Freeze-dried composition of lipid nanoparticles having nucleic acid encapsulated therein

Abstract

Provided is a freeze-dried composition containing nucleic acid-encapsulated lipid nanoparticles in which an ionic lipid represented by formula (1) has a pH of 4.5-6.9 (inclusive) (wherein the symbols are as defined in the description). # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a freeze-dried composition of lipid nanoparticles encapsulating nucleic acid and a method for producing the same, a method for producing lipid nanoparticles encapsulating nucleic acid obtained by rehydrating the freeze-dried composition, a method for producing a pharmaceutical composition containing the freeze-dried composition or lipid nanoparticles encapsulating nucleic acid obtained by rehydrating the freeze-dried composition, and a method for introducing nucleic acid into cells or target cells. Background Art

[0002] In order to put nucleic acid therapy using oligonucleic acids such as siRNA and gene therapy using mRNA, pDNA, etc. into practical use, an effective and safe nucleic acid delivery vector is needed. Viral vectors are nucleic acid delivery vectors with good expression efficiency, but the development of non-viral nucleic acid delivery vectors that can be used more safely is also underway.

[0003] Since the cationic liposomes using cationic lipids having quaternary amines are positively charged, they can form a complex (lipoplex) with negatively charged nucleic acids through electrostatic interaction, and can deliver nucleic acids into cells. Since a freeze-dried composition of cationic liposomes not containing nucleic acids is prepared by further utilizing quaternary amines to interact electrostatically with nucleic acids, and a lipoplex can be formed by rehydrating with an aqueous solution of nucleic acids, it has been shown that it can also be used as a gene transfer agent (for example, see Patent Documents 1 and 2).

[0004] However, it is difficult to control the particle size of the liposome complex prepared by such a method, and cytotoxicity derived from the positively charged cationic lipids is problematic.

[0005] Therefore, lipid nanoparticles (LNPs) using ionic lipids with tertiary amines that are positively charged under acidic conditions and uncharged near neutrality have been developed. These lipid nanoparticles have now become the most commonly used non-viral nucleic acid delivery vectors (see, for example, Non-Patent Document 1).

[0006] As an example of lipid nanoparticles using an ionic lipid having such a tertiary amine in the molecule, lipid nanoparticles in which a degradable group is imparted to the ionic lipid are known (for example, see Patent Document 3).

[0007] As described above, various non-viral vectors have been developed. However, since nucleic acids are generally unstable compounds, there are still problems with the stability of the formulations. Prior art literature Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2008-5801 Patent Document 2: Japanese Patent Application Publication No. 2001-2565 Patent Document 3: International Publication No. 2013 / 073480 Patent Document 4: International Publication No. 2017 / 218704 Non-patent literature

[0009] Non-patent literature 1: Gene Therapy 6: 271-281, 1999 Non-patent document 2: Supplementary materials of Biol. Pharm. Bull. 41, 1291-1294 (2018) Summary of the invention Problem that the invention aims to solve

[0010] However, as a method for improving the stability of the preparation, an attempt is made to freeze-dry the lipid nanoparticles encapsulated with nucleic acid, and then rehydrate them during use to reconstitute the lipid nanoparticles (e.g., with reference to patent document 4 and non-patent document 2). In view of this, it is necessary that when the lipid nanoparticles encapsulated with nucleic acid are freeze-dried, even after the freeze-drying process and the rehydration process after storage, the physical properties of the particles and the gene delivery efficiency do not change.

[0011] For example, Non-Patent Document 2 discloses a method of using t-BuOH as a solvent for lipids, mixing with a pH 4.0 malic acid buffer to prepare lipid nanoparticles, and freeze-drying the mixture.

[0012] Furthermore, for example, Patent Document 4 discloses a method in which lipid nanoparticles are prepared using EtOH as a solvent for lipids, the solvent is replaced with a buffer solution of pH 7.4, and an amphiphilic polymer is further added to the mixture, followed by freeze-drying.

[0013] However, in any of the examples of Non-Patent Document 2 and Patent Document 4, no comparison is made between the gene delivery efficiency of lipid nanoparticles before freeze drying and the gene delivery efficiency after storage and rehydration. That is, there is no technology for maintaining the gene delivery efficiency before and after freeze drying. In addition, since the ratio of the ionic lipids and other components constituting the lipid nanoparticles is different, the physical properties of the lipid nanoparticles are also very different, so it is necessary to study the freeze drying conditions for each ionic lipid.

[0014] In view of the above problems, the present invention aims to provide a freeze-dried composition of lipid nanoparticles encapsulating nucleic acids, which has improved storage stability that has not been achieved in the prior art, and a method for producing a pharmaceutical composition containing the freeze-dried composition of lipid nanoparticles encapsulating nucleic acids, and a method for introducing nucleic acids into cells or target cells. Means of solving the problem

[0015] Lipid nanoparticles have strong interactions with nucleic acids under acidic conditions, but when administered to cells or organisms, they are preferably neutral from the viewpoint of toxicity. The present inventors have made intensive efforts in view of the above-mentioned problems, and have found that when lipid nanoparticles encapsulating nucleic acids are prepared in an acidic buffer, the storage stability can be improved by replacing the buffer with another buffer having a buffer region at pH 4.5 to 6.9 and then freeze-drying, thereby completing the present invention.

[0016] That is, the present invention includes the following contents. [1] A freeze-dried composition of lipid nanoparticles encapsulating nucleic acid, comprising an ionic lipid represented by formula (1) and having a pH of 4.5 or more and 6.9 or less:

[0017] [Chemistry 1]

[0018] (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 residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, A residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, The group represented by formula (3), A group having 50 or less carbon atoms represented by formula (4), or The group represented by formula (5) having 50 or less carbon atoms, R 9 -O-CO-(CH 2 ) a -*(3) (In formula (3), * indicates the bonding position, R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. )

[0019] [Chemistry 2]

[0020] (In formula (4), * indicates bonding position, and R 10 represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R 10 It may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0021] [Chemistry 3]

[0022] (In formula (5), * indicates the bonding position, R 11 represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynyl group having 2 to 9 carbon atoms, and R 11 may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group and a hydrocarbon ring group having 3 to 12 carbon atoms, R 12 and R 13 Each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, and R 12 At least one ethylene group or at least one trimethylene group in the group may be substituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 13 At least one ethylene group or at least one trimethylene group in R may be substituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 12 and R 13may be substituted independently by a substituent selected from the group consisting of a halogen atom, a hydroxyl group and a hydrocarbon ring group having 3 to 12 carbon atoms, and X 4 represents an oxygen atom, NH or a sulfur atom. ). [2] The freeze-dried composition according to [1], wherein R 3a and R 3b Each independently is: A residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, A residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, 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 The group represented by formula (3) Formula (3): R 9 -O-CO-(CH 2 ) a -*(3) (The definitions of symbols in formula (3) are as described in [1]). [3] The freeze-dried composition according to [1], wherein the ionic lipid is a compound represented by the following formula:

[0023] [Chemistry 4]

[0024] [4] The freeze-dried composition according to [3], further comprising a compound represented by the following formula as the ionic lipid:

[0025] [Chemistry 5]

[0026] [5] The freeze-dried composition according to [1], wherein the ionic lipid is a compound represented by the following formula:

[0027] [Chemistry 6]

[0028] [6] The freeze-dried composition according to [1], wherein the ionic lipid is a compound represented by the following formula:

[0029] [Chemistry 7]

[0030] [7] The freeze-dried composition according to any one of [1] to [6], wherein the nucleic acid encapsulated in the lipid nanoparticles is mRNA. [8] A method for producing a freeze-dried composition of lipid nanoparticles encapsulating nucleic acid, comprising the following steps: a) preparing a suspension of lipid nanoparticles encapsulating nucleic acid by mixing an alcohol solution containing an ionic lipid represented by formula (1) described in [1], a sterol, a phospholipid and a PEG lipid with a nucleic acid solution in an acidic buffer having a buffering effect at pH 1.0 to 6.5, b) replacing the external aqueous phase of the lipid nanoparticles encapsulating nucleic acid with another buffer having a buffering effect at pH 4.5 to 6.9 to obtain a mixture containing the lipid nanoparticles encapsulating nucleic acid at pH 4.5 to 6.9, c) mixing the obtained mixture with a cryoprotectant to prepare a mixture containing 80 to 320 mg / mL of the cryoprotectant and lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9, d) a step of freeze-drying the mixture obtained in step c) to obtain a freeze-dried composition. [9] A method for producing lipid nanoparticles encapsulating nucleic acid, comprising the following steps: a) preparing a suspension of lipid nanoparticles encapsulating nucleic acid by mixing an alcohol solution containing an ionic lipid represented by formula (1) described in [1], a sterol, a phospholipid and a PEG lipid with a nucleic acid solution in an acidic buffer having a buffering effect at pH 1.0 to 6.5, b) replacing the external aqueous phase of the lipid nanoparticles encapsulating nucleic acids with another buffer having a buffering effect at pH 4.5 to 6.9 to obtain a mixture containing lipid nanoparticles encapsulating nucleic acids at pH 4.5 to 6.9, c) mixing the obtained mixture with a cryoprotectant to prepare a mixture containing 80 to 320 mg / mL of the cryoprotectant and lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9, d) freeze-drying the mixture obtained in step c) to obtain a freeze-dried composition, e) A step of mixing the freeze-dried composition with water and incubating the mixture at 0 to 95° C. for 0 to 60 minutes to obtain lipid nanoparticles encapsulating nucleic acid.

[10] A method for introducing a nucleic acid into a cell, comprising the step of contacting a lipid nanoparticle encapsulating the nucleic acid produced by the method described in [9] with the cell in vitro.

[11] A method for introducing a nucleic acid into a target cell, comprising the step of administering to a living body lipid nanoparticles encapsulating the nucleic acid produced by the method described in [9].

[12] A method for producing a pharmaceutical composition, comprising the method described in [8].

[13] A method for producing a pharmaceutical composition, comprising the method described in [9]. Effects of the Invention

[0031] The freeze-dried composition of the lipid nanoparticles encapsulated with nucleic acid of the present invention can be stored for a long time because the reduction in gene delivery efficiency before and after freeze drying is small, and the nucleic acid delivery efficiency does not change during storage. In addition, the lipid nanoparticles encapsulated with nucleic acid prepared by the manufacturing method of the present invention have a small reduction in nucleic acid introduction efficiency before and after freeze drying compared to the prior art, and the nucleic acid delivery efficiency does not change during storage, so it is conducive to introducing genes into cells and organisms, and is particularly useful as a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] [ Figure 1 ] is a graph for evaluating the nucleic acid introduction efficiency of lipid nanoparticles encapsulating nucleic acids in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3. [ Figure 2 ] is a graph for evaluating the temporal changes in the nucleic acid introduction efficiency of lipid nanoparticles encapsulating nucleic acids in Comparative Example 4. [ Figure 3 ] is a graph for evaluating the effects of storage temperature and the presence or absence of incubation on nucleic acid introduction efficiency. [ Figure 4 ] is a diagram for evaluating the effect of the composition of ionic lipids in lipid nanoparticles encapsulating nucleic acids on the efficiency of nucleic acid introduction. [ Figure 5 ] is a graph for evaluating the effects of the type of ionic lipid and the type of buffer in lipid nanoparticles encapsulating nucleic acids on the efficiency of nucleic acid introduction. DETAILED DESCRIPTION

[0033] Embodiments of the present invention will be described below, but the present invention is not limited to these.

[0034] The present invention provides a freeze-dried composition of lipid nanoparticles encapsulating nucleic acid at a pH of 4.5 or higher and 6.9 or lower.

[0035] The pH of the freeze-dried composition can be measured by rehydrating the freeze-dried composition, but since it does not change after the freeze-drying process and the rehydration process, the "freeze-dried composition of lipid nanoparticles encapsulating nucleic acids having a pH of 4.5 or more and 6.9 or less" in the present invention means that the pH measured after the freeze-dried composition is rehydrated is 4.5 or more and 6.9 or less, or the pH of the buffer used before freeze-drying is 4.5 or more and 6.9 or less. More specifically, the "freeze-dried composition of lipid nanoparticles encapsulating nucleic acids having a pH of 4.5 or more and 6.9 or less" means that the pH of the suspension of lipid nanoparticles encapsulating nucleic acids obtained after the freeze-dried composition is rehydrated is 4.5 or more and 6.9 or less, or the pH of the buffer used as the dispersion medium of the suspension of lipid nanoparticles encapsulating nucleic acids before freeze-drying is 4.5 or more and 6.9 or less (in other words, the pH of the suspension of the lipid nanoparticles encapsulating nucleic acids dispersed in the buffer before freeze-drying is 4.5 or more and 6.9 or less). The pH is preferably pH 5.0 or higher and pH 6.9 or lower, and more preferably pH 5.5 or higher and pH 6.9 or lower.

[0036] "Lipid nanoparticles" refer to particles in which the hydrophilic groups of amphiphilic lipids have a membrane structure arranged on the water phase side facing the interface. "Amphiphilic lipids" refer to lipids having both a hydrophilic group showing hydrophilicity and a hydrophobic group showing hydrophobicity. Examples of amphiphilic lipids include ionic lipids, phospholipids, and PEG lipids.

[0037] The lipid nanoparticles used in the present invention contain ionic lipids, sterols and PEG lipids as membrane constituents, and may further contain phospholipids. The particle size of the lipid nanoparticles is not particularly limited, but is preferably 10nm to 500nm, more preferably 30nm to 300nm. The particle size can be measured, for example, using a particle size distribution measuring device such as ZetasizerNano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted according to the method for producing the lipid nanoparticles. In the present invention, "particle size" refers to the average particle size (number average) measured by dynamic light scattering.

[0038] In the present invention, "total lipids" refers to the total amount of lipids. Examples of lipids include ionic lipids, sterols, PEG lipids, and phospholipids.

[0039] In the present invention, "nucleic acid-encapsulating lipid nanoparticles" refers to lipid nanoparticles in which nucleic acid is encapsulated inside the lipid nanoparticles.

[0040] Ionic lipids The ionic lipid that can be used in the present invention is a compound represented by the following formula (1), and a plurality of ionic lipids may be used in combination.

[0041] [Chemistry 8]

[0042] (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 residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, A residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, The group represented by formula (3), A group having 50 or less carbon atoms represented by formula (4), or A group having 50 or less carbon atoms represented by formula (5), R 9 -O-CO-(CH 2 ) a -*(3) (In formula (3), * indicates the bonding position, R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. )

[0043] [Chemistry 9]

[0044] (In formula (4), * indicates bonding position, and R 10 represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R 10 It may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0045] [Chemistry 10]

[0046] (In formula (5), * indicates the bonding position, R 11 represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynyl group having 2 to 9 carbon atoms, and R 11 may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group and a hydrocarbon ring group having 3 to 12 carbon atoms, R 12 and R 13 Each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, and R 12 At least one ethylene group or at least one trimethylene group in the group may be substituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 13 At least one ethylene group or at least one trimethylene group in R may be substituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 12 and R 13 may be substituted independently by a substituent selected from the group consisting of a halogen atom, a hydroxyl group and a hydrocarbon ring group having 3 to 12 carbon atoms, and X 4 represents an oxygen atom, NH or a sulfur atom. )

[0047] The following is a description of the definitions of the symbols in formula (1). R 1a and R 1bEach independently represents an alkylene group having 1 to 6 carbon atoms, which may be straight-chain or branched, but is preferably straight-chain. The number of carbon atoms in the alkylene group is preferably 1 to 4, and 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.

[0048] R 1a With R 1b They can be the same or different, but preferably R 1a For R 1b Same group.

[0049] 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.

[0050] 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.

[0051] A 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 In addition, X 1 Both ends of the following formulae represent bonding positions rather than carbon atoms.

[0052] [Chemistry 11]

[0053] 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.

[0054] 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.

[0055] 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 In addition, X 2 Both ends of the following formulae represent bonding positions rather than carbon atoms.

[0056] [Chemistry 12]

[0057] 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.

[0058] 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 In addition, X 3 Both ends of the following formulae represent bonding positions rather than carbon atoms.

[0059] [Chemistry 13]

[0060] X 3 w is 1 or 2. When w is 1, X 3 When w is 2, X 3 It is piperazinylene.

[0061] X a With X b Can be the same or different, but preferably X a For X b Same group.

[0062] R 2a and R 2b Each 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.

[0063] 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.

[0064] 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, but 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.

[0065] R 2a With R 2b They can be the same or different, but preferably R 2a For R 2b Same group.

[0066] 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.

[0067] Y a With Y b Can be the same or different, but preferably Y aFor Y b Same group.

[0068] Z a 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.

[0069] 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 furothiphene 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.

[0070] 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.

[0071] As Z a and Z b The preferred specific structure of Z 1 In addition, Z 1 Both ends of the following formulae represent bonding positions rather than carbon atoms.

[0072] [Chemistry 14]

[0073] 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.

[0074] 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.

[0075] 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.

[0076] Z a With Z b Can be the same or different, preferably Z a For Z b Same group.

[0077] n a and n b Each independently is 0 or 1. n a With n b Can be the same or different, preferably n a With n b same.

[0078] R 3a and R 3b Each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, a group represented by the formula (3), a group having 50 or less carbon atoms represented by the formula (4), or a group having 50 or less carbon atoms represented by the formula (5).

[0079] In one embodiment of the present invention, R 3a and R 3b Preferably, each independently is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, 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 a group represented by the formula (3). More preferably, each independently is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, More preferably, each independently is an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0080] In another embodiment of the present invention, R 3a and R 3b Preferably, each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group having 50 or less carbon atoms represented by the formula (4), or a group having 50 or less carbon atoms represented by the formula (5).

[0081] In another embodiment of the present invention, more preferably R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or R 3a and R 3b are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or R 3a and R 3b One of them is an aliphatic hydrocarbon group having 12 to 22 carbon atoms and the remaining one is a group having 50 or less carbon atoms represented by the formula (4), or R 3a and R 3bOne of them is an aliphatic hydrocarbon group having 12 to 22 carbon atoms and the remaining one is a group having 50 or less carbon atoms represented by the formula (5).

[0082] In another embodiment of the present invention, it is further preferred that R 3a and R 3b Each independently is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or R 3a and R 3b Each independently is an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0083] The “residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride” means that the hydroxyl group of the fat-soluble vitamin having a hydroxyl group is substituted with *-O-CO-CH 2 -CH 2 - or *-O-CO-CH 2 -CH 2 -CH 2 - The group of the structure of. * indicates the bonding position with the fat-soluble vitamin.

[0084] The “residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride” means that the hydroxyl group of the sterol derivative having a hydroxyl group is substituted with *—O—CO—CH 2 -CH 2 - or *-O-CO-CH 2 -CH 2 -CH 2 -structure. * indicates the bonding position to the sterol derivative.

[0085] Examples of the fat-soluble vitamin having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0086] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and cholesterol or cholestanol is preferred.

[0087] 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. 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. The 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.

[0088] 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 is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is contained 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 a heptadecadienyl group; when oleic acid is used as the fatty acid, it is a heptadecenyl group.

[0089] R 3a and R 3bThe "alkyl group having 3 to 40 carbon atoms and a cyclopropane ring" in the formula 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 in is preferably a group represented by the formula (6).

[0090] [Chemistry 15]

[0091] (In formula (6), * represents a bonding position, b and c each independently represent 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, more preferably an integer of 3 to 17, and more preferably an integer of 4 to 12. More preferably, c is an integer of 3 to 15, more preferably an integer of 3 to 11, and more preferably an integer of 3 to 9. Examples of the group represented by formula (6) include 7-(2-octylcyclopropyl)heptyl and the like.

[0092] In the formula (3), * represents a bonding position other than a carbon atom as described above. In formula (3), R 9 The aliphatic hydrocarbon group having 2 to 20 carbon atoms represented may be linear or branched.

[0093] The aliphatic hydrocarbon group may be saturated or unsaturated. When the aliphatic hydrocarbon group 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. The aliphatic hydrocarbon group includes alkyl, alkenyl, alkynyl, etc., preferably includes alkyl or alkenyl, and more preferably alkyl. Specific examples of the aliphatic hydrocarbon group having 2 to 20 carbon atoms include 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, 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, nonadecenyl, 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, and 1-hexylnonyl, and particularly preferably tridecyl, heptadecyl, heptadecenyl, heptadecadienyl, and heptadecadienyl.

[0094] In formula (3), 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.

[0095] In the formula (4), * represents a bonding position other than a carbon atom as described above. In formula (4), R 10 represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R 10 The group may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0096] In this manual, “R 10 "may be substituted by a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms" (hereinafter referred to as "the above expression") means that R10 The alkyl, alkenyl, alkynyl or hydrocarbon ring group may be substituted independently by a substituent selected from the group consisting of a 3-14-membered heterocyclic group and a hydrocarbon ring group having 3-12 carbon atoms. Other expressions identical to the above expressions also have the same meanings as the above expressions. In addition, in this specification, "alkyl", "alkenyl", "alkynyl", "alkylene", "alkenediyl" and "alkynediyl" which are not particularly described with respect to substituents represent unsubstituted groups.

[0097] In the present specification, the alkyl group may be any type of straight chain or branched chain. Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, 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, nonadecyl, triacontyl, tritriacontyl, tritriacontyl, tetratriacontyl, pentacontyl, hexatriacontyl, tetratetracontyl, tetratetracontyl, tetratetracontyl, tetratetracontyl, tetratetracontyl, tetratetracontyl.

[0098] In this specification, alkenyl can be any kind of straight chain or branched. In addition, in this specification, the number of olefinic carbon-carbon double bonds in alkenyl can be only 1, or more than 2. As alkenyl, for example, vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icodecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicodecenyl, hexadecenyl, dodecenyl, tricadecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, triacontenyl, triacontenyl, triacontenyl.

[0099] In this specification, alkynyl can be any kind of straight chain or branched. In addition, in this specification, the number of carbon-carbon triple bonds of alkynyl can be only 1, or more than 2. As alkynyl, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, 11 carbon alkynyl, 12 carbon alkynyl, 13 carbon alkynyl, 14 carbon alkynyl, 15 carbon alkynyl, 16 carbon alkynyl, 17 carbon alkynyl, 18 carbon alkynyl, 19 carbon alkynyl, 20 carbon alkynyl, 21 carbon alkynyl, 22 carbon alkynyl, 23 carbon alkynyl, 24 carbon alkynyl, 25 carbon alkynyl, 26 carbon alkynyl, 27 carbon alkynyl, 28 carbon alkynyl, 29 carbon alkynyl, 30 carbon alkynyl, 31 carbon alkynyl, 32 carbon alkynyl.

[0100] In the present specification, "a hydrocarbon ring group having 3 to 12 carbon atoms" refers to a group which is a cyclic group and the ring consists of 3 to 12 carbon atoms. Examples of the hydrocarbon ring group having 3 to 12 carbon atoms include a cycloalkyl group having 3 to 8 carbon atoms, a phenyl group, a naphthyl group, and an adamantyl group. Examples of the cycloalkyl group having 3 to 8 carbon atoms include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group. The hydrocarbon ring group having 3 to 12 carbon atoms is preferably a non-aromatic hydrocarbon ring group having 3 to 12 carbon atoms, more preferably a cycloalkyl group or an adamantyl group having 3 to 8 carbon atoms, and further preferably a cyclohexyl group or an adamantyl group.

[0101] In the present specification, "3- to 14-membered heterocyclic group" refers to a heterocyclic group having 3 to 14 ring atoms. Examples of the 3- to 14-membered heterocyclic group include 5- to 14-membered aromatic heterocyclic groups and 3- to 14-membered non-aromatic heterocyclic groups.

[0102] In the present specification, examples of the 5- to 14-membered aromatic heterocyclic group include the following groups: (i) 5- to 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, and triazinyl; (ii) benzothienyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridyl, thienopyridyl, furopyridyl, pyrrolopyridyl, pyrazolopyridyl, oxazolopyridyl, thiazolopyridyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, 8-14 membered fused polycyclic aromatic heterocyclic groups such as pyridyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl and phenoxazinyl.

[0103] In the present specification, examples of the 3- to 14-membered non-aromatic heterocyclic group include the following groups: (i) aziridinyl, oxirane, thiirane, azetidinyl, oxetanyl, thietanyl, tetrahydrothiophenyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepanyl, 3-8 membered monocyclic non-aromatic heterocyclic groups such as azepinyl, oxepanyl, azocanyl, and diazacyclooctanyl; (ii) a 9- to 14-membered condensed polycyclic non-aromatic heterocyclic group such as dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolizinyl, indolyl, isoindolyl, tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl, tetrahydroquinoxalinyl, tetrahydrophenanthridinyl, hexahydrophenothiazinyl, hexahydrophenoxazinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbolinyl, tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl and octahydroisoquinolyl.

[0104] R 10 Preferred are alkyl groups having 1 to 20 carbon atoms, alkenyl groups having 2 to 20 carbon atoms, alkynyl groups having 2 to 20 carbon atoms, or hydrocarbon ring groups having 3 to 12 carbon atoms. The alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms.

[0105] R 10 More preferably, it is an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a cyclohexyl group, and the alkyl group may be substituted with an adamantyl group.

[0106] R 10 More preferably, it is an alkyl group having 1 to 20 carbon atoms, particularly preferably, it is an alkyl group having 1 to 10 carbon atoms, and most preferably, it is a heptyl group.

[0107] In formula (5), * represents a bonding position other than a carbon atom as described above. In formula (5), R 11 represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynyl group having 2 to 9 carbon atoms, and R 11 The group may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0108] In the present specification, the alkylene group may be any type of linear or branched. Examples of the alkylene group include methylene, ethylene, trimethylene (-(CH 2 ) 3 -), propylene (-CH(CH 3 )CH 2 -、-CH 2 CH(CH 3 )-), tetramethylene (-(CH 2 ) 4 -), butylene (-CH(C 2 H 5 )CH 2 -、-CH 2 CH(C 2 H 5 )-), pentamethylene (-(CH 2 ) 5 -), hexamethylene (-(CH 2 ) 6 -), heptamethylene (-(CH 2 ) 7 -), octamethylene (-(CH 2 ) 8 -), nonamethylene (-(CH 2 ) 9 -), decamethylene (-(CH 2 ) 9 -) ("-" in the above formula represents a single bond.).

[0109] In this specification, "alkenediyl" refers to a divalent group having a structure obtained by removing 2 hydrogen atoms from an alkene. In this specification, alkenediyl can be any type of straight chain or branched. In addition, in this specification, the number of olefinic carbon-carbon double bonds in an alkene or alkenediyl can be only 1, or more than 2. As alkenediyl, for example, ethylenediyl, propylenediyl, butenediyl, pentenediyl, hexenediyl, heptenediyl, octenyl, nonenediyl, decenediyl. In addition, in this specification, "compound name + diyl (e.g., ethylenediyl)" refers to a divalent group having a structure obtained by removing 2 hydrogen atoms from the compound.

[0110] In this specification, "alkynediyl" refers to a divalent group having a structure obtained by removing 2 hydrogen atoms from alkynes. In this specification, the alkynediyl can be any type of straight chain or branched. In addition, in this specification, the number of carbon-carbon triple bonds in alkynes or alkynediyl can be only 1, or more than 2. As alkynediyl, for example, ethynediyl, propynediyl, butynediyl, pentynediyl, hexynediyl, heptynediyl, octynediyl, nonynediyl, decynediyl can be enumerated.

[0111] In formula (5), R 11 It is preferably an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, more preferably an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms, further preferably an alkylene group having 2 to 9 carbon atoms, particularly preferably an alkylene group having 2 or 3 carbon atoms, and most preferably a trimethylene group.

[0112] In formula (5), R 12 and R 13 Each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, and R 12 At least one ethylene group or at least one trimethylene group in the group may be substituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 13 At least one ethylene group or at least one trimethylene group in R may be substituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 12 and R 13 Each of the groups may be substituted independently by a substituent selected from the group consisting of a halogen atom, a hydroxyl group and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0113] R 12 and R 13Preferably, each independently is an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, more preferably, each independently is an alkyl group having 1 to 17 carbon atoms, further preferably, an alkyl group having 1 to 10 carbon atoms, and particularly preferably, a hexyl group.

[0114] In formula (5), X 4 represents an oxygen atom, NH or a sulfur atom. 4 It is preferably an oxygen atom or NH, and more preferably an oxygen atom.

[0115] In formula (5), it is most preferred that: R 11 is trimethylene, R 12 and R 13 At the same time, hexyl, and X 4 For oxygen atoms.

[0116] R 3a With R 3b Can be the same or different, preferably R 3a For R 3b Same group.

[0117] 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.

[0118] Preferred examples of the ionic lipid represented by formula (1) (may be simply referred to as "ionic lipid (1)" in this specification) include the following ionic lipids.

[0119] [Ionic lipids (1-1a)] An ionic lipid (1), 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 independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., -N(CH 3 )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidinyl); R2a 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 (e.g., -C 6 H 4 -CH 2 -、-CH 2 -C 6 H 4 -CH 2 -); n a and n b Each is independently 0 or 1; R 3a and R 3b Each of them is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl), a group having 50 or less carbon atoms represented by the formula (4) (R in the formula (4) 10 is an alkyl group having 1 to 20 carbon atoms. ) or a group having 50 or less carbon atoms represented by the formula (5) (R in the formula (5) 11 is an alkylene group having 2 to 9 carbon atoms, R 12 and R 13 are each independently an alkyl group having 1 to 17 carbon atoms, and X 4 is an oxygen atom or NH. ).

[0120] [Ionic lipids (1-1b)] An ionic lipid (1), 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 independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., -N(CH 3 )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidinyl); R 2a and R 2bEach 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 (e.g., -C 6 H 4 -CH 2 -、-CH 2 -C 6 H 4 -CH 2 -); n a and n b Each is independently 0 or 1; R 3a and R 3b Each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0121] [Ionic lipids (1-2a)] An ionic lipid (1), 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 independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g., -N(CH 3 )-), 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 independently is an ester bond or an amide bond; Z a and Z b Each is independently a divalent group (e.g., -C 6 H 4 -CH2 -、-CH 2 -C 6 H 4 -CH 2 -); n a and n b Each is independently 0 or 1; R 3a and R 3b Each of them is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl), a group having 50 or less carbon atoms represented by the formula (4) (R in the formula (4) 10 is an alkyl group having 1 to 10 carbon atoms. ), or a group having 50 or less carbon atoms represented by the formula (5) (R in the formula (5) 11 is an alkylene group having 2 or 3 carbon atoms, R 12 and R 13 are each independently an alkyl group having 1 to 10 carbon atoms, and X 4 is an oxygen atom. ).

[0122] [Ionic lipids (1-2b)] An ionic lipid (1), 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 independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g., -N(CH 3 )-), 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 independently is an ester bond or an amide bond; Z a and Z b Each is independently a divalent group (e.g., -C 6 H 4 -CH 2 -、-CH 2 -C 6 H4 -CH 2 -); n a and n b Each is independently 0 or 1; R 3a and R 3b Each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (eg, tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (eg, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0123] [Ionic lipids (1-3a)] An ionic lipid (1), 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 :

[0124] [Chemistry 16]

[0125] (Where R 5 An alkyl group having 1 to 3 carbon atoms (e.g., methyl group).

[0126] [Chemistry 17]

[0127] (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 :

[0128] [Chemistry 18]

[0129] (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 nb Each is independently 0 or 1; R 3a and R 3b Each of them is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl), a group having 50 or less carbon atoms represented by the formula (4) (R in the formula (4) 10 is an alkyl group having 1 to 10 carbon atoms. ), or a group having 50 or less carbon atoms represented by the formula (5) (R in the formula (5) 11 is an alkylene group having 2 or 3 carbon atoms, R 12 and R 13 are each independently an alkyl group having 1 to 10 carbon atoms, and X 4 is an oxygen atom. ).

[0130] [Ionic lipids (1-3b)] An ionic lipid (1), 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 :

[0131] [Chemistry 19]

[0132] (Where R 5 An alkyl group having 1 to 3 carbon atoms (e.g., methyl group).

[0133] [Chemistry 20]

[0134] (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 :

[0135] [Chemistry 21]

[0136] (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 is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (eg, tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (eg, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0137] Specific examples of ionic lipids (1) include the following: O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, B-2, B-2-5, TS-P4C2, L-P4C2, O-P4C2, PiP9, and PiP15.

[0138] [Table 1-1]

[0139] [Table 1-2]

[0140] [Table 1-3] [Table 1-4]

[0141] The ionic lipid (1) is preferably at least one selected from the group consisting of the compounds described in Table 1-1 to Table 1-4, and more preferably at least one selected from the group consisting of SS-OP, SS-EC, PiP9 and PiP15.

[0142] The ionic lipid (1) is more preferably (i) SS-OP, or (ii) SS-OP and SS-EC, or (iii) PiP9, or (iv) PiP15.

[0143] The ionic lipid (1) is particularly preferably (i) SS-OP, or (ii) SS-OP and SS-EC. The ionic lipid (1) is most preferably SS-OP.

[0144] Ionic lipids (1) can be produced according to known methods (e.g., methods described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, and WO2021 / 195529A2). In addition, PiP9 and PiP15 used in the examples were produced by the following synthesis methods. Ionic lipids (1) other than PiP9 or PiP15 can also be produced in the same manner as the synthesis methods of PiP9 or PiP15.

[0145] (I) Synthesis of PiP9 <Synthesis of Intermediate 1> Intermediate 1 was synthesized according to the following synthetic route.

[0146] [Chemistry 22]

[0147] <Synthesis of Intermediate 1-A> 30.0 g (197 mmol) of 4-hydroxyphenylacetic acid, 5.00 g (19.9 mmol) and pyridine p-toluenesulfonate were dissolved in 120 mL of dichloromethane at room temperature. A mixed solution of 83.0 g (987 mmol) of 3,4-dihydro-2H-pyran and 31.1 mL of dichloromethane was added dropwise to the obtained mixture at 20°C or below, and the mixture was reacted at room temperature for 2 hours. Thereafter, 12.0 g (98.2 mmol) of DMAP was added to the reaction mixture for neutralization. 301 mL of 2-propanol and 160 g of a 100 g / L NaOH aqueous solution were added to the obtained mixture, and the mixture was reacted at room temperature for 1 hour. After the mixed solution was concentrated using an evaporator, the concentrate was washed with chloroform, and 6 M hydrochloric acid was added for neutralization. After the obtained mixture was extracted with chloroform, sodium sulfate was added to the organic layer for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 47.0 g of intermediate 1-A.

[0148] <Synthesis of Intermediate 1-B> 36.0g (95.6mmol) of intermediate 57, 49.7g (210mmol) of intermediate 1-A, and 4.68mg (38.3mmol) of DMAP synthesized according to the method described in International Publication No. 2016 / 121942 were dissolved in 240mL of chloroform at room temperature. 55.1g (287mmol) of EDC hydrochloride was added to the resulting mixture and reacted at room temperature for 2 hours. After that, the reaction solution was washed with 5% by weight sodium dihydrogen phosphate aqueous solution, 9% by weight sodium bicarbonate solution, and 20% by weight saline, and sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 77.9g of intermediate 1-B.

[0149] <Synthesis of Intermediate 1> 77.7 g (95.6 mmol) of intermediate 1-B was dissolved in 325 mL of THF at room temperature. 319 mL of 2-propanol and 38.2 g (201 mmol) of p-toluenesulfonic acid monohydrate were added to the resulting mixture, and the mixture was reacted at 25°C or below for 1 hour. Thereafter, 25.0 g (205 mmol) of DMAP was added to the reaction mixture for neutralization. After removing DMAP by filtration, the filtrate was concentrated using an evaporator. The resulting residue was dissolved in 627 mL of chloroform, washed with 0.5 M phosphate buffer (pH = 6.5) and 0.5 M glycine buffer (pH = 9.5), and then dehydrated by adding sodium sulfate. After removing sodium sulfate by filtration, 1.56 L of toluene was added for crystallization. The resulting crude product was washed with hexane and dried in vacuo to obtain 40.3 g of intermediate 1.

[0150] <Synthesis of Intermediate 1-1> 5.11 g (18.1 mmol) of oleic acid, 16.6 g (25.8 mmol) of intermediate 1, and 630 mg (5.16 mmol) of DMAP were dissolved in 166 mL of chloroform at room temperature. 5.94 g (31.0 mmol) of EDC hydrochloride was added to the resulting mixture at room temperature and reacted for 2 hours. After that, the reaction solution was washed with 5 wt% sodium hydrogen phosphate aqueous solution, 0.5 M phosphate buffer (pH = 2.0), and 20 wt% saline, and then sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator, and the resulting residue was purified using silica gel column chromatography (chloroform / ethanol = 88 / 12) to obtain 8.21 g of intermediate 1-1.

[0151] [Chemistry 23]

[0152] <Synthesis of Intermediate 5-1> 5.00 g (16.0 mmol) of ricinoleic acid methyl ester, 2.54 g (17.6 mmol) of octanoic acid, and 391 mg (3.20 mmol) of DMAP were dissolved in 50.0 g of chloroform at room temperature. 4.60 g (24.0 mmol) of EDC hydrochloride was added to the resulting mixture and reacted for 1 hour at room temperature. After that, the reaction solution was washed with 0.5 M phosphate buffer (pH 4.0), 7 wt% sodium bicarbonate water, and 20 wt% saline, and then sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 6.78 g of intermediate 5-1.

[0153] [Chemistry 24]

[0154] <Synthesis of Intermediate 5> Dissolve 6.40 g (14.6 mmol) of Intermediate 5-1 in tert-butanol at room temperature to make Intermediate 5-1 0.3 M. Add 8.17 g of 2 M aqueous NaOH solution to the resulting mixture and react it at room temperature for 8 hours. Then, add 1 M hydrochloric acid, extract the resulting mixture with hexane, wash the organic layer with 20 wt% brine, and then add sodium sulfate for dehydration. After removing sodium sulfate by filtration, concentrate the filtrate using an evaporator. Purify the resulting residue by silica gel column chromatography (chloroform / ethanol = 94 / 6 (v / v)) to obtain 5.93 g of Intermediate 5.

[0155] [Chemical Formula 25]

[0156] <Synthesis of PiP9> Dissolve 492 mg (0.541 mmol) of Intermediate 1-1 in 2.91 g of chloroform, then add 230 mg (0.541 mmol) of Intermediate 5, 13.2 mg (0.108 mmol) of DMAP, and 207 mg (1.08 mmol) of EDC hydrochloride and react at room temperature for 2 hours. Wash the reaction solution with 5 wt% brine, then concentrate the filtrate using an evaporator, and perform silica gel column purification using ethanol / chloroform after concentration to obtain 532 mg of PiP9.

[0157] (II) Synthesis Method of PiP15 <Synthesis of Intermediate 11-1> Dissolve 25.0 g (189 mmol) of (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol and 19.3 g (284 mmol) of imidazole in 251 g of dimethylformamide at room temperature. Add 57.3 g (208 mmol) of TBDPS-Cl to the resulting mixture and react at room temperature for 1 hour. Then, add chloroform to the reaction solution, wash it with 5 wt% aqueous sodium hydrogen phosphate solution and ion-exchanged water respectively, and then add sodium sulfate for dehydration. After removing sodium sulfate by filtration, concentrate the filtrate using an evaporator to obtain 56.0 g of Intermediate 11-1.

[0158] [Chemical Formula 26]

[0159] <Synthesis of Intermediate 11-2> 55.9 g (151 mmol) of intermediate 11-1 and 503 g of 0.5 M phosphate buffer (pH 1.0) were dissolved in 530 g of THF at room temperature and reacted at 50 ° C for 12 hours. 1.0 M NaOH aqueous solution was added to pH 7.0. Chloroform was added to the resulting mixture, and after washing with 20% by weight saline, sodium sulfate was added for dehydration. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 44.9 g of intermediate 11-2.

[0160] [Chemistry 27]

[0161] <Synthesis of Intermediate 14-1> 6.90 g (20.9 mmol) of intermediate 11-2, 6.84 g (46.0 mmol) of heptanoic acid, and 0.511 g (4.18 mmol) of DMAP were dissolved in 69.5 g of chloroform at room temperature. 12.0 g (62.7 mmol) of EDC hydrochloride was added to the resulting mixture and reacted at room temperature for 1 hour. After that, the reaction solution was washed with 0.5 M phosphate buffer (pH 4.0), 7 wt% sodium bicarbonate water, and 20 wt% saline, and sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 9.03 g of intermediate 14-1.

[0162] [Chemistry 28]

[0163] <Synthesis of Intermediate 14-2> 9.41 g (17.9 mmol) of intermediate 14-1 and 3.92 g (65.3 mmol) of acetic acid were dissolved in 27.2 g of THF at room temperature. 59.9 g (65.2 mmol) of 1M TBAF in THF solution was added to the resulting mixture and reacted at room temperature for 18 hours. Thereafter, 94.3 g of ethyl acetate was added to the reaction solution. After that, the reaction solution was washed with 0.5 M phosphate buffer (pH 4.0) and 20 wt % saline, and sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 4.90 g of intermediate 14-2.

[0164] [Chemistry 29]

[0165] <Intermediate 14> At room temperature, 4.90 g (15.4 mmol) of Intermediate 14-2, 3.73 g (32.7 mmol) of glutaric anhydride, 4.95 g (48.9 mmol) of triethylamine, and 0.398 g (3.26 mmol) of DMAP were dissolved in 51.6 g of chloroform and reacted at room temperature for 1 hour. Thereafter, the reaction solution was washed with 0.5 M phosphate buffer (pH 4.0), 7 wt% sodium bicarbonate solution, and 20 wt% saline, and then sodium sulfate was added for dehydration. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 3.16 g of Intermediate 14.

[0166] [Chemical Formula 30]

[0167] <Synthesis of PiP15> Using Intermediate 14 and Intermediate 1-1, 0.215 g of PiP15 was synthesized in the same manner as the synthesis of PiP9 described above.

[0168] From the viewpoints of the encapsulation efficiency of nucleic acids, the release efficiency of nucleic acids in cells, and the stability of lipid nanoparticles, the content of the ionic lipid (1) in the lyophilized composition is preferably 20 to 75 mol%, more preferably 30 to 70 mol%, and further preferably 35 to 60 mol% based on the total lipids.

[0169] Nucleic acid Examples of the nucleic acid encapsulated by the lipid nanoparticles include the nucleic acids described below. This nucleic acid is preferably mRNA.

[0170] From the viewpoints of the encapsulation efficiency of nucleic acids and toxicity, the molar ratio of the total amino groups of the ionic lipid (1) to the phosphate groups of the nucleic acid (total amino groups of the ionic lipid / phosphate groups of the nucleic acid) (in this specification, there are cases where it is described as "N / P ratio") is preferably 5 to 280, more preferably 10 to 140, and further preferably 15 to 70.

[0171] Phospholipid Phospholipids can be used as components constituting the lipid membrane of lipid nanoparticles. Examples of phospholipids include: 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), and lysosomes of these substances.

[0172] 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-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).

[0173] 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), and 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA) which are obtained by replacing the phosphorylcholine part of the above specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) with phosphorylethanolamine, phosphorylserine, phosphorylglycerol, or phosphatidic acid, respectively, can also be used.

[0174] The phospholipids used in the present invention are preferably PC or PE, more preferably DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), and particularly preferably DOPC, DSPC, and DEPC.

[0175] From the viewpoint of nucleic acid internalization efficiency, nucleic acid release efficiency in cells and stability of lipid nanoparticles, the phospholipid content in the freeze-dried composition is preferably 0 to 35 mol %, more preferably 2.5 to 30 mol %, and further preferably 5 to 30 mol % relative to the total lipids.

[0176] Sterols Sterol can be used as a component for regulating the fluidity of the lipid membrane of lipid nanoparticles. Examples of sterol include cholesterol, lanosterol, phytosterol, zimosterol, zymostenol, streptosterol, stigmasterol, dihydrolanosterol and 7-dehydrocholesterol. Sterol is preferably cholesterol, lanosterol, phytosterol, and more preferably cholesterol.

[0177] When sterol is used, the content in the freeze-dried composition is preferably 5 to 60 mol %, more preferably 7 to 55 mol %, and further preferably 10 to 50 mol %, from the viewpoint of nucleic acid internalization efficiency, nucleic acid release efficiency in cells, and stability of lipid nanoparticles.

[0178] PEG lipids PEG lipids are lipid nanoparticles whose surface is coated with hydrophilic polyethylene glycol (PEG). They can be used as stabilizers to inhibit aggregation between particles and to inhibit the interaction between biological components and particles when administered to a living body.

[0179] The PEG region can be of any molecular weight. In some embodiments, the PEG region has a number average molecular weight of 200 to 10,000 and can be linear or branched.

[0180] Examples of PEG lipids include PEG-phospholipids, PEG-ceramides, PEG-diacylglycerols, and PEG-cholesterols. Diacylglycerol PEG having a number average molecular weight of 1,000 to 10,000 is preferred, and dimyristoylglycerol PEG or distearoylglycerol PEG having a number average molecular weight of 1,000 to 10,000 is more preferred.

[0181] When PEG lipids are used, from the viewpoint of the internal sealing efficiency of nucleic acids, the release efficiency of nucleic acids in cells and the stability of lipid nanoparticles, the content in the freeze-dried composition is preferably 0.1 to 6.0 mol%, more preferably 0.5 to 5.0 mol%, and further preferably 0.7 to 3.0 mol% relative to the total of other lipids. Among them, "other lipids" refer to lipids other than PEG lipids. For example, when ionic lipids (1) and PEG lipids are used as lipids, "the total of other lipids" refers to ionic lipids (1). In addition, for example, when ionic lipids (1), phospholipids, sterols and PEG lipids are used as lipids, "the total of other lipids" refers to the total of ionic lipids (1), phospholipids and sterols. In addition, in the present specification, "the content of B relative to A (mol%)" refers to "100×the amount of B (mol) / the amount of A (mol)".

[0182] Other Ingredients The freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of the present invention may contain other components besides nucleic acid and lipid, within the range not inhibiting the effect of the present invention. Other components may be used alone or in combination of two or more.

[0183] Cryoprotectants As other components, one or more cryoprotectants may be used. Examples of cryoprotectants include sugars such as monosaccharides, sugar alcohols, disaccharides, oligosaccharides, and polysaccharides. The sugar is preferably a disaccharide, and more preferably sucrose.

[0184] When a cryoprotectant is used, from the viewpoint of the stability, cryoprotective ability and particle formation of the lipid nanoparticles, the content of the cryoprotectant in the freeze-dried composition is preferably 90.0 to 99.9% by weight, more preferably 95.0 to 99.9% by weight, and even more preferably 97.0 to 99.9% by weight relative to the entire freeze-dried composition.

[0185] Method for producing freeze-dried composition The present invention provides a method for producing a freeze-dried composition of lipid nanoparticles encapsulating nucleic acid, comprising the following steps. a) mixing an alcohol solution containing an ionic lipid represented by formula (1), a sterol, a phospholipid and a PEG lipid with a nucleic acid solution in an acidic buffer having a buffering effect at pH 1.0 to 6.5 to prepare a suspension of lipid nanoparticles encapsulating nucleic acids, b) replacing the external aqueous phase of the lipid nanoparticles encapsulating nucleic acids with another buffer having a buffering effect at pH 4.5 to 6.9 to obtain a mixture containing lipid nanoparticles encapsulating nucleic acids at pH 4.5 to 6.9, c) mixing the obtained mixture with a cryoprotectant to prepare a mixture containing 80 to 800 mg / mL of the cryoprotectant and lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9, d) a step of freeze-drying the mixture obtained in step c) to obtain a freeze-dried composition.

[0186] a) Step of preparing a suspension of lipid nanoparticles encapsulating nucleic acid In the method of the present invention, a suspension of lipid nanoparticles encapsulating nucleic acids is prepared by mixing an alcohol solution (hereinafter referred to as "alcohol solution") containing ionic lipid (1), phospholipid, sterol and PEG lipid with a nucleic acid solution (hereinafter referred to as "nucleic acid solution") in an acidic buffer having a buffering effect at pH 1.0 to 6.5 (hereinafter referred to as "acidic buffer") . In order to prepare lipid nanoparticles, an operation of inducing arbitrary organization can be adopted. Examples of alcohols include ethanol and tert-butyl alcohol.

[0187] From the viewpoint of lipid solubility and nucleic acid encapsulation, the concentration of total lipids (i.e., the total of ionic lipids (1), phospholipids, sterols and PEG lipids) in the alcohol solution used in step a) is preferably 0.1 to 30 mM, more preferably 0.5 to 25 mM, and even more preferably 1 to 20 mM.

[0188] From the viewpoint of lipid solubility, the concentration of the ionic lipid (1) in the alcohol solution used in step a) is preferably 0.005 to 30 mM, more preferably 0.05 to 25 mM, and even more preferably 0.1 to 20 mM.

[0189] From the viewpoint of the solubility of the lipid, the sterol concentration in the alcohol solution used in step a) is preferably 0.001 to 25 mM, more preferably 0.005 to 20 mM, and even more preferably 0.01 to 15 mM.

[0190] From the viewpoint of the solubility of the lipid, the phospholipid concentration in the alcohol solution used in step a) is preferably 0.001 to 25 mM, more preferably 0.001 to 15 mM, and even more preferably 0.01 to 10 mM.

[0191] From the viewpoint of lipid solubility, the PEG lipid concentration in the alcohol solution used in step a) is preferably 0.00001 to 10 mM, more preferably 0.0001 to 5.0 mM, and even more preferably 0.001 to 3.0 mM.

[0192] In step a), an acidic buffer having a buffering effect at pH 1.0 to 6.5 (preferably pH 3 to 6.5, more preferably pH 3 to 5.0) is used. In addition, in this specification, "an acidic buffer having a buffering effect at pH 1.0 to 6.5" means an acidic buffer having a buffering effect to maintain the pH within the range of pH 1.0 to 6.5. The same expression as "an acidic buffer having a buffering effect at pH 1.0 to 6.5" also has the same meaning as this.

[0193] Examples of the acidic buffer include tartaric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / Na formate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1 M NaCl buffer, phenylacetic acid / Na phenylacetic acid buffer, acetic acid / Na acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, dimethylarsonic acid Na / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH 2 PO 4 / NaOH buffer, 2-morpholineethanesulfonic acid (MES) buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartaric acid buffer, citric acid buffer, Bis-Tris buffer, glycylglycine buffer, etc.

[0194] The acidic buffer is preferably a malic acid buffer, a citric acid buffer or a MES buffer (more preferably a malic acid buffer or a MES buffer) having a buffering effect at pH 1.0 to 6.5 (more preferably pH 3 to 6.5, and further preferably pH 3 to 5.0).

[0195] From the viewpoint of nucleic acid stability, the total concentration of the acid and its salt in the acidic buffer used in step a) (hereinafter sometimes abbreviated as "buffer concentration") is preferably 10 to 100 mM, more preferably 10 to 80 mM.

[0196] In the present specification, "a nucleic acid solution in an acidic buffer having a buffering effect at pH 1.0 to 6.5" means a nucleic acid solution containing an acidic buffer having a buffering effect at pH 1.0 to 6.5 as a solvent.

[0197] From the viewpoint of nucleic acid solubility, the nucleic acid concentration in the nucleic acid solution used in step a) is preferably 0.1 to 720 μg / mL, more preferably 0.5 to 350 μg / mL, and even more preferably 1 to 250 μg / mL.

[0198] From the perspective of the particle size of lipid nanoparticles encapsulating nucleic acids and the nucleic acid encapsulation rate, when mixing a nucleic acid solution and an alcoholic lipid solution, the volume ratio of these (nucleic acid solution: alcoholic lipid solution) is preferably 1:1 to 20:1, more preferably 1.5:1 to 15:1, and further preferably 2:1 to 8:1.

[0199] Examples of the "organization-inducing procedure" for preparing lipid nanoparticles include: alcohol dilution method using microfluidics or vortex, simple hydration method (pure water method), ultrasonic treatment, heating, vortexing, ether injection method, French Press method, bile acid method, Ca 2+ method, etc. 2+ The known methods such as fusion method, freeze-thaw method, reverse phase evaporation method, etc. are preferably the alcohol dilution method using microfluid or vortex, and more preferably the alcohol dilution method using microfluid. The particle preparation in the alcohol dilution method using microfluid, for example, can be carried out using NanoAssemblr (Precision NanoSystems company). The lipid nanoparticles prepared can replace the buffer of the outer aqueous phase by operations such as ultrafiltration, dialysis, dilution.

[0200] b) Step of preparing a mixture containing lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9 In step b), the external aqueous phase of the lipid nanoparticles encapsulating nucleic acids is replaced with other buffer solutions (hereinafter referred to as "other buffer solutions") having a buffering effect at pH 4.5 to 6.9 (preferably pH 5.0 to 6.9, preferably pH 5.5 to 6.9) by ultrafiltration, dialysis, dilution and the like. The "external aqueous phase of the lipid nanoparticles encapsulating nucleic acids" refers to the dispersion medium of the suspension of the lipid nanoparticles encapsulating nucleic acids. In addition, "other buffer solutions having a buffering effect at pH 4.5 to 6.9" refers to a buffer solution different from the acidic buffer solution used in step a), which is a buffer solution having a pH of 4.5 to 6.9 and having a buffering effect to maintain the said pH. The same expression as "other buffer solutions having a buffering effect at pH 4.5 to 6.9" also has the same meaning as this.

[0201] Specific examples of other buffers include tartaric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / Na formate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer, phenylacetic acid / Na phenylacetic acid buffer, acetic acid / Na acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, dimethyl cacodylate Na / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH 2 PO 4 / NaOH buffer, MES buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartaric acid buffer, citric acid buffer, Bis-Tris buffer, glycylglycine buffer, etc.

[0202] The other buffer is preferably a malic acid buffer, a citrate buffer, a MES buffer or a maleic acid / Tris / NaOH buffer (more preferably a malic acid buffer or a MES buffer) having a buffering effect at pH 4.5 to 6.9 (more preferably pH 5.0 to 6.9, and further preferably pH 5.5 to 6.9).

[0203] From the viewpoint of nucleic acid internalization rate and lipid nanoparticle formation, the total concentration of the acid and its salt in other buffers (hereinafter referred to as "buffer concentration") is preferably 10 to 100 mM, more preferably 20 to 50 mM.

[0204] The nucleic acid concentration in the mixture obtained in step b) is not particularly limited as long as it is a concentration that can form lipid nanoparticles after replacement with other buffers. From the viewpoint of the particle size of the lipid nanoparticles encapsulating the nucleic acid, it is preferably 0.25 to 3300 μg / mL, more preferably 2.5 to 1500 μg / mL, and even more preferably 3 to 800 μg / mL. In step b), other buffers can be used in an amount that can obtain the above-mentioned nucleic acid concentration.

[0205] c) mixing the obtained mixture with a cryoprotectant to obtain a mixture containing 80 to 320 mg / mL of the cryoprotectant and lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9; As a cryoprotectant, for example, monosaccharides, sugar alcohols, disaccharides, oligosaccharides, polysaccharides and the like can be added to the suspension of lipid nanoparticles. As the sugar, disaccharides are preferred, and sucrose is more preferred. As the concentration before freeze drying, the concentration of the cryoprotectant (especially sugar) in the mixture is 80 to 320 mg / mL, preferably 80 to 240 mg / mL.

[0206] d) freeze-drying the mixture obtained in step c) to obtain a freeze-dried composition The freeze-drying process can be carried out in any suitable container known in the field of pharmaceutical technology, such as a glass container (or, for example, a glass vial) or a two-chamber container.

[0207] The stabilized lipid nanoparticle composition of the present invention containing a cryoprotectant can be introduced into a glass container. The volume of the composition added to the container can be 0.1 to 20 mL or 1 to 10 mL.

[0208] Any freeze-drying technique may be used, including techniques known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Penn. (1990).

[0209] The freeze-drying process may include freezing the lipid nanoparticle composition stabilized by a cryoprotectant at a temperature of about -55° C. to about -30° C. The frozen composition may be a freeze-dried composition in a dried form.

[0210] In some embodiments, the freezing process can slowly reduce the temperature from room temperature to the final temperature over several minutes. The temperature gradient can be about 1°C / minute.

[0211] In some embodiments, the drying process can be carried out at a pressure of about 0 to 250 mTorr or 50 to 150 mTorr and a temperature of about -55°C to about 40°C. The drying process can be continued at a high temperature range up to room temperature for a specified period of up to several days. The level of residual water in the solid freeze-dried composition can be about less than 5 w / v%, or less than 4% w / v, or less than 3 w / v%, or less than 2 w / v%, or less than 1% w / v.

[0212] Method for producing lipid nanoparticles encapsulating nucleic acid The present invention also provides a method for producing lipid nanoparticles encapsulating nucleic acid, comprising rehydrating the freeze-dried composition obtained in the above steps a) to d) in the step e) described below.

[0213] e) mixing the freeze-dried composition with water, and incubating the mixture at 0 to 95° C. for 0 to 60 minutes to obtain lipid nanoparticles encapsulating nucleic acids. Step e) is a step of adding water to the freeze-dried composition of the lipid nanoparticles encapsulated with nucleic acids of the present invention to prepare the lipid nanoparticles encapsulated with nucleic acids. The lipid nanoparticles encapsulated with nucleic acids can be prepared by adding water to the freeze-dried composition and mixing by a dropper and a vortex. In addition, in order to increase the nucleic acid encapsulation rate, alcohols can be added during rehydration. As alcohols, methanol, ethanol, n-butanol, tert-butanol, preferably ethanol, can be used. The concentration of alcohols in the water added to the freeze-dried composition is 0 to 50 v / v%, preferably 0 to 30 v / v%, and more preferably 0 to 25 v / v%. In addition, further in the rehydration step, in order to improve the nucleic acid encapsulation efficiency, the mixture can be arbitrarily incubated after adding water to the freeze-dried composition, or alcohols. The incubation conditions are, for example, 0 to 120 minutes at 0 to 100 ° C, or 0 to 60 minutes at 0 to 95 ° C, preferably 0 to 60 minutes at 30 to 100 ° C.

[0214] In step e), a suspension of lipid nanoparticles encapsulating nucleic acids can be obtained by mixing the freeze-dried product with water. The concentration of nucleic acids in the suspension is not particularly limited as long as it is within the range in which a suspension can be formed by mixing the freeze-dried product with water. From the viewpoint of the particle size of the lipid nanoparticles encapsulating nucleic acids, it is preferably 0.1 to 4000 μg / mL, more preferably 0.5 to 3000 μg / mL, and further preferably 12000 μg / mL. In step e), water can be used in an amount such as to obtain the above-mentioned nucleic acid concentration.

[0215] The external aqueous phase of the lipid nanoparticles encapsulating nucleic acids obtained by step e) can also be replaced with a neutral buffer. As a method for replacing the external aqueous phase with a neutral buffer, there can be mentioned: a method using dialysis, ultrafiltration or dilution. As a neutral buffer, for example, there can be mentioned: phosphate buffered saline (PBS), Tris buffered saline (TBS), Tris-HCl buffer, N-(2-acetylamino)-iminodiacetic acid (ADA) buffer, 1,4-piperazine diethanesulfonic acid (PIPES) buffer, 1,4-piperazine diethanesulfonic acid sesquisodium salt (PIPES sesquisodium) buffer, N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES) buffer, 3(N-morpholino)propanesulfonic acid (MOPS) buffer, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO) buffer, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid (HEPES) buffer, 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO) buffer, piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (POPSO) buffer, 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (HEPSO) buffer. The pH of the neutral buffer is 6 to 8.

[0216] Methods for introducing nucleic acids into cells By contacting the lipid nanoparticles containing nucleic acid prepared in the method of the present invention with cells, the nucleic acid can be introduced into the cells in vivo and / or in vitro. Therefore, the present invention provides a method for introducing the nucleic acid into the cells in vivo and / or in vitro.

[0217] Any nucleic acid can be introduced into a cell according to the method of the present invention. As the type of nucleic acid, there can be mentioned: DNA, RNA, chimeric nucleic acid of RNA, hybrid chain of DNA / RNA, etc., but it is not limited to these. In addition, any type of nucleic acid with 1 to 3 chains can be used, preferably single-stranded or double-stranded. For example, the nucleic acid can also be an oligomer of other types of nucleotides or non-nucleotide backbones containing N-glycosides as purine or pyrimidine bases (such as commercially available peptide nucleic acids (PNA)), or an oligomer with special bonds (wherein the oligomer contains nucleotides with a configuration that allows base pairing and base adhesion as found in DNA and RNA), etc. 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 within the molecule, 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.

[0218] 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. For example, plasmid DNA, cDNA, antisense strand DNA, chromosomal DNA, PAC, BAC, CpG oligonucleotide, etc. can be mentioned, but plasmid DNA, cDNA, antisense strand DNA, and plasmid DNA are preferred. Circular DNA such as plasmid DNA can also be digested with appropriate restriction enzymes and used as linear DNA.

[0219] 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. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. are mentioned, preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon.

[0220] The nucleic acid used in the present invention is preferably purified by a method commonly used by those skilled in the art.

[0221] The lipid nanoparticles enclosed with nucleic acid manufactured by the method of the present invention can, for example, be administered in vivo for the purpose of preventing and / or treating a disease. Therefore, the nucleic acid used in the present invention is preferably a nucleic acid (preventive / therapeutic nucleic acid) having preventive and / or therapeutic activity for a certain specific disease. As such nucleic acid, for example, nucleic acids used for so-called gene therapy, etc. can be enumerated.

[0222] The lipid nanoparticles encapsulated with nucleic acids produced by the method of the present invention can be used as a drug delivery system for selectively delivering nucleic acids to specific cells, for example, DNA vaccines for introducing antigen genes into dendritic cells, gene therapy drugs for tumors, nucleic acid drugs for inhibiting target gene expression using RNA interference, etc. Therefore, the present invention provides a method for producing a pharmaceutical composition.

[0223] The particle size of the lipid nanoparticles encapsulating nucleic acid is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. For the determination of the particle size, for example, a particle size distribution measuring device such as Zetasizer Nano (Malvern Company) can be used. The particle size of the lipid nanoparticles can be appropriately adjusted by the method for manufacturing the lipid nanoparticles.

[0224] The surface potential (ZETA potential) of the lipid nanoparticles encapsulated with nucleic acid is not particularly limited, but is preferably -15 to +15mV, more preferably -10 to +10mV. In the past gene introduction, particles with positive surface potential were mainly used. This is useful as a method for promoting the electrostatic interaction with the negatively charged heparan sulfate on the cell surface to promote the uptake into the cell, but the positive surface charge exists to suppress the release of nucleic acid by the carrier caused by the interaction with the delivery nucleic acid in the cell, and the possibility of suppressing the synthesis of proteins caused by the interaction between mRNA and the delivery nucleic acid. This problem can be solved by adjusting the surface charge to the above range. For the determination of surface charge, for example, a Zetasizer Nano and other ZETA potential measuring devices can be used to carry out. The surface charge of lipid nanoparticles can be adjusted by the composition of the constituent components of lipid nanoparticles.

[0225] The following specifically describes the step of bringing the lipid nanoparticles encapsulating nucleic acid into contact with cells in vitro.

[0226] Several days before contacting with the lipid nanoparticles, the cells are suspended in an appropriate culture medium and cultured under suitable conditions. When contacting with the lipid nanoparticles, the cells may or may not be in a proliferation phase.

[0227] The culture fluid during the contact can be a culture medium containing serum or a culture medium without serum. The serum concentration in the culture medium is preferably below 30% by weight, more preferably below 20% by weight. When the culture medium contains excessive serum or other proteins, there is a possibility that the contact of the lipid nanoparticle with the cell is inhibited.

[0228] The cell density during contact is not particularly limited and can be appropriately set in consideration of the type of cells, etc., but is generally 1×10 4 ~1×10 7 Cells / mL range.

[0229] To the cells thus prepared, for example, a suspension of lipid nanoparticles enclosed with the above-mentioned nucleic acid is added. The amount of the suspension added is not particularly limited, and can be appropriately set considering the number of cells, etc. As long as it is possible to achieve the introduction of the target nucleic acid into the cell, the concentration of the lipid nanoparticles when contacting the cell is not particularly limited, and as a lipid concentration, it is usually 1 to 300 nmol / mL, preferably 10 to 200 nmol / mL. As the concentration of nucleic acid, it is usually 0.01 to 100 μg / mL, preferably 0.05 to 10 μg / mL.

[0230] After adding the above suspension to the cells, the cells are cultured. The temperature, humidity, and CO 2 The concentration and the like can be appropriately set in consideration of the type of cells. When the cells are derived from mammals, the temperature is generally about 37°C, the humidity is about 95%, and the CO 2 The concentration is about 5%. In addition, the culture time can also be appropriately set considering the conditions such as the cell type used, but is usually in the range of 0.1 to 96 hours, preferably in the range of 0.2 to 72 hours, and more preferably in the range of 0.5 to 48 hours. When the above-mentioned culture time is too short, the nucleic acid is not fully introduced into the cells, and when the culture time is too long, there is a situation where the cells are weak.

[0231] The nucleic acid is introduced into the cells by the above culture, and the culture medium is preferably replaced with a fresh one, or a fresh one is added to the culture medium and the culture is further continued. When the cells are mammalian cells, the fresh culture medium preferably contains serum or nutritional factors.

[0232] In addition, as mentioned above, by using the lipid nanoparticles encapsulated with nucleic acid, it is not only in vitro (invitro), but also in vivo (in vivo) nucleic acid can be introduced into cells. That is, by administering the lipid nanoparticles encapsulated with nucleic acid to the object, the lipid nanoparticles reach the target cell and contact it, and the nucleic acid encapsulated in the lipid nanoparticles is introduced into the cell in vivo. As the object that can be administered to the lipid nanoparticles, there is no particular limitation, for example, it can be enumerated: vertebrates such as mammals (such as people, monkeys, mice, rats, hamsters, cattle, etc.), birds (such as chickens, ostriches, etc.), amphibians (such as frogs, etc.), fish (such as zebrafish, killifish, etc.), invertebrates such as insects (such as silkworms, moths, fruit flies, etc.), plants, etc. As the administration object of the lipid nanoparticles encapsulated with nucleic acid, it is preferably people or other mammals.

[0233] The type of target cells is not particularly limited. By using lipid nanoparticles encapsulating nucleic acids, nucleic acids can be introduced into cells in various tissues (e.g., liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0234] For the method of administering lipid nanoparticles introduced with nucleic acids and / or compounds other than nucleic acids to a subject (e.g., vertebrates, invertebrates, etc.), there is no particular limitation as long as the lipid nanoparticles reach the target cells and come into contact with them, and the compound introduced into the lipid nanoparticles can be introduced into the cells. Considering the type of the introduced compound, the type and site of the target cells, etc., a known administration method (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.) can be appropriately selected. The dose of the lipid nanoparticles is not particularly limited as long as it is within the range that can achieve the introduction of the compound into the cells, and can be appropriately selected considering the type of the administration subject, the administration method, the type of the introduced compound, the type and site of the target cells, etc.

[0235] When lipid nanoparticles encapsulating nucleic acids are used as nucleic acid-transferring agents, they can be formulated by conventional means.

[0236] When providing the nucleic acid introduction agent as a research reagent, the nucleic acid introduction agent of the present invention can be provided directly as lipid nanoparticles encapsulating nucleic acids, or using, for example, a sterile solution or suspension with water or a physiologically acceptable liquid other than water (e.g., a water-soluble solvent (e.g., malic acid buffer, etc.), an organic solvent (e.g., ethanol, methanol, DMSO, tert-butyl alcohol, etc.), or a mixture of a water-soluble solvent and an organic solvent, etc.). The nucleic acid introduction agent of the present invention may appropriately contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0237] In addition, when the nucleic acid introduction agent is provided as a drug, the nucleic acid introduction agent of the present invention can be used directly as lipid nanoparticles encapsulating nucleic acids, or can be used together with known pharmaceutically acceptable additives (such as carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.), and can be mixed in the form of unit dosage required in the generally recognized formulation implementation, thereby manufacturing as an oral preparation (such as tablets, capsules, etc.) or a non-oral preparation (such as injections, sprays, etc.), preferably as a non-oral preparation (more preferably an injection).

[0238] In addition to being used for adults, the nucleic acid-transfer agent of the present invention can also be used as a preparation for children. Example

[0239] The following is a further detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments.

[0240] In the following examples, the ionic lipids represented by formula (1) are referred to as the names in the above table. Lipid nanoparticles encapsulated with nucleic acids may be referred to as "nucleic acid encapsulated nanoparticles". In addition, the abbreviations used in the following examples have the following meanings. Chol: Cholesterol DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (PEG number average molecular weight (Mn): 2000) DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine MES: 2-morpholineethanesulfonic acid PBS: Phosphate buffered saline TBS: Tris buffered saline DDW: Deionized distilled water tBuOH: tert-butyl alcohol EtOH: ethanol

[0241] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Comparative Example 1 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 10 mM.

[0242] 2840 μL of an aqueous solution of mRNA (CleanCap (registered trademark) FLuc mRNA (L-7602), hereinafter referred to as "FLuc mRNA") diluted with acid malic acid buffer (buffer concentration 20 mM, pH 3.0) with an mRNA concentration of 12.5 μg / mL and 710 μL of a lipid tert-butanol solution were measured into a syringe. LNPs were prepared using a NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by PrecisionNanoSystems) at a flow ratio of nucleic acid solution: lipid tert-butanol solution of 4:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. Sucrose solution was added to the recovered LNPs to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a volume of 600 μL each time and freeze-dried to prepare a freeze-dried composition (ionic lipid (1) content: 0.77 wt %, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9 wt %).

[0243] For freeze drying, the solution was first frozen at normal pressure and -55°C for 14 to 21 hours, then the pressure was reduced to 200 mTorr (26 Pa), and the temperature was increased by 10°C at a time. The temperature increase program was set as follows: 10°C to -20°C over 3 hours, 10°C over 2 hours at -10°C or above, and then allowed to stand at this temperature for 1 hour. When the temperature was raised to 30°C, it was allowed to stand at this temperature for 3 hours and then returned to normal pressure to recover the freeze-dried composition.

[0244] 300 μL of DDW was added to the freeze-dried composition, and the mixture was stirred using a vortex mixer to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration: 20 μg / mL).

[0245] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 1 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 7.25 mM.

[0246] 2919 μL of a solution of 12.1 μg / mL FLuc mRNA diluted with acid malic acid buffer (buffer concentration 20 mM, pH 3.0) and 973 μL of lipid ethanol solution were measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine production device (manufactured by Precision NanoSystems), LNP was prepared under the conditions of a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 4000 μL of MES buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The concentrate was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The concentrate was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1000 μL, and diluted using MES buffer (buffer concentration 20 mM, pH 6.0), and a mixture containing lipid nanoparticles encapsulated with nucleic acids (3300 μL, nucleic acid concentration 10 μg / mL) was recovered, and sucrose solution was added to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a volume of 600 μL each time and freeze-dried to prepare a freeze-dried composition (ionic lipid (1) content: 0.77 wt %, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9 wt %).

[0247] For freeze drying, the solution was first frozen at normal pressure and -55°C for 14 to 21 hours, then the pressure was reduced to 200 mTorr (26 Pa) and the temperature was increased by 10°C at a time. The temperature increase program was set as follows: 10°C to -20°C over 3 hours, 10°C over 2 hours at temperatures above -10°C, and then allowed to stand at this temperature for 1 hour. When the temperature was raised to 30°C, it was allowed to stand at this temperature for 3 hours and then returned to normal pressure to recover the freeze-dried composition.

[0248] 600 μL of DDW was added to the freeze-dried composition, and the mixture was stirred using a vortex mixer to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration: 10 μg / mL).

[0249] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of comparative example 2 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 7.25 mM.

[0250] 2919 μL of a solution of 12.1 μg / mL FLuc mRNA diluted with acid malic acid buffer (buffer concentration 20 mM, pH 3.0) and 973 μL of lipid ethanol solution were measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine production device (manufactured by Precision NanoSystems), LNP was prepared under the conditions of a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 4000 μL of MES buffer (buffer concentration 20 mM, pH 6.5) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The concentrate obtained was diluted to 15 mL with PBS (pH 7.4), and ultrafiltration was performed again under centrifugal conditions (25°C, 1000g, 5 minutes) to concentrate to about 1500 μL. The concentrate obtained was diluted to 15 mL with PBS (pH 7.4), and ultrafiltration was performed again under centrifugal conditions (25°C, 1000g, 5 minutes) to concentrate to about 1000 μL, and diluted with PBS (pH 7.4) to recover a mixture containing lipid nanoparticles encapsulating nucleic acids (3300 μL, nucleic acid concentration 10 μg / mL), and sucrose solution was added to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a time of 600 μL and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.77% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0251] For freeze drying, the solution was first frozen at normal pressure and -55°C for 14 to 21 hours, then the pressure was reduced to 200 mTorr (26 Pa) and the temperature was increased by 10°C at a time. The temperature increase program was set as follows: 10°C to -20°C over 3 hours, 10°C over 2 hours at temperatures above -10°C, and then allowed to stand at this temperature for 1 hour. When the temperature was raised to 30°C, it was allowed to stand at this temperature for 3 hours and then returned to normal pressure to recover the freeze-dried composition.

[0252] 600 μL of DDW was added to the freeze-dried composition, and the mixture was stirred using a vortex mixer to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration: 10 μg / mL).

[0253] Preparation method of lipid nanoparticles of Comparative Example 3 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 3.0 mM.

[0254] 788 μL of a 5 μg / mL solution of FLuc mRNA diluted with an acidic malic acid buffer (buffer concentration 20 mM, pH 3.0) and 263 μL of an ethanol solution of lipids were measured into a syringe. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine production device (manufactured by Precision NanoSystems), LNP was prepared under the conditions of a flow ratio of nucleic acid solution: ethanol solution of lipids of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 1000 μL of MES buffer (buffer concentration 20 mM, pH 6.5) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. The concentrate was diluted to 15 mL using PBS (pH 7.4), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. The above operation was repeated twice, and the suspension was concentrated to 300 μL to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 10 μg / mL).

[0255] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by Ribogreen (registered trademark) analysis. The pH of the suspension of nucleic acid-encapsulated nanoparticles after DDW melting of the freeze-dried compositions of Comparative Examples 1 and 2 and Example 1 was measured using a pH meter (hereinafter referred to as "pH after dissolution") (Table 2). The particle size is represented by Z-Ave (Z-average) and Number Mean (number average) (hereinafter the same).

[0256] The obtained nucleic acid-encapsulated nanoparticles were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. 1.5×10 4 HeLa cells were seeded in a 48-well flat-bottom transparent plate at 1:1 cells / 300 μL. The resulting suspension of nucleic acid-encapsulated nanoparticles was added in such a way that the nucleic acid concentration in the culture medium became 0.06 μg / 30 μL. 24 hours after the addition, 300 μL of Steady-Glo (registered trademark) luciferase assay system was added, and after incubation for 5 minutes using aluminum foil to shield the light, the luminescence at 560 nm was measured using a plate reader. As a result, among the nucleic acid-encapsulated nanoparticles based on freeze-drying technology, the nucleic acid-encapsulated nanoparticles based on freeze-drying technology produced in Example 1 had the highest gene expression activity, and had the same activity as the nucleic acid-encapsulated nanoparticles produced by the non-freeze-drying method of Comparative Example 3 (reference Figure 1 ).

[0257] [Table 2]

[0258] Preparation method of lipid nanoparticles of Comparative Example 4 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 4.0 mM.

[0259] 1986 μL of a solution of 6.7 μg / mL FLuc mRNA diluted with acid malic acid buffer (buffer concentration 20 mM, pH 3.0) and 663 μL of lipid ethanol solution were measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine production device (manufactured by Precision NanoSystems), LNP was prepared under the conditions of a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 10600 μL of MES buffer (buffer concentration 20 mM, pH 6.5) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The concentrate was diluted to 15 mL using PBS (pH 7.4), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The above operation was repeated twice, and the suspension was concentrated to 600 μL to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 10 μg / mL).

[0260] The preparation day of the suspension of the obtained nucleic acid encapsulated nanoparticles, the particle diameter of the 1st week and the 2nd week after preparation, the polydispersity index (PdI), and the encapsulation rate of mRNA are analyzed. Particle diameter and PdI are measured by the dynamic light scattering method using Zetasizer (registered trademark). The encapsulation rate of mRNA is measured by Ribogreen (registered trademark). As a result, no change in the physical properties of the particles is observed, and particle diameter and encapsulation rate are maintained in the 2nd week after preparation, indicating that the storage stability is high (refer to Table 3).

[0261] [Table 3]

[0262] The obtained nucleic acid-encapsulated nanoparticles were applied to HeLa cells to evaluate the efficiency of nucleic acid introduction. 5×10 3HeLa cells were seeded in a 35mm culture dish at 1:1 cells / 100μL. The resulting suspension of nucleic acid-encapsulated nanoparticles was added in such a way that the nucleic acid concentration in the culture medium became 0.4μg / 2000μL. Luciferin was added to the culture medium to 0.1mM, and the amount of luciferase protein introduced was evaluated over time using the luminescence. HeLa cells cultured in a culture medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence was measured once every hour for 2 minutes. As a result, a decrease in gene expression activity was observed from just after preparation to the first week, and the activity further decreased in the second week, indicating that the storage stability was low (refer to Figure 2 ).in addition, Figure 2 The "1.4.E+07" in the text means "1.4×10 7 "wait.

[0263] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 2 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 16.0 mM.

[0264] 2918 μL of an acidic malic acid buffer (buffer concentration 20 mM, pH 3.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding human erythropoietin (CleanCap (registered trademark) hEPO mRNA (L-7209)) and 973 μL of a lipid ethanol solution were measured into syringes. LNPs were prepared using a NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) with a flow ratio of nucleic acid solution: lipid ethanol solution set to 3:1, a total flow rate set to 4 mL / min, and a syringe holder temperature of 25°C. After adding 8400 μL of MES buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was divided into 5 equal portions in an Amicon Ultra 15. The mixture was diluted to 15 mL with MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL. The concentrate was diluted to 15 mL with MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL to obtain a mixture of lipid nanoparticles containing nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a rate of 600 μL each time and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.76% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight). The obtained freeze-dried composition was stored at -20°C, 4°C, and 25°C.

[0265] For freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 390 minutes. After that, raise the temperature by 10°C each time. The temperature rise program is set as follows: raise the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. Set as follows: raise the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is raised to 30°C, let it stand at this temperature for 3 hours and then return to normal pressure to recover the freeze-dried composition.

[0266] 600 μL of DDW was added to the freeze-dried composition and rehydrated by stirring with a vortex stirrer. In order to investigate the effect of incubation after rehydration, a portion was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator after rehydration. After the incubated suspension was cooled at 20°C to 25°C, 600 μL of PBS (pH 7.4) was added to prepare a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0267] Preparation of lipid nanoparticles of Comparative Example 4 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0268] 2288 μL of an acidic malic acid buffer (buffer concentration 20 mM, pH 3.0, nucleic acid concentration 6.7 μg / mL) containing mRNA encoding human erythropoietin (CleanCap (registered trademark) hEPO mRNA (L-7209)) and 863 μL of a lipid ethanol solution were measured into a syringe. LNP was prepared using a NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) with a flow ratio of nucleic acid solution: lipid ethanol solution set to 3:1, a total flow rate set to 4 mL / min, and a syringe holder temperature of 25°C. After adding 2400 μL of MES buffer (buffer concentration 20 mM, pH 6.5) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The concentrate was diluted to 15 mL using PBS (pH 7.4), and then ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1000 μL. The above operation was repeated twice, and the suspension was concentrated to 800 μL to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 10 μg / mL).

[0269] The particle diameter, polydispersity index (PdI), and mRNA encapsulation rate of the nucleic acid encapsulated nanoparticles of Example 2 obtained at each time point of the 0th day, 2 weeks, 1 month, and 3 months were analyzed. Particle diameter and PdI were measured by the dynamic light scattering method using Zetasizer (registered trademark). The mRNA encapsulation rate was measured by Ribogreen (registered trademark). As a result, in the 3-month storage, no change in particle properties was observed at each storage temperature, and particle properties were maintained, indicating that storage stability was high (see Table 4).

[0270] [Table 4]

[0271] The suspension of nucleic acid-encapsulated nanoparticles was administered to the tail vein of mice (Balb / c, 6 weeks old, female). 15 μL of blood was collected from the tail vein of the mice 6 hours and 24 hours after administration and immediately mixed with 0.3 μL of heparin solution (5000U / 5mL). Each blood sample was centrifuged under centrifugal conditions (25°C, 2000g, 20 minutes) to recover the supernatant. Using the mouse erythropoietin Quantikine ELISA kit (manufactured by R&D Systems), the erythropoietin concentration in the supernatant was measured according to the method described in the specifications of the kit. As a result, even after storage at 25°C for 3 months, the gene expression activity was maintained, indicating that the storage stability is high (refer to Figure 3 ).

[0272] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 3 SS-OP and SS-EC were used as ionic lipids (1), and DOPC, Chol, and DMG-PEG2000 were further used as other lipids. The molar ratio of SS-OP:SS-EC:DOPC:Chol used was 42.5:10:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, SS-EC, DOPC, and Chol. The total amount of the mixed lipids was 16 mM.

[0273] 1688 μL of an acidic malic acid buffer (buffer concentration 20 mM, pH 3.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 563 μL of a lipid ethanol solution were measured into a syringe. LNP was prepared using a NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 2000 μL of MES buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL. The concentrate was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL to obtain a mixture of lipid nanoparticles containing nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The mixture was placed in a screw tube at a rate of 600 μL each time and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.80% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0274] For freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 390 minutes. After that, raise the temperature by 10°C each time. The temperature rise program is set as follows: raise the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. Set as follows: raise the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is raised to 30°C, let it stand at this temperature for 3 hours and then return to normal pressure, and recover the freeze-dried composition.

[0275] The DDW added to the freeze-dried composition was stirred using a vortex agitator to dissolve the freeze-dried composition. The resulting mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. After the incubated suspension was water-cooled at 20°C to 25°C, 600 μL of PBS (pH 7.4) was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0276] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 4 SS-OP and SS-EC were used as ionic lipids (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:SS-EC:DOPC:Chol used was 32.5:20:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, SS-EC, DOPC, and Chol. The total amount of the mixed lipids was 16 mM.

[0277] 1688 μL of an acidic malic acid buffer (buffer concentration 20 mM, pH 3.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 563 μL of a lipid ethanol solution were measured into a syringe. LNP was prepared using a NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 2000 μL of MES buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL. The concentrate was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered twice again under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL to obtain a mixture of lipid nanoparticles containing nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a rate of 600 μL each time and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.83% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0278] For freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 390 minutes. After that, raise the temperature by 10°C each time. The temperature rise program is set as follows: raise the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. Set as follows: raise the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is raised to 30°C, let it stand at this temperature for 3 hours and then return to normal pressure, and recover the freeze-dried composition.

[0279] The DDW added to the freeze-dried composition was stirred using a vortex stirrer to dissolve the freeze-dried composition. The resulting mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. The incubated suspension was water-cooled at 20°C to 25°C, and 600 μL of PBS was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0280] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 5 SS-OP and SS-EC were used as ionic lipids (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:SS-EC:DOPC:Chol used was 22.5:30:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, SS-EC, DOPC, and Chol. The total amount of the mixed lipids was 10.7 mM.

[0281] 1688 μL of an acidic malic acid buffer (buffer concentration 20 mM, pH 3.0, nucleic acid concentration 17.8 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 563 μL of a lipid ethanol solution were measured into a syringe. LNP was prepared using a NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. After adding 2000 μL of MES buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL. The concentrate was diluted to 15 mL using MES buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1300 μL, thereby obtaining a mixture of lipid nanoparticles containing nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The mixture was placed in a screw tube at a rate of 600 μL each time, and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.86% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0282] For freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 390 minutes. After that, raise the temperature by 10°C each time. The temperature rise program is set as follows: raise the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. Set as follows: raise the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is raised to 30°C, let it stand at this temperature for 3 hours and then return to normal pressure, and recover the freeze-dried composition.

[0283] The DDW added to the freeze-dried composition was stirred using a vortex stirrer to dissolve the freeze-dried composition. The mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. The incubated suspension was water-cooled at 20°C to 25°C, and 600 μL of PBS was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0284] The pH of the suspension of nucleic acid-encapsulated nanoparticles after melting with DDW of each freeze-dried composition was measured (hereinafter referred to as "pH after dissolution"). In addition, the particle size, polydispersity index (PdI), and mRNA encapsulation rate of the obtained nucleic acid-encapsulated nanoparticles on the 4th and 21st day after preparation were analyzed. The particle size and PdI were measured by dynamic light scattering using Zetasizer (registered trademark). The mRNA encapsulation rate was measured by Ribogreen (registered trademark) analysis. As a result, even if the composition ratio of SS-EC was changed, no change in the physical properties of the particles was observed, and the particle size and encapsulation rate were maintained in the 3rd week after preparation, indicating that the storage stability is high. In addition, the particle size and mRNA encapsulation rate are equivalent to those of nucleic acid-encapsulated nanoparticles prepared using only SS-OP as the ionic lipid (1) (see Table 5).

[0285] [Table 5]

[0286] The obtained nucleic acid-encapsulated nanoparticles were applied to HeLa cells to evaluate the efficiency of nucleic acid introduction. 5×10 3 HeLa cells were seeded in a 35 mm culture dish at 1:1 cells / 100 μL. The resulting suspension of nucleic acid-encapsulated nanoparticles was added in such a manner that the nucleic acid concentration in the culture medium was 0.4 μg / 2000 μL. Luciferin was added to the culture medium to 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using the luminescence. HeLa cells cultured in a culture medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence was measured once every hour for 2 minutes. As a result, the highest gene introduction efficiency was obtained in Example 4, in which the content of SS-EC was 20 mol% relative to the total of ionic lipids (1), phospholipids and sterols, and at the same time, its introduction rate was maintained even after 21 days of storage, indicating that the storage stability is high (refer to Figure 4 ).in addition, Figure 4 The "1.2E+07" in the above example means "1.2×10 7 "wait.

[0287] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 6 SS-OP was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and Chol. The mixture was mixed until the total lipid content reached 16 mM.

[0288] 2325 μL of acidic citric acid buffer (buffer concentration 20 mM, pH 5.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 775 μL of lipid ethanol solution were measured into syringes. LNP was prepared using the NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 12 mL / min, and a syringe holder temperature of 25°C. After adding 2600 μL of citric acid buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The concentrate obtained was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), and ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1300 μL, thereby obtaining a mixture of lipid nanoparticles containing nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a rate of 600 μL each time, and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.77% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0289] Regarding freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 360 minutes. After that, reduce the pressure to 50 mTorr (6.7 Pa) and let it stand for 840 minutes. After that, increase the temperature by 10°C each time. The temperature increase program is set as follows: increase the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. It is set as follows: increase the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is increased to 30°C, it is allowed to stand at this temperature for 3 hours before returning to normal pressure and recovering the freeze-dried composition.

[0290] The DDW added to the freeze-dried composition was stirred using a vortex agitator to dissolve the freeze-dried composition. The mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. After the incubated suspension was cooled at 20°C to 25°C, 600 μL of TBS was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0291] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 7 SS-OP and SS-EC were used as ionic lipids (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:SS-EC:DOPC:Chol used was 32.5:20:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, SS-EC, DOPC, and Chol. The total amount of the mixed lipids was 16 mM.

[0292] 2325 μL of acidic citric acid buffer (buffer concentration 20 mM, pH 5.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 775 μL of lipid ethanol solution were measured into syringes. LNP was prepared using the NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 12 mL / min, and a syringe holder temperature of 25°C. After adding 2600 μL of citric acid buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 2000 μL. The concentrate was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered twice again under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1300 μL, thereby obtaining a mixture of lipid nanoparticles encapsulating nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The mixture was placed in a screw tube at a rate of 600 μL each time, and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.83% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0293] Regarding freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 360 minutes. After that, reduce the pressure to 50 mTorr (6.7 Pa) and let it stand for 840 minutes. After that, increase the temperature by 10°C each time. The temperature increase program is set as follows: increase the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. It is set as follows: increase the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is increased to 30°C, it is allowed to stand at this temperature for 3 hours before returning to normal pressure and recovering the freeze-dried composition.

[0294] The DDW added to the freeze-dried composition was stirred using a vortex stirrer to dissolve the freeze-dried composition. The mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. After the incubated suspension was water-cooled at 20°C to 25°C, 600 μL of TBS was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0295] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 8 PiP9 was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of PiP9:DOPC:Chol used was 52.5:7.5:40, and DMG-PEG2000 was used at 1.5 mol% relative to the total of PiP9, DOPC, and Chol. The total mixed lipid content was 16 mM.

[0296] 2325 μL of acidic citric acid buffer (buffer concentration 20 mM, pH 5.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 775 μL of lipid ethanol solution were measured into syringes. LNP was prepared using the NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 12 mL / min, and a syringe holder temperature of 25°C. After adding 2600 μL of citric acid buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 2000 μL. The concentrate obtained was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), and ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1300 μL, thereby obtaining a mixture of lipid nanoparticles containing nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The obtained mixture was placed in a screw tube at a rate of 600 μL each time, and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.86% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0297] Regarding freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 360 minutes. After that, reduce the pressure to 50 mTorr (6.7 Pa) and let it stand for 840 minutes. After that, increase the temperature by 10°C each time. The temperature increase program is set as follows: increase the temperature by 10°C to 0°C over 10 minutes, and let it stand at this temperature for 6 hours. It is set as follows: increase the temperature by 10°C over 10 minutes at a temperature above 0°C, and let it stand at this temperature for 3 hours. If the temperature is increased to 30°C, it is allowed to stand at this temperature for 3 hours before returning to normal pressure and recovering the freeze-dried composition.

[0298] The DDW added to the freeze-dried composition was stirred using a vortex agitator to dissolve the freeze-dried composition. The mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. After the incubated suspension was cooled at 20°C to 25°C, 600 μL of TBS was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0299] Preparation method of freeze-dried composition of lipid nanoparticles encapsulating nucleic acid of Example 9 PiP15 was used as the ionic lipid (1), and DOPC, Chol, and DMG-PEG2000 were used as other lipids. The molar ratio of PiP15:DOPC:Chol used was 52.5:7.5:40, and DMG-PEG2000 was used at 1.5 mol% relative to the total of PiP15, DOPC, and Chol. The total mixed lipid content was 16 mM.

[0300] 2325 μL of acidic citric acid buffer (buffer concentration 20 mM, pH 5.0, nucleic acid concentration 26.7 μg / mL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA (L-7602)) and 775 μL of lipid ethanol solution were measured into syringes. LNP was prepared using the NanoAssmblr (registered trademark) ultra-high-speed nanodrug preparation device (manufactured by Precision NanoSystems) at a flow ratio of nucleic acid solution: lipid ethanol solution of 3:1, a total flow rate of 12 mL / min, and a syringe holder temperature of 25°C. After adding 2600 μL of citric acid buffer (buffer concentration 20 mM, pH 6.0) to the recovered product, the resulting mixture was transferred to an Amicon Ultra 15. The mixture was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 2000 μL. The concentrate was diluted to 15 mL using a citric acid buffer (buffer concentration 20 mM, pH 6.0), ultrafiltered twice under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1300 μL, thereby obtaining a mixture of lipid nanoparticles encapsulating nucleic acids (nucleic acid concentration 10 μg / mL). Sucrose solution was added to a final concentration of 160 mg / mL. The mixture was placed in a screw tube at a rate of 600 μL each time, and freeze-dried to obtain a freeze-dried composition (ionic lipid (1) content: 0.87% by weight, nucleic acid content: 6 μg, N / P ratio: 67.5, sucrose content: 99.9% by weight).

[0301] Regarding freeze drying, first freeze the solution at normal pressure and -40°C for 150 minutes, reduce the pressure to 80 mTorr (11 Pa), and let it stand for 360 minutes. After that, reduce the pressure to 50 mTorr (6.7 Pa) and let it stand for 840 minutes. After that, increase the temperature by 10°C each time. The temperature increase program is set to: increase the temperature by 10°C to 0°C in 10 minutes, and let it stand at this temperature for 6 hours. Set it to increase the temperature by 10°C in 10 minutes above 0°C, and let it stand at this temperature for 3 hours. If the temperature is increased to 30°C, it is returned to normal pressure after standing at this temperature for 3 hours, and the freeze-dried composition is recovered.

[0302] The DDW added to the freeze-dried composition was stirred using a vortex agitator to dissolve the freeze-dried composition. The mixture was incubated at 95°C for 5 minutes using an ALUBLOCKS incubator. After the incubated suspension was cooled at 20°C to 25°C, 600 μL of TBS was added to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration 5 μg / mL).

[0303] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the nucleic acid encapsulated nanoparticles of Examples 6 to 9 obtained at each time point on the 0th day, the 7th day, and the 14th day were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by Ribogreen (registered trademark) analysis. As a result, no changes in the physical properties of the particles were observed at each storage temperature during the 2-week storage, and the physical properties of the particles were maintained, indicating that the storage stability was high (see Table 6).

[0304] [Table 6]

[0305] The obtained nucleic acid-encapsulated nanoparticles were applied to HeLa cells to evaluate the efficiency of nucleic acid introduction. 3×10 4 HeLa cells were seeded in a 24-well flat-bottom transparent black plate at 1:1 cells / 600 μL. The resulting suspension of nucleic acid-encapsulated nanoparticles was added in a manner such that the nucleic acid concentration in the culture medium was 0.12 μg / 60 μL. Luciferin was added to the culture medium to 0.1 mM, and the luminescence was used to evaluate the amount of luciferase protein introduced over time. HeLa cells cultured in a culture medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence was measured every hour. As a result, the introduction rate was maintained even after 14 days of storage due to the overall composition, indicating that the storage stability is high (refer to Figure 5 ).in addition, Figure 5 The "1.8E+08" in the above example means "1.8×10 8 "wait. Industrial Applicability

[0306] According to the present invention, nucleic acids can be efficiently introduced into cells, which is useful for nucleic acid drugs, gene therapy, and biochemical experiments.

[0307] This application is based on Japanese Patent Application No. 2022-114394 filed in Japan, the entire contents of which are incorporated herein by reference.

Claims

1. A freeze-dried composition of lipid nanoparticles encapsulating nucleic acid, comprising an ionic lipid represented by formula (1) and having a pH of 4.5 or more and 6.9 or less, 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 is 0 or 1, R 3a and R 3b Each independently represents: A residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, A residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, The group represented by formula (3), A group having 50 or less carbon atoms represented by formula (4), or A group having 50 or less carbon atoms represented by formula (5), R 9 -O-CO-(CH2) a -*(3) In formula (3), * indicates the bonding position, R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10; In formula (4), * indicates bonding position, and R 10 represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R 10 It may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms; In formula (5), * indicates the bonding position, R 11 represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynyl group having 2 to 9 carbon atoms, and R 11 is substituted or unsubstituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group and a hydrocarbon ring group having 3 to 12 carbon atoms, R 12 and R 13 Each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, and R 12 At least one ethylene group or at least one trimethylene group in the group is substituted or unsubstituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 13 At least one ethylene group or at least one trimethylene group in the group is substituted or unsubstituted by at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R 12 and R 13 are each independently substituted or unsubstituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, and X 4 represents an oxygen atom, NH or a sulfur atom.

2. The freeze-dried composition according to claim 1, R 3a and R 3b Each independently is: A residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, A residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, 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 The group represented by formula (3): R 9 -O-CO-(CH2) a -*(3) The symbols in formula (3) are defined as described in claim 1.

3. The freeze-dried composition according to claim 1, wherein the ionic lipid is a compound represented by the following formula:

4. The freeze-dried composition according to claim 3, further comprising a compound represented by the following formula as the ionic lipid:

5. The freeze-dried composition according to claim 1, wherein the ionic lipid is a compound represented by the following formula:

6. The freeze-dried composition according to claim 1, wherein the ionic lipid is a compound represented by the following formula: 7 . The freeze-dried composition according to claim 1 , wherein the nucleic acid encapsulated in the lipid nanoparticles is mRNA.

8. A method for producing a freeze-dried composition of lipid nanoparticles encapsulating nucleic acids, comprising the following steps: a) mixing an alcohol solution containing the ionic lipid represented by formula (1) according to claim 1, sterol, phospholipid and PEG lipid with a nucleic acid solution in an acidic buffer having a buffering effect at pH 1.0 to 6.5 to prepare a suspension of lipid nanoparticles encapsulating nucleic acid, b) replacing the external aqueous phase of the lipid nanoparticles encapsulating nucleic acids with another buffer having a buffering effect at pH 4.5 to 6.9 to obtain a mixture containing lipid nanoparticles encapsulating nucleic acids at pH 4.5 to 6.9, c) mixing the obtained mixture with a cryoprotectant to prepare a mixture containing 80 to 320 mg / mL of the cryoprotectant and lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9, d) a step of freeze-drying the mixture obtained in step c) to obtain a freeze-dried composition.

9. A method for producing lipid nanoparticles encapsulating nucleic acid, comprising the following steps: a) mixing an alcohol solution containing the ionic lipid represented by formula (1) according to claim 1, sterol, phospholipid and PEG lipid with a nucleic acid solution in an acidic buffer having a buffering effect at pH 1.0 to 6.5 to prepare a suspension of lipid nanoparticles encapsulating nucleic acid, b) replacing the external aqueous phase of the lipid nanoparticles encapsulating nucleic acids with another buffer having a buffering effect at pH 4.5 to 6.9 to obtain a mixture containing lipid nanoparticles encapsulating nucleic acids at pH 4.5 to 6.9, c) mixing the obtained mixture with a cryoprotectant to prepare a mixture containing 80 to 320 mg / mL of the cryoprotectant and lipid nanoparticles encapsulating nucleic acids at a pH of 4.5 to 6.9, d) freeze-drying the mixture obtained in step c) to obtain a freeze-dried composition, e) A step of mixing the freeze-dried composition with water and incubating the mixture at 0 to 95° C. for 0 to 60 minutes to obtain lipid nanoparticles encapsulating nucleic acid.

10. A method for introducing a nucleic acid into a cell, comprising the step of contacting the lipid nanoparticles encapsulating the nucleic acid produced by the method according to claim 9 with the cell in vitro.

11. A method for introducing a nucleic acid into a target cell, comprising the step of administering to a living body lipid nanoparticles encapsulating the nucleic acid produced by the method according to claim 9.

12. A method for producing a pharmaceutical composition, comprising the method according to claim 8.

13. A method for producing a pharmaceutical composition, comprising the method according to claim 9.

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