that the LNPs of Examples 4 to 6 improve acquired immunity compared to Comparative Example 1 of the prior art.

[0220] [Test Example 4] Evaluation of CTL activity using LNP containing TS (Study of TS addition amount) Subcutaneous administration to mice LNPs encapsulating OVA-mRNA were subcutaneously administered to mice according to the method described in [Test Example 2] except that the mRNA concentration of the prepared LNPs was halved (the dosage of mRNA was 0.05 μg per mouse). Evaluation of CTL activity The CTL activity was evaluated according to the method described in [Test Example 2]. Figure 3 When any one of the LNPs of Examples 7 to 9 was used, high CTL activity was shown relative to Comparative Example 1. In particular, the LNP of Example 9 to which 16.7 mol% TS was added showed about 5.4 times the CTL activity relative to the LNP of Comparative Example 1. This result shows that the LNPs of Examples 7 to 9 further improve acquired immunity compared to Comparative Example 1 of the prior art.

[0221] [Test Example 5] CTL evaluation using various LNPs (comparison of pre-addition method and post-addition method) Subcutaneous administration to mice [Manufacturing example] LNPs encapsulating OVA-mRNA were subcutaneously administered to mice according to the method described in [Test Example 2] (mRNA dosage: 0.025 μg per mouse), except that the mRNA concentration of the prepared LNPs was reduced to 1 / 4. Evaluation of CTL activity The CTL activity was evaluated according to the method described in [Test Example 2]. Figure 4 As shown. When using the LNPs of Example 6 and Examples 9 to 13, they all showed high CTL activity compared to Comparative Example 1, and it was determined that the CTL activity was improved regardless of the addition method. This shows that no matter which addition method is used, it is possible to further improve acquired immunity compared to Comparative Example 1 of the prior art of LNP. In particular, the LNPs of Examples 12 and 13 to which 9.1 mol% SS-EC was added further improved CTL activity compared to the conditions of adding 16.7 mol% VE and TS.

[0222] [Test Example 6] Evaluation of gene expression activity using LNP containing SS-EC (Study of SS-EC addition amount) Subcutaneous administration to mice The Luc-mRNA encapsulated LNP prepared according to the method described in [Manufacturing Example 2] was diluted with PBS to an mRNA concentration of 5.0 μg / mL. The diluted mRNA encapsulated LNP was subcutaneously administered to 6-week-old female C57BL / 6J mice at 200 μL per mouse (mRNA dosage, 1.0 μg per mouse). Determination of gene expression activity 5.5 hours after LNP administration, Luciferin solution was intraperitoneally administered to mice. 30 minutes after administration, the gene expression activity at the administration site was evaluated using IVIS. Figure 5 It was found that any one of the LNPs of Examples 14 to 17 exhibited the same gene expression activity as the LNP of Comparative Example 2. This result shows that the LNPs of Examples 14 to 17 have the same nucleic acid delivery efficiency as that of Comparative Example 2.

[0223] [Test Example 7] Evaluation of CLT activity using LNP containing SS-EC (Study of SS-EC addition amount) Subcutaneous administration to mice LNPs encapsulating OVA-mRNA were subcutaneously administered to mice (mRNA dosage: 0.05 μg per mouse) according to the method described in [Test Example 2] except that the mRNA concentration of the prepared LNPs was halved. CTL activity evaluation The CTL activity was evaluated according to the method described in [Test Example 2]. Figure 6 As shown. When any one of the LNPs of Examples 18 to 21 was used, high CTL activity was shown relative to Comparative Example 1. This shows that any addition amount can further improve acquired immunity compared to Comparative Example 1 of the prior art LNP. In particular, the LNP of Comparative Example 21 to which 30 mol% SS-EC was added showed 4.3 times the CTL activity of Comparative Example 1. Although the nucleic acid delivery efficiency in [Test Example 6] was equivalent to that of the LNP of the Comparative Example, since the LNPs of Examples 18 to 21 improved the CTL activity, it was shown that these LNPs efficiently improved acquired immunity.

[0224] [Test Example 8] Evaluation of in vivo antibody production using LNP containing SS-EC (Study of SS-EC addition amount) Intramuscular administration to mice The LNPs encapsulating OVA-mRNA prepared by the method described in [Manufacturing Example 2] were diluted to a concentration of 30 μg / mL using PBS. The LNPs were administered intramuscularly to 6-week-old female Balb / c mice at a dose of 50 μL per mouse (mRNA dosage, 1.5 μg per mouse). After 14 days of administration, the mice were administered LNPs of the same composition. Evaluation of in vivo antibody production 14 days after the second administration, the blood of the mice was collected to recover the serum, and the amount of anti-OVA antibodies contained in the obtained serum was quantified by ELISA. The results are as follows Figure 7 As shown. When any one of the LNPs of Examples 19 to 21 was used, a high antibody production amount was shown relative to Comparative Example 1. Antibody production against the antigen (OVA) is an indicator of whether acquired immunity is obtained, and the higher the antibody production amount, the more efficient the acquired immunity is. Therefore, it is shown that the LNPs of Examples 19 to 21 improve acquired immunity compared to Comparative Example 1 of the prior art.

[0225] [Test Example 9] Evaluation of in vivo antibody production using LNP containing SS-EC 2 Intramuscular administration to mice According to the method described in [Test Example 8], LNPs encapsulating OVA-mRNA were intramuscularly administered to mice (the dosage of mRNA was 1.5 μg per mouse). Evaluation of in vivo antibody production According to the method described in [Test Example 8], the amount of anti-OVA antibodies contained in the serum was quantified. Figure 8It is shown that: in Comparative Example 3, no antibody was produced, but the LNP of Example 22 produced antibody.

[0226] [Test Example 10] Evaluation of in vivo antibody production using LNP containing SS-EC 3 (Investigation of the influence of the ratio of nucleic acid amount to total lipids) Intramuscular administration to mice According to the method described in [Test Example 8], OVA-mRNA-encapsulated LNPs with different ratios of nucleic acid amount to total lipid amount were prepared and intramuscularly administered to mice (mRNA administration amount: 1.5 μg per mouse). Evaluation of in vivo antibody production According to the method described in [Test Example 8], the amount of anti-OVA antibodies contained in the serum was quantified. Fig. 9 It was shown that the amount of antibody produced was high when the LNPs of any of the Examples (Example 20 and Examples 23 to 27) having different ratios of the amount of nucleic acid to the amount of total lipids were used.

[0227] [Test Example 11] Evaluation of cytokine production using LNP containing SS-EC Subcutaneous administration to mice Empty LNPs without encapsulating nucleic acid were prepared according to the method described in [Production Example 2] and subcutaneously administered to mice (200 nmol in terms of lipid content). Evaluation of cytokine production Six hours after LNP administration, blood was collected and serum was recovered. The amount of IL-6 contained in the obtained serum was quantified by ELISA. The results are as follows: Fig.10 As shown. It is shown that: compared with Comparative Example 4, the IL-6 concentration in the serum of LNPs of Examples 28 to 31 is high. IL-6 is a cytokine produced by cells such as T cells and macrophages, and is responsible for enhancing acquired immunity. That is, a high IL-6 concentration in serum indicates that acquired immunity is efficiently enhanced. Industrial Applicability

[0228] The lipid nanoparticles of the present invention are useful as components of nucleic acid vaccines for obtaining adaptive immunity.

[0229] This application is based on Japanese Patent Application No. 2022-114395 filed in Japan, the entire contents of which are incorporated herein by reference.

Claims

1. A lipid nanoparticle comprising: (A) an ionic lipid represented by formula (1), (B) vitamin E and / or its derivatives, (C) phospholipids, (D) cholesterol, and (E) PEG lipids, In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and having or not having a heteroatom, n a and n b Each independently represents 0 or 1, R 3a and R 3b Each independently represents: (a)R 3a -COO- and R 3b -COO- represents a group derived from a residue of a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, or (b) an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or (c) A group represented by formula (2): R 9 -O-CO-(CH2) a -(2) In formula (2), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer of 2-10. 2 . The lipid nanoparticle according to claim 1 , wherein the vitamin E is any one selected from the group consisting of α-tocopherol, β-tocopherol, α-tocotrienol and β-tocotrienol.

3. The lipid nanoparticle according to claim 1, wherein the vitamin E derivative is any one selected from the group consisting of α-tocopherol succinate, α-tocopherol glutarate, β-tocopherol succinate, β-tocopherol glutarate, α-tocotrienol succinate, α-tocotrienol glutarate, β-tocotrienol succinate and β-tocotrienol glutarate.

4. The lipid nanoparticle according to claim 1, wherein the vitamin E derivative is a compound represented by formula (3): In formula (3), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and having or not having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3b Each independently is R 3a -COO- and R 3b -COO- represents a group derived from a residue of a reaction product of a hydroxyl group of vitamin E and succinic anhydride or glutaric anhydride.

5. The lipid nanoparticle according to claim 1, wherein the vitamin E derivative is any one of the compounds represented by formula (4) or formula (5): In formula (4), R is RCOO(CH2)2COO-, which represents a residue formed by the reaction of the hydroxyl group of α-tocopherol, β-tocopherol, α-tocotrienol or β-tocotrienol with succinic anhydride; In formula (5), R is RCOO(CH2)2COO-, which represents a residue obtained by reacting a hydroxyl group of α-tocopherol, β-tocopherol, α-tocotrienol or β-tocotrienol with succinic anhydride.

6. The lipid nanoparticle according to any one of claims 1 to 5, wherein the ionic lipid is a compound represented by the following formula:

7. The lipid nanoparticles according to claim 5, relative to the total of the ionic lipids, the vitamin E and / or its derivatives, the phospholipids and the cholesterol, the ionic lipids are 20-55 mol%, the vitamin E and / or its derivatives are 4.5-35 mol%, the phospholipids are 5-35 mol%, the cholesterol is 10-50 mol%, and the PEG lipids are 0.5-4 mol%.

8. The lipid nanoparticles according to claim 6, relative to the total of the ionic lipids, the vitamin E and / or its derivatives, the phospholipids and the cholesterol, the ionic lipids are 20-55 mol%, the vitamin E and / or its derivatives are 4.5-35 mol%, the phospholipids are 5-35 mol%, the cholesterol is 10-50 mol%, and the PEG lipids are 0.5-4 mol%. 9 . The lipid nanoparticle according to claim 8 , wherein the phospholipid is 1,2-diacyl-sn-glycero-3-phosphocholine or 1,2-diacyl-sn-glycero-phosphoethanolamine.

10. The lipid nanoparticle according to claim 8, wherein the phospholipid is any one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

11. The lipid nanoparticle according to claim 8, wherein the PEG lipid is dimyristoylglycerol PEG represented by formula (6): CH2(OR 6 )-CH(OR 7 )-CH2(OR 8 )(6) In formula (6), R 6 , R 7 and R 8 Any two of them represent myristoyl groups, and the remaining one represents an alkyl group having 1 to 6 carbon atoms connected via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000.

12. The lipid nanoparticle according to claim 10, wherein the PEG lipid is dimyristoylglycerol PEG represented by formula (6): CH2(OR 6 )-CH(OR 7 )-CH2(OR 8 )(6) In formula (6), R 6 , R 7 and R 8 Any two of them represent myristoyl groups, and the remaining one represents an alkyl group having 1 to 6 carbon atoms connected via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000. 13 . A nucleic acid-encapsulated lipid nanoparticle, comprising the lipid nanoparticle according to claim 1 in which a nucleic acid is encapsulated.

14. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 13, comprising a vitamin adding step of adding the vitamin E and / or its derivatives to the ionic lipid, the phospholipid, the cholesterol and the PEG lipid.

15. The production method according to claim 14, further comprising a step of mixing with a buffer containing nucleic acid after adding the vitamin E and / or its derivative to the alcohol solution containing the ionic lipid, the phospholipid, the cholesterol and the PEG lipid in the vitamin adding step.

16. The production method according to claim 14, wherein in the vitamin adding step, after the alcohol solution containing the ionic lipid, the phospholipid, the cholesterol and the PEG lipid is mixed with the buffer containing the nucleic acid, the vitamin E and / or its derivative is added.

17. A method for imparting acquired immunity to an antigen to an organism, comprising the step of administering to the organism the lipid nanoparticle according to claim 1 encapsulating a nucleic acid expressing the antigen protein.

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