Method for producing ligand-modified lipid nanoparticles encapsulating nucleic acid

Abstract

The present invention provides a method for producing ligand-modified lipid nanoparticles encapsulating a nucleic acid, said method comprising the following steps a) to c), etc.　Step a) Mixing an alcohol solution comprising an ionic lipid, a sterol, and a PEG lipid with an acidic buffer solution having a pH of 1-6.5, thereby obtaining a suspension of lipid nanoparticles that do not include a nucleic acid. Step b) Mixing the lipid nanoparticles that do not include a nucleic acid with a nucleic acid solution, thereby obtaining a suspension of lipid nanoparticles that encapsulate a nucleic acid. Step c) Mixing the suspension of lipid nanoparticles that encapsulate a nucleic acid with a ligand-bonded lipid, thereby obtaining a suspension of ligand-modified lipid nanoparticles that encapsulate a nucleic acid.

Description

Method for producing ligand-modified lipid nanoparticles encapsulating nucleic acids

[0001] The present invention relates to a method for producing ligand-modified lipid nanoparticles encapsulating nucleic acids.

[0002] Effective and safe nucleic acid delivery carriers are needed to commercialize nucleic acid therapy using oligonucleic acids such as siRNA and gene therapy using mRNA, pDNA, etc. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but development of non-viral nucleic acid delivery carriers that can be used more safely is underway, and among these, lipid nanoparticles, which are carriers using ionic lipids, are currently the most commonly used non-viral nucleic acid delivery carriers.

[0003] Ionic lipids are broadly composed of an amine moiety and a lipid moiety. The amine moiety, which becomes protonated under acidic conditions, interacts electrostatically with nucleic acids, which are polyanions, to form lipid nanoparticles, which promote uptake into fat and deliver nucleic acids into cells.

[0004] A commonly used known ionic lipid is 1,2-dioleoyl-3-dimethylammonium propane (DODAP). It is known that by combining a known ionic lipid with a phospholipid, cholesterol, or PEG lipid, lipid nanoparticles can be formed, and nucleic acids can be delivered into cells (see, for example, Non-Patent Document 1).

[0005] In addition, Patent Document 1 describes an ionic lipid having a structure in which compounds consisting of one or two amine moieties and one lipid moiety are linked by a biodegradable disulfide bond.This document shows that this ionic lipid can improve the pharmacokinetics such as blood stability and tumor targeting, and by changing the structure around the amine moiety, the pKa of the lipid membrane structure can be adjusted to a value favorable for endosomal escape in cells, and furthermore, by utilizing the cleavage of disulfide bonds in cells, it has the effect of dissociating nucleic acid from the lipid membrane structure.In fact, compared with the known ionic lipid DODAP, it shows a high nucleic acid delivery efficiency, and it has been revealed that this ionic lipid can improve intracellular pharmacokinetics such as improving the delivery efficiency of nucleic acid into the cytoplasm.

[0006] Furthermore, Patent Document 2 discloses a lipid membrane structure that uses an ionic lipid having an aromatic ring introduced near the lipid moiety in addition to a tertiary amine moiety and a disulfide bond, thereby enhancing the ability to fuse with an endosomal membrane and further increasing the efficiency of nucleic acid transfer into the cytoplasm.

[0007] As mentioned above, lipid nanoparticles with improved intracellular dynamics have been developed by increasing endosomal escape efficiency and membrane fusion ability. However, for lipid nanoparticles made of ionic lipids to be more effective as nucleic acid delivery carriers in vivo, they must be able to target organs and cells.

[0008] A method for improving the target organ or cell directionality is to modify lipid nanoparticles with ligands.

[0009] For example, Non-Patent Document 2 discloses lipid nanoparticles modified with GalNAc. In this document, in addition to ionic lipids, phospholipids, and cholesterol, ligand-bound PEG lipids and nucleic acids for cell targeting are mixed to produce ligand-modified lipid nanoparticles encapsulating nucleic acids. This document also shows that intravenous injection of the ligand-modified lipid nanoparticles encapsulating nucleic acids produced in this manner improves gene silencing efficiency.

[0010] Furthermore, Non-Patent Document 3 discloses lipid nanoparticles modified with RGD peptide. In this document, a PEG lipid is first conjugated to a ligand to synthesize a ligand-conjugated PEG lipid, and lipid nanoparticles containing nucleic acid are produced from other components. The resulting lipid nanoparticles containing nucleic acid are then incubated with the ligand-conjugated PEG lipid to produce ligand-modified lipid nanoparticles containing nucleic acid. This document also shows that intravenous injection of the ligand-modified lipid nanoparticles containing nucleic acid produced in this manner improves the efficiency of nucleic acid delivery to tumors.

[0011] Furthermore, Patent Document 3 discloses lipid nanoparticles modified with anti-CD3 IgG antibodies. In this document, ligand-modified lipid nanoparticles encapsulating nucleic acids are produced by adding a ligand to lipid nanoparticles encapsulating nucleic acids produced using activated PEG lipids. This document also discloses that intravenous administration of the ligand-modified lipid nanoparticles encapsulating nucleic acids produced in this manner improves the efficiency of nucleic acid delivery to T cells in the spleen.

[0012] International Publication No. WO 2013 / 073480 International Publication No. WO 2019 / 188867 International Publication No. WO 2023 / 054243

[0013] Biomaterials 29 (2008) 3477-3496Molecular Therapy vol. 18 no. 7, 1357-1364 july 2010Journal of Controlled Release 173 (2014) 110-118

[0014] The method of Non-Patent Document 2 above involves mixing the components of lipid nanoparticles, nucleic acid, and ligand-bound PEG lipids all at once to obtain ligand-modified lipid nanoparticles encapsulating nucleic acid. The methods of Non-Patent Document 3 and Patent Document 3 involve producing lipid nanoparticles encapsulating nucleic acid and then modifying them with ligands. Both of these methods involve simultaneously forming lipid nanoparticles and encapsulating nucleic acid into them to produce lipid nanoparticles encapsulating nucleic acid, but there is room for improvement in terms of encapsulating nucleic acid into lipid nanoparticles.

[0015] The present invention has been made in light of the above-mentioned circumstances, and its purpose is to provide a method for producing ligand-modified lipid nanoparticles encapsulating nucleic acids, which allows nucleic acids to be encapsulated in lipid nanoparticles using a procedure different from that of conventional techniques.

[0016] As a result of intensive research in view of the above problems, the present inventors have found that, unlike conventional techniques, nucleic acids can be encapsulated in lipid nanoparticles by first preparing lipid nanoparticles that do not contain nucleic acids and then encapsulating nucleic acids in these lipid nanoparticles. The present invention based on this finding is as follows.

[0017] [1] A method for producing ligand-modified lipid nanoparticles encapsulating nucleic acid, comprising: a) mixing an alcohol solution containing an ionic lipid, a sterol, and a PEG lipid with an acidic buffer solution having a pH of 1 to 6.5 to obtain a suspension of lipid nanoparticles free of nucleic acid; and b) mixing the nucleic acid-free lipid nanoparticles with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulating nucleic acid, and c) mixing the suspension of lipid nanoparticles encapsulating nucleic acid with a ligand-binding lipid to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid; or c') mixing the nucleic acid-free lipid nanoparticles with water and a ligand-binding lipid to obtain a suspension of ligand-modified lipid nanoparticles free of nucleic acid, and b') mixing the nucleic acid-free ligand-modified lipid nanoparticles with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid; or d) mixing the nucleic acid-free lipid nanoparticles with a nucleic acid solution and a ligand-binding lipid to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.

[0018] [2] The method according to [1] above, wherein in step b), a suspension of lipid nanoparticles not containing nucleic acid is mixed with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulating nucleic acid, or in step c'), a suspension of lipid nanoparticles not containing nucleic acid is mixed with a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles not containing nucleic acid, and in step b'), a suspension of ligand-modified lipid nanoparticles not containing nucleic acid is mixed with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid, or in step d), a suspension of lipid nanoparticles not containing nucleic acid is mixed with a nucleic acid solution and a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.

[0019] [3] The method according to [1] or [2], comprising steps a), b), and c), or comprising steps a), c′), and b′).

[0020] [4] The method according to any one of [1] to [3] above, comprising maintaining a mixture of nucleic acid-free lipid nanoparticles of step b) and a nucleic acid solution, or a mixture of nucleic acid-free ligand-modified lipid nanoparticles of step b') and a nucleic acid solution at 0 to 95°C for 1 minute to 2 hours.

[0021] [5] The method according to any one of [1] to [4], comprising maintaining a mixture of a suspension of lipid nanoparticles encapsulating nucleic acid in step c) and a ligand-conjugated lipid, or a mixture of lipid nanoparticles not containing nucleic acid in step c'), water, and a ligand-conjugated lipid, at 0 to 95°C for 1 minute to 5 hours.

[0022] [6] The method according to any one of [1], [2], and [4] to [5], comprising maintaining the mixture of the nucleic acid-free lipid nanoparticles of step d), the nucleic acid solution, and the ligand-bound lipid at 0 to 95°C for 1 minute to 2 hours.

[0023] [7] The method according to any one of [1] to [6] above, wherein in step c), a suspension of lipid nanoparticles encapsulating nucleic acid is mixed with a ligand-conjugated lipid and a lipid to which no ligand is bound, to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid; or in step c'), lipid nanoparticles not containing nucleic acid are mixed with water, a ligand-conjugated lipid and a lipid to which no ligand is bound, to obtain a suspension of ligand-modified lipid nanoparticles not containing nucleic acid; or in step d), lipid nanoparticles not containing nucleic acid are mixed with a nucleic acid solution, a ligand-conjugated lipid and a lipid to which no ligand is bound, to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.

[0024] [8] The method according to any one of [1] to [7], wherein the alcohol solution in step a) further contains a phospholipid. [9] The ionic lipid is represented by the formula (1):

[0025]

[0026] (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two 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 carbamate 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, at least one aromatic ring, and optionally a heteroatom; ａ and n ｂ are each independently 0 or 1; ３ａ and R３ｂ 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, 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 a cyclopropane ring and having 3 to 40 carbon atoms, Formula (3): R ９ —O—CO—(CH ２ ) ａ -* (3) (In formula (3), * represents a bonding position, R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.

[0027]

[0028] (In formula (4), * represents a 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 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.

[0029]

[0030] (In formula (5), * represents a 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 alkynediyl 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; R １２At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; １３ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and １２ and R １３ may each independently 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, and X ４ represents an oxygen atom, NH, or a sulfur atom.) represents a group having 50 or less carbon atoms, represented by the formula: (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate.

[0031]

[10] The method according to [9] above, wherein the ionic lipid comprises a compound represented by formula (1).

[11] The method according to any one of [1] to

[10] above, wherein the ligand in the ligand-binding lipid is a ligand having brain cell tropism or a ligand having T cell tropism.

[0032]

[12] A method for introducing a nucleic acid into a cell, comprising a step of contacting the cell ex vivo with a ligand-modified lipid nanoparticle encapsulating the nucleic acid, obtained by the method described in any one of [1] to

[11] above.

[13] A method for introducing the nucleic acid into a target cell in the living body, comprising a step of administering to the living body ligand-modified lipid nanoparticles encapsulating the nucleic acid, obtained by the method described in any one of [1] to

[11] above.

[14] A method for producing a pharmaceutical composition, comprising the method described in any one of [1] to

[11] above.

[0033] According to the method of the present invention for producing ligand-modified lipid nanoparticles encapsulating nucleic acids, nucleic acids can be encapsulated in lipid nanoparticles by a procedure different from that of conventional techniques.

[0034] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these. Note that the descriptions in this specification can be combined with each other unless it is clear that they cannot be combined.

[0035] <Method for producing ligand-modified lipid nanoparticles encapsulating nucleic acids> The present invention comprises a step a) of mixing an alcohol solution containing ionic lipids, sterols, and PEG lipids (hereinafter sometimes abbreviated as "lipid solution") with an acidic buffer solution having a pH of 1 to 6.5 (hereinafter sometimes abbreviated as "acidic buffer") to obtain a suspension of lipid nanoparticles that do not contain nucleic acids, and a step b) of mixing lipid nanoparticles that do not contain nucleic acids with a nucleic acid solution (preferably mixing a suspension of lipid nanoparticles that do not contain nucleic acids with a nucleic acid solution) to obtain a suspension of lipid nanoparticles that encapsulate nucleic acids, and a step c) of mixing the suspension of lipid nanoparticles encapsulating nucleic acids with a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles that encapsulate nucleic acids, or The present invention provides a method for producing nucleic acid-encapsulated ligand-modified lipid nanoparticles (hereinafter sometimes abbreviated as "target lipid nanoparticles") (hereinafter sometimes abbreviated as "the production method of the present invention"), which comprises: step c') of mixing nucleic acid-free lipid nanoparticles, water, and ligand-conjugated lipids (preferably, mixing a suspension of nucleic acid-free lipid nanoparticles with ligand-conjugated lipids) to obtain a suspension of nucleic acid-free, ligand-modified lipid nanoparticles; and step b') of mixing nucleic acid-free, ligand-modified lipid nanoparticles with a nucleic acid solution (preferably, mixing a suspension of nucleic acid-free, ligand-modified lipid nanoparticles with a nucleic acid solution) to obtain a suspension of nucleic acid-encapsulated ligand-modified lipid nanoparticles.

[0036] The production method of the present invention includes "step a), step b), and step c")," "step a), step c'), and step b'")," or "step a) and step d)". Other steps (e.g., exchange of a dispersion medium, etc.) may be performed between these steps as needed. The production method of the present invention preferably includes "step a), step b), and step c") or "step a), step c'), and step b'"), and more preferably includes "step a), step b), and step c)".

[0037] In this specification, "lipid nanoparticles" refers to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are arranged toward the aqueous phase side of the interface. "Amphipathic lipids" refers to lipids having both hydrophilic groups that exhibit hydrophilicity and hydrophobic groups that exhibit hydrophobicity. Examples of amphipathic lipids include ionic lipids, phospholipids, PEG lipids, etc.

[0038] The target lipid nanoparticles obtained by the production method of the present invention contain ionic lipids, sterols, and PEG lipids as membrane constituents, and may further contain phospholipids. The particle size of the target lipid nanoparticles is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Measurement of particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). In this specification, "particle size" refers to the average particle size (zeta mean) measured by dynamic light scattering.

[0039] As used herein, the term "total lipids" refers to the total amount of lipids, including, for example, ionic lipids, sterols, PEG lipids, and phospholipids.

[0040] As used herein, the term "(ligand-modified) lipid nanoparticles that do not contain nucleic acids" refers to (ligand-modified) lipid nanoparticles whose nucleic acid content is below the detection limit.

[0041] As used herein, the term "(ligand-modified) lipid nanoparticles encapsulating nucleic acids" refers to (ligand-modified) lipid nanoparticles in which nucleic acids are encapsulated inside the (ligand-modified) lipid nanoparticles.

[0042] As used herein, the term "ligand" refers to a substance that has specific affinity for a molecule expressed on the surface of a target cell to which the lipid nanoparticle should be delivered (e.g., a cell (e.g., cancer cell, etc.) to which a therapeutic effect against a disease is exerted by introducing a drug (preferably, a nucleic acid) encapsulated in the lipid nanoparticle into the cell).

[0043] As used herein, the term "ligand-modified lipid nanoparticle" refers to a lipid nanoparticle having a ligand, which is displayed on the surface of the lipid nanoparticle so that the ligand interacts with a molecule expressed on the target cell to which it has a specific affinity, and is delivered to the target cell.

[0044] <Step a)> In step a), a lipid solution is mixed with an acidic buffer solution to obtain a suspension of nucleic acid-free lipid nanoparticles. The acidic buffer solution used in step a) contains water as a solvent, and the suspension of nucleic acid-free lipid nanoparticles obtained in step a) contains water as a dispersion medium.

[0045] Examples of alcohols for the lipid solution include ethanol and tert-butanol. Only one type of alcohol may be used, or two or more types may be used in combination. The alcohol is preferably ethanol.

[0046] The lipid solution and the acidic buffer solution in step a) are preferably mixed using a device with a microchannel or a vortex mixer, more preferably using a device with a microchannel. Examples of devices with a microchannel include "NanoAssemblr Ignite" (manufactured by Precision NanoSystems) and the nanoparticle manufacturing device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.).

[0047] The flow rates of the lipid solution and the acidic buffer solution when mixing the lipid solution and the acidic buffer solution in step a) using a device containing a microchannel, and the flow rate ratio thereof, are not particularly limited as long as they are capable of forming lipid nanoparticles, and can be appropriately selected depending on the performance of the microchannel used and the device. The flow rates of the lipid solution and the acidic buffer solution are preferably 100 to 200,000 μL / min, more preferably 1,000 to 200,000 μL / min. The ratio of the flow rate of the lipid solution to the flow rate of the acidic buffer solution (flow rate of the lipid solution / flow rate of the acidic buffer solution) is preferably 1 / 10 to 1 / 1, more preferably 1 / 8 to 1 / 2. Note that the "ratio of the flow rate of the lipid solution to the flow rate of the acidic buffer solution (flow rate of the lipid solution / flow rate of the acidic buffer solution)" is equal to the "ratio of the volume of the lipid solution to the volume of the acidic buffer solution (volume of the lipid solution / volume of the acidic buffer solution)."

[0048] The temperature at which the lipid solution and the acidic buffer solution are mixed in step a) is not particularly limited as long as it is a temperature at which lipid nanoparticles can be formed. From the viewpoint of ease of formation of lipid nanoparticles, the temperature is preferably 4 to 50°C, more preferably 20 to 30°C.

[0049] The alcohol solution (i.e., lipid solution) of step a) contains an ionic lipid. In one embodiment of the present invention, the ionic lipid preferably contains at least one selected from the group consisting of a compound represented by the following formula (1) (sometimes abbreviated as "ionic lipid (1)" in this specification), [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and more preferably contains ionic lipid (1).

[0050] In another embodiment of the present invention, the ionic lipid is preferably at least one selected from the group consisting of ionic lipid (1), [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and more preferably ionic lipid (1), [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and more preferably ionic lipid (1). Ionic lipid (1) may be used alone or in combination of two or more.

[0051]

[0052] (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two 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 carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂeach 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; ａ and n ｂ are each independently 0 or 1; 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, 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 a cyclopropane ring and having 3 to 40 carbon atoms, Formula (3): R ９ —O—CO—(CH ２ ) ａ -* (3) (In formula (3), * represents a bonding position, R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.

[0053]

[0054] (In formula (4), * represents a 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 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.

[0055]

[0056] (In formula (5), * represents a 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 alkynediyl 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 １３each 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; R １２ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; １３ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and １２ and R １３ may each independently 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, and X ４ represents an oxygen atom, NH, or sulfur atom.) represents a group having 50 or less carbon atoms represented by the following formula:

[0057] The definition of each symbol in formula (1) will be explained below. １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 4, and more preferably 1 to 2. Specific examples of alkylene groups having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. R １ａ and R １ｂ are preferably each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each are an ethylene group.

[0058] R １ａ is R １ｂ may be the same as or different from, but preferably, R １ａ is R １ｂ is the same group as

[0059] Xａ and X ｂ each independently represent a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups, and preferably each independently represent a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups.

[0060] The alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0061] A preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is: X １ It is shown by X １ The ends of the following formula indicate the bond positions, not carbon atoms.

[0062]

[0063] X １ R ５represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0064] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups is preferably 4 or 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups include aziridinediyl, azetidinediyl, pyrrolidinediyl, piperidinediyl, imidazolidinediyl, and piperazinediyl groups, preferably pyrrolidinediyl, piperidinediyl, and piperazinediyl groups, and most preferably piperidinediyl groups. In this specification, the term "compound name + diyl group (e.g., aziridinediyl group)" refers to a divalent group having a structure obtained by removing two hydrogen atoms from the compound (e.g., aziridine).

[0065] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is X ２ It is shown by X ２ The ends of the following formula indicate the bond positions, not carbon atoms.

[0066]

[0067] X ２ In the formula, p is 1 or 2. When p is 1, X ２ is a pyrrolidinediyl group, and when p is 2, X ２ is a piperidinediyl group. Preferably, p is 2.

[0068] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X ３ It is shown by X ３ The ends of the following formula indicate the bond positions, not carbon atoms.

[0069]

[0070] X ３ The w in is 1 or 2. When w is 1, X ３ is an imidazolidinediyl group, and when w is 2, X ３ is a piperazinediyl group.

[0071] X ａ is X ｂ may be the same as or different from, but preferably, X ａ is X ｂ is the same group as

[0072] R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, and preferably each independently represents an alkylene group having 8 or less carbon atoms.

[0073] The alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear. 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 a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group. Preferred are a methylene group, an ethylene group, a trimethylene group, and a tetramethylene group, and most preferably an ethylene group.

[0074] In this specification, an "oxydialkylene group having 8 or less carbon atoms" refers to an alkylene group (alkylene-O-alkylene, in other words, an "alkyleneoxyalkylene group") bonded via an ether bond, in which the total number of carbon atoms in the two alkylene groups is 8 or less. Here, the two alkylene groups may be the same or different, but are preferably the same. Specific examples of oxydialkylene groups having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydi(trimethylene) group (i.e., a trimethyleneoxytrimethylene group), and an oxydi(tetramethylene) group (i.e., a tetramethyleneoxytetramethylene group). Preferred are an oxydimethylene group, an oxydiethylene group, and an oxydi(trimethylene) group, and most preferred is an oxydiethylene group.

[0075] R ２ａ is R ２ｂ may be the same as or different from, but preferably, R ２ａ is R ２ｂ is the same group as

[0076] Y ａ and Y ｂ are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or a carbamate bond, more preferably each independently an ester bond or an amide bond, and most preferably each an ester bond. ａ and Y ｂ The bond direction of Y is not restricted. ａ and Y ｂ is an ester bond, preferably -Z ａ -CO-O-R ２ａ - and -Z ｂ -CO-O-R ２ｂ - structure.

[0077] Y ａ Is Y ｂ may be the same as or different from, but preferably, Y ａ Is Y ｂ is the same group as

[0078] 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 aromatic compound preferably contains 6 to 12 carbon atoms, and most preferably 6 to 7 carbon atoms. The aromatic compound preferably contains one aromatic ring.

[0079] Examples of the aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms include, for example, a benzene ring, a naphthalene ring, and an anthracene ring as aromatic hydrocarbon rings, and 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 furanthiophene 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, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring as aromatic hetero rings. Of these, a benzene ring, a naphthalene ring, and an anthracene ring are preferred, and a benzene ring is most preferred.

[0080] 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 alkylsulfanyl 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 ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl 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 ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, a methyl group having 1 to 4 ... Examples of the alkyl group include an aryl group having 0 carbon atoms and an aryloxy group having 6 to 10 carbon atoms, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl 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.

[0081] Z ａ and Z ｂ Preferred specific structures of Z １ In addition, Z １ The ends of the following formula indicate the bond positions, not carbon atoms.

[0082]

[0083] 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.

[0084] Z １ In the formula, s is preferably an integer of 0 to 1, and more preferably 0. １ t is preferably an integer of 0 to 2, more preferably 1. １ The u in the formula (1) is preferably an integer of 0 to 2, and more preferably an integer of 0 to 1.

[0085] Z １ R ４ is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that 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 alkylsulfanyl 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 ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, Examples of such an aryloxy group include those having 6 to 10 carbon atoms, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl 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. ４ When there are multiple R ４ may be the same or different.

[0086] Z ａ is Z ｂ may be the same as or different from, but preferably, Z ａ is Z ｂ is the same group as

[0087] n ａ and n ｂ are each independently 0 or 1. ａ is n ｂ may be the same as or different from, but preferably, n ａ is n ｂ is the same as

[0088] R３ａ 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, 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 a cyclopropane ring, a group represented by the formula (3) above, a group represented by the formula (4) above and having 50 or less carbon atoms, or a group represented by the formula (5) above and having 50 or less carbon atoms.

[0089] In one embodiment of the present invention, R ３ａ and R ３ｂ are preferably each 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 a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (3); more preferably, 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 an aliphatic hydrocarbon group having 12 to 22 carbon atoms; and even more preferably, are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0090] In another embodiment of the present invention, R ３ａ and R ３ｂ are preferably each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with 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).

[0091] In another embodiment of the present invention, R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or R ３ａ and R ３ｂare each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or R ３ａ and R ３ｂ are each independently a group having 50 or less carbon atoms and represented by the formula (4), or ３ａ and R ３ｂ one of R is an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and the other is a group having 50 or less carbon atoms and represented by the formula (4), or ３ａ and R ３ｂ It is more preferable that one of the above is an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and the remaining one is a group represented by the above formula (5) having 50 or less carbon atoms.

[0092] In another embodiment of the present invention, R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with 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 R is an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and the other is a group having 50 or less carbon atoms and represented by the formula (4), or ３ａ and R ３ｂ It is more preferable that one of the above is an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and the remaining one is a group represented by the above formula (5) having 50 or less carbon atoms.

[0093] In another embodiment of the present invention, R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or R ３ａ and R ３ｂ It is particularly preferred that each of the groups is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0094] In yet another embodiment of the present invention, R ３ａ and R ３ｂare each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or R ３ａ and R ３ｂ are each independently a group having 50 or less carbon atoms and represented by the formula (4), or ３ａ and R ３ｂ It is preferable that one of the above is an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and the remaining one is a group represented by the above formula (4) having 50 or less carbon atoms.

[0095] In yet another embodiment of the present invention, R ３ａ and R ３ｂ and are more preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0096] "Residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride" means a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group, wherein the hydroxyl group of the fat-soluble vitamin is *-O-CO-CH ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure where a - is replaced by *. The symbol * represents the bonding position with a fat-soluble vitamin.

[0097] The "residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride" means a residue derived from the reaction product of a sterol derivative having a hydroxyl group, wherein the hydroxyl group is *-O-CO-CH ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure where a - is replaced with *. The symbol * represents the bonding position with the sterol derivative.

[0098] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0099] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and preferably cholesterol or cholestanol.

[0100] The aliphatic hydrocarbon group having 1 to 40 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, with carbon-carbon double bonds being preferred. 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. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., with alkyl or alkenyl groups being preferred. Specific examples of the aliphatic hydrocarbon group having 1 to 40 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a henicosyl group, a docosyl group, a tricosyl group, a tetracosyl group, a pentacosyl group, a hexacosyl group, a heptacosyl group, an octacosyl group, a nonacosyl group, a triacontyl group, a tetracontyl group, a dodecenyl group, a tridecenyl group, a tetracont ... Adecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosenyl group, docosenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosenyl group Examples of such groups include a 1-hexyl group, a 1-hexylnonyl group, a 1-octylundecyl group, a 1-decylundecyl group, and a 1-decylundecyl group.The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0101] In one embodiment of the present invention, R ３ａ and R ３ｂ The aliphatic hydrocarbon group having 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) represented by the formula (1) is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). Specific examples of the aliphatic hydrocarbon group include a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.

[0102] R ３ａ and R ３ｂ The "alkyl group having 3 to 40 carbon atoms and a cyclopropane ring" in the above means an alkyl group having 3 to 40 carbon atoms and having at least one cyclopropane ring in the alkyl chain. The number of carbon atoms of the alkyl group, 3 to 40, does not include the number of carbon atoms of the cyclopropane ring. The number of cyclopropane rings that the alkyl group has is preferably one. R ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the formula (6):

[0103]

[0104] (In formula (6), * represents a bonding position, b and c each independently represent an integer, and the sum 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, even more preferably an integer of 3 to 17, and even more preferably an integer of 4 to 12. More preferably, c is an integer of 3 to 15, even more preferably an integer of 3 to 11, and even more preferably an integer of 3 to 9. Examples of the group represented by formula (6) include 7-(2-octylcyclopropyl)heptyl.

[0105] In formula (3), * represents a bonding position, not a carbon atom, as described above. ９ The aliphatic hydrocarbon group having 2 to 20 carbon atoms represented by the formula (I) may be linear or branched.

[0106] 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, with carbon-carbon double bonds being preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, even more preferably 13 to 19, and most preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., with alkyl groups or alkenyl groups being preferred, and alkyl groups being more preferred. Specific examples of the aliphatic hydrocarbon group having 2 to 20 carbon atoms include an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, Examples thereof include an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosenyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group, a tetradecadienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-ethylnonyl group, a 1-butylnonyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, and a 3-octylundecyl group. The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0107] In formula (3), a is preferably an integer of 3 to 9, more preferably an integer of 3 to 7, even more preferably an integer of 5 to 7, and most preferably 5 or 7.

[0108] In formula (4), * represents a bonding position, not a carbon atom, as described above. 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 may be substituted with a substituent selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms.

[0109] In this specification, "R １０ 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" (hereinafter abbreviated as "the expression") means that R １０ This means that the alkyl group, alkenyl group, alkynyl group, or hydrocarbon ring group may each independently 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. Other expressions similar to the above expression have the same meaning as the above expression. In this specification, "alkyl group," "alkenyl group," "alkynyl group," "alkylene group," "alkenediyl group," and "alkynediyl group" without any particular description of the substituent represent unsubstituted groups.

[0110] In this specification, the alkyl group may be either linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, Examples of such groups include a henicosyl group, a docosyl group, a tricosyl group, a tetracosyl group, a pentacosyl group, a hexacosyl group, a heptacosyl group, an octacosyl group, a nonacosyl group, a triacontyl group, a hentriacontyl group, a dotriacontyl group, a tritriacontyl group, a tetratriacontyl group, a pentatriacontyl group, a hexatriacontyl group, a tetracontyl group, a hentetracontyl group, a dotetracontyl group, a tritetracontyl group, and a tetratetracontyl group.

[0111] In this specification, the alkenyl group may be either linear or branched, and the number of olefinic carbon-carbon double bonds in the alkenyl group may be one or two or more. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, henicosenyl, docosenyl, tricosenyl, tetracosenyl, pentacosenyl, hexacosenyl, heptacosenyl, octacosenyl, nonacosenyl, triacontenyl, hentriacontenyl, and dotriacontenyl groups.

[0112] In this specification, the alkynyl group may be either linear or branched, and the number of carbon-carbon triple bonds in the alkynyl group may be one or two or more. Examples of the alkynyl group include an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tridecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecinyl group, a heptadecynyl group, an octadecynyl group, a nonadecinyl group, an icosinyl group, a henicosinyl group, a docosinyl group, a tricosinyl group, a tetracosinyl group, a pentacosinyl group, a hexacosinyl group, a heptacosinyl group, an octacosinyl group, a nonacosinyl group, a triacontinyl group, a hentriacontinyl group, and a dotriacontinyl group.

[0113] In this specification, the term "hydrocarbon ring group having 3 to 12 carbon atoms" refers to a cyclic group whose ring is composed of 3 to 12 carbon atoms. Examples of hydrocarbon ring groups having 3 to 12 carbon atoms include cycloalkyl groups having 3 to 8 carbon atoms, phenyl groups, naphthyl groups, and adamantyl groups. Examples of cycloalkyl groups having 3 to 8 carbon atoms include cyclopropyl groups, cyclobutyl groups, cyclopentyl groups, cyclohexyl groups, cycloheptyl groups, and cyclooctyl groups. 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 having 3 to 8 carbon atoms or an adamantyl group, and even more preferably a cyclohexyl group or an adamantyl group.

[0114] As used herein, the term "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 a 5- to 14-membered aromatic heterocyclic group and a 3- to 14-membered non-aromatic heterocyclic group. The 3- to 14-membered heterocyclic group is preferably a 3- to 14-membered non-aromatic heterocyclic group.

[0115] In the present specification, examples of the 5- to 14-membered aromatic heterocyclic group include the following: (i) 5- to 6-membered monocyclic aromatic heterocyclic groups such as a thienyl group, a furyl group, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a 1,2,4-oxadiazolyl group, a 1,3,4-oxadiazolyl group, a 1,2,4-thiadiazolyl group, a 1,3,4-thiadiazolyl group, a triazolyl group, a tetrazolyl group, and a triazinyl group; (ii) a benzothiophenyl group, a benzofuranyl group, a benzimidazolyl group, a benzoxazolyl group, a benzisoxazolyl group, a benzothiazolyl group, a benzisothiazolyl group, a benzotriazolyl group, an imidazopyridinyl group, a thienopyridinyl group, a furopyridinyl group, a pyrrolopyridinyl group, a pyrazolopyridinyl group, an oxazolopyridinyl group, a thiazolopyridinyl group, an imidazopyrazinyl group, an imidazopyrimidinyl group, a thienopyrimidinyl group, a furopyrimidinyl group, a pyrrolopyrimidinyl group, a pyrazolopyrimidinyl group, an oxazolopyridinyl group, and 8- to 14-membered fused polycyclic aromatic heterocyclic groups such as azolopyrimidinyl group, thiazolopyrimidinyl group, pyrazolotriazinyl group, naphtho[2,3-b]thienyl group, phenoxathiinyl group, indolyl group, isoindolyl group, 1H-indazolyl group, purinyl group, isoquinolyl group, quinolyl group, phthalazinyl group, naphthyridinyl group, quinoxalinyl group, quinazolinyl group, cinnolinyl group, carbazolyl group, β-carbolinyl group, phenanthridinyl group, acridinyl group, phenazinyl group, phenothiazinyl group, and phenoxazinyl group.

[0116] In the present specification, examples of the 3- to 14-membered non-aromatic heterocyclic group include the following: (i) an aziridinyl group, an oxiranyl group, a thiiranyl group, an azetidinyl group, an oxetanyl group, a thietanyl group, a tetrahydrothienyl group, a tetrahydrofuranyl group, a pyrrolinyl group, a pyrrolidinyl group, an imidazolinyl group, an imidazolidinyl group, an oxazolinyl group, an oxazolidinyl group, a pyrazolinyl group, a pyrazolidinyl group, a thiazolinyl group, a thiazolidinyl group, a tetrahydroisothiazolyl group, a dithiolanyl group (e.g., a 1,2-dithiolan-3-yl group), and a tetrahydrooxazolyl group. 3- to 8-membered monocyclic non-aromatic heterocyclic groups such as a tetrahydroisoxazolyl group, a piperidinyl group, a piperazinyl group, a tetrahydropyridinyl group, a dihydropyridinyl group, a dihydrothiopyranyl group, a tetrahydropyrimidinyl group, a tetrahydropyridazinyl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a morpholinyl group, a thiomorpholinyl group, an azepanyl group, a diazepanyl group, an azepinyl group, an oxepanyl group, an azocanyl group, or a diazocanyl group; (ii) a dihydrobenzofuranyl group, a dihydrobenzimidazolyl group, a dihydrobenzoxazolyl group, a dihydrobenzothiazolyl group, a dihydrobenzisothiazolyl group, a dihydronaphtho[2,3-b]thienyl group, a tetrahydroisoquinolyl group, a tetrahydroquinolyl group, a 4H-quinolidinyl group, an indolinyl group, an isoindolinyl group, a tetrahydrothieno[2,3-c]pyridinyl group, a tetrahydrobenzazepinyl group, a tetrahydroquinoxalinyl group, a tetrahydro and 9- to 14-membered fused polycyclic non-aromatic heterocyclic groups such as a tetrahydrophenanthridinyl group, a hexahydrophenothiazinyl group, a hexahydrophenoxazinyl group, a tetrahydrophthalazinyl group, a tetrahydronaphthyridinyl group, a tetrahydroquinazolinyl group, a tetrahydrocinnolinyl group, a tetrahydrocarbazolyl group, a tetrahydro-β-carbolinyl group, a tetrahydroacridinyl group, a tetrahydrophenazinyl group, a tetrahydrothioxanthenyl group, and an octahydroisoquinolyl group.

[0117] In one embodiment of the present invention, R １０is preferably 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 the alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms.

[0118] In one embodiment of the present invention, R １０ is more preferably 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.

[0119] In one embodiment of the present invention, R １０ is more preferably an alkyl group having 1 to 20 carbon atoms, particularly preferably an alkyl group having 1 to 10 carbon atoms, and most preferably a heptyl group.

[0120] In another embodiment of the present invention, R １０ is preferably 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 the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group).

[0121] In another embodiment of the present invention, R １０ is more preferably an alkyl group having 1 to 20 carbon atoms or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group).

[0122] In one embodiment of the present invention, R １０ is more preferably an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 1 to 10 carbon atoms, and the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group).

[0123] In formula (5), * represents a bonding position, not a carbon atom, as described above. １１ represents 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, and R １１may 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.

[0124] In this specification, the alkylene group may be either linear or branched. Examples of the alkylene group include a methylene group, an ethylene group, and a trimethylene group (-(CH ２ ) ３ -), propylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), tetramethylene group (-(CH ２ ) ４ -), butylene group (-CH(C ２ H ５ ) CH ２ -, -CH ２ CH(C ２ H ５ )-), pentamethylene group (-(CH ２ ) ５ -), hexamethylene group (-(CH ２ ) ６ -), heptamethylene group (-(CH ２ ) ７ -), octamethylene group (-(CH ２ ) ８ -), nonamethylene group (-(CH ２ ) ９ -), decamethylene group (-(CH ２ ) １０ -) (in the above formula, "-" represents a single bond).

[0125] As used herein, the term "alkenediyl group" refers to a divalent group having a structure obtained by removing two hydrogen atoms from an alkene. The alkenediyl group may be linear or branched. The number of olefinic carbon-carbon double bonds in the alkene or alkenediyl group may be one or two or more. Examples of alkenediyl groups include ethenediyl, propenediyl, butenediyl, pentenediyl, hexenediyl, heptenediyl, octenediyl, nonenediyl, and decenediyl. The term "compound name + diyl group (e.g., ethenediyl)" as used herein refers to a divalent group having a structure obtained by removing two hydrogen atoms from the compound.

[0126] As used herein, the term "alkynediyl group" refers to a divalent group having a structure obtained by removing two hydrogen atoms from an alkyne. As used herein, the alkynediyl group may be either linear or branched. Furthermore, as used herein, the number of carbon-carbon triple bonds in either the alkyne or the alkynediyl group may be either one or two or more. Examples of the alkynediyl group include an ethynediyl group, a propynediyl group, a butynediyl group, a pentynediyl group, a hexynediyl group, a heptynediyl group, an octynediyl group, a nonynediyl group, and a decynediyl group.

[0127] In formula (5), R １１ 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, even more 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.

[0128] 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; R １２At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; １３ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and １２ and R １３ may each independently 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.

[0129] R １２ and R １３ are preferably each independently 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 an alkyl group having 1 to 17 carbon atoms, even more preferably an alkyl group having 1 to 10 carbon atoms, and particularly preferably a hexyl group.

[0130] In formula (5), X ４ represents an oxygen atom, NH, or sulfur atom. ４ is preferably an oxygen atom or NH, more preferably an oxygen atom.

[0131] In formula (5), R １１ is a trimethylene group, and R １２ and R １３ are both hexyl groups, and X ４ is most preferably an oxygen atom.

[0132] R ３ａ is R ３ｂ may be the same as or different from, but preferably, R ３ａ is R ３ｂ is the same group as

[0133] In one embodiment of the present invention, R １ａ is R １ｂ is the same as X ａ is Xｂ is the same as R ２ａ is R ２ｂ is identical to Y ａ Is Y ｂ is identical to Z ａ is Z ｂ is the same as R ３ａ is R ３ｂ is the same as

[0134] Suitable examples of the ionic lipid (1) include the following ionic lipids:

[0135] [Ionic lipid (1-1a)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂeach independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), a group having 50 or less carbon atoms represented by the formula (4) (R in formula (4) １０ is an alkyl group having 1 to 20 carbon atoms or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group), or a group having 50 or less carbon atoms represented by the formula (5) (R in formula (5) １１ is an alkylene group having 2 to 9 carbon atoms, and 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. ); Ionic lipid (1).

[0136] [Ionic lipid (1-1b)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), a group having 50 or less carbon atoms represented by the formula (4) (R in 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 １１ is an alkylene group having 2 to 9 carbon atoms, and 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. ); Ionic lipid (1).

[0137] [Ionic lipid (1-1c)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂare each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0138] [Ionic lipid (1-1d)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group) or a group having 50 or less carbon atoms represented by the formula (4) (R １０ is an alkyl group having 1 to 20 carbon atoms or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group); an ionic lipid (1).

[0139] [Ionic lipid (1-2a)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); nａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), a group having 50 or less carbon atoms represented by the formula (4) (R １０ is an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 1 to 10 carbon atoms, and the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group), or a group having 50 or less carbon atoms represented by the formula (5) (R in formula (5) １１ is an alkylene group having 2 or 3 carbon atoms, and R １２ and R １３ are each independently an alkyl group having 1 to 10 carbon atoms, and X ４ is an oxygen atom.) Ionic lipid (1).

[0140] [Ionic lipid (1-2b)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂare each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), a group having 50 or less carbon atoms represented by the formula (4) (R 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 １１ is an alkylene group having 2 or 3 carbon atoms, and R １２ and R １３ are each independently an alkyl group having 1 to 10 carbon atoms, and X ４ is an oxygen atom.) Ionic lipid (1).

[0141] [Ionic lipid (1-2c)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a trimethylene group); ａ and Yｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0142] [Ionic lipid (1-2d)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group) or a group having 50 or less carbon atoms represented by the formula (4) (R １０ is an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 1 to 10 carbon atoms, and the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group); an ionic lipid (1).

[0143] [Ionic lipid (1-3a)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ However, each independently, X １ :

[0144]

[0145] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0146]

[0147] (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ However, each independently, Z １:

[0148]

[0149] (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; ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), a group having 50 or less carbon atoms represented by the formula (4) (R １０ is an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 2 to 10 carbon atoms, and the alkyl group may be substituted with a 3- to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group), or a group having 50 or less carbon atoms represented by the formula (5) (R in formula (5) １１ is an alkylene group having 2 or 3 carbon atoms, and R １２ and R １３ are each independently an alkyl group having 1 to 10 carbon atoms, and X ４ is an oxygen atom.) Ionic lipid (1).

[0150] [Ionic lipid (1-3b)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ However, each independently, X １ :

[0151]

[0152] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0153]

[0154] (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ However, each independently, Z １ :

[0155]

[0156] (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; ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), a group having 50 or less carbon atoms represented by the formula (4) (R 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 １１ is an alkylene group having 2 or 3 carbon atoms, and R １２ and R １３ are each independently an alkyl group having 1 to 10 carbon atoms, and X ４ is an oxygen atom.) Ionic lipid (1).

[0157] [Ionic lipid (1-3c)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ However, each independently, X１ :

[0158]

[0159] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0160]

[0161] (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ However, each independently, Z １ :

[0162]

[0163] (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; ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0164] [Ionic lipid (1-3d)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ However, each independently, X １ :

[0165]

[0166] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0167]

[0168] (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ However, each independently, Z １ :

[0169]

[0170] (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; ａ and n ｂ are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group) or a group having 50 or less carbon atoms represented by the formula (4) (R １０ is an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 2 to 10 carbon atoms, and the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., a dithiolanyl group); an ionic lipid (1).

[0171] Specific examples of the ionic lipid (1) include the compounds listed in Tables 1-1 to 1-5 below.

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] In one embodiment of the present invention, the ionic lipid (1) (i.e., the compound represented by formula (1)) is preferably at least one selected from the group consisting of the compounds listed in Tables 1-1 to 1-5, and more preferably at least one selected from the group consisting of SS-OP, SS-EC, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6.

[0178] In another embodiment of the present invention, the ionic lipid (1) (i.e., the compound represented by formula (1)) is preferably at least one selected from the group consisting of the compounds listed in Tables 1-1 to 1-4, more preferably at least one selected from the group consisting of SS-OP, SS-EC, Compound 1, and Compound 2, and even more preferably SS-OP.

[0179] In one embodiment of the present invention, the ionic lipid is preferably at least one selected from the group consisting of SS-OP, compound 3, compound 4, compound 5, compound 6, [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and more preferably SS-OP, compound 3, compound 4, compound 5, compound 6, [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, more preferably SS-OP, Compound 3, Compound 4, Compound 5, or Compound 6, and particularly preferably SS-OP.

[0180] Ionic lipids can be commercially available products (e.g., "ALC-0315" manufactured by Jenkem Technology USA; "SM-102" and "DLin-MC3-DMA" manufactured by Cayman Chemical). In addition, ionic lipid (1) (i.e., the compound represented by formula (1)) can be produced by known methods (e.g., the methods described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, WO2021 / 195529A2). In addition, compound 1 and compound 2 can be produced by the following synthesis method. Ionic lipids other than compound 1 or compound 2 (1) can also be produced in the same manner as the synthesis method of compound 1 or compound 2.

[0181] (I) Method for Synthesizing Compound 1 <Synthesis of Intermediate 1> Intermediate 1 was synthesized according to the following synthetic route.

[0182]

[0183] <Synthesis of Intermediate 1-A> 30.0 g (197 mmol) of 4-hydroxyphenylacetic acid and 5.00 g (19.9 mmol) of pyridinium 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 resulting mixture at 20°C or below, and the mixture was allowed to react at room temperature for 2 hours. Subsequently, 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 aqueous NaOH solution were added to the resulting mixture, and the mixture was allowed to react at room temperature for 1 hour. The mixed solution was concentrated using an evaporator, and the concentrate was washed with chloroform and neutralized by adding 6 M hydrochloric acid. The resulting mixture was extracted with chloroform, and the organic layer was dehydrated by adding sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 47.0 g of intermediate 1-A.

[0184] <Synthesis of Intermediate 1-B> 36.0 g (95.6 mmol) of intermediate 57 synthesized according to the method described in WO 2016 / 121942, 49.7 g (210 mmol) of intermediate 1-A, and 4.68 mg (38.3 mmol) of DMAP were dissolved in 240 mL of chloroform at room temperature. 55.1 g (287 mmol) of EDC hydrochloride was added to the resulting mixture and allowed to react at room temperature for 2 hours. The reaction solution was then washed with a 5 wt% aqueous solution of sodium dihydrogen phosphate, a 9 wt% sodium bicarbonate solution, and a 20 wt% saline solution, followed by dehydration by the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 77.9 g of intermediate 1-B.

[0185] <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 reacted at 25°C or below for 1 hour. 25.0 g (205 mmol) of DMAP was then added to the reaction mixture for neutralization. After removing the 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 glycinate buffer (pH = 9.5), and then dehydrated by adding sodium sulfate. After removing the sodium sulfate by filtration, 1.56 L of toluene was added to allow crystallization. The resulting crude product was washed with hexane and then vacuum-dried to obtain 40.3 g of Intermediate 1.

[0186] <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 and allowed to react at room temperature for 2 hours. The reaction solution was then washed with a 5 wt% aqueous solution of sodium hydrogen phosphate, 0.5 M phosphate buffer (pH = 2.0), and 20 wt% saline, followed by dehydration with the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator. The resulting residue was purified by silica gel column chromatography (chloroform / ethanol = 88 / 12) to obtain 8.21 g of Intermediate 1-1.

[0187]

[0188] Synthesis of Intermediate 5-1: 5.00 g (16.0 mmol) of methyl ricinoleate, 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 allowed to react at room temperature for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt % sodium bicarbonate water, and 20 wt % saline, followed by dehydration with the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 6.78 g of Intermediate 5-1.

[0189]

[0190] <Synthesis of Intermediate 5> 6.40 g (14.6 mmol) of Intermediate 5-1 was dissolved at room temperature in tert-butanol to a concentration of 0.3 M. 8.17 g of a 2 M aqueous solution of NaOH was added to the resulting mixture, and the mixture was allowed to react at room temperature for 8 hours. 1 M hydrochloric acid was then added, and the resulting mixture was extracted with hexane. The organic layer was washed with 20 wt % saline, and then sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator. The resulting residue was purified by silica gel column chromatography (chloroform / ethanol = 94 / 6 (volume ratio)), and 5.93 g of Intermediate 5 was obtained.

[0191]

[0192] Synthesis of Compound 1: 2.91 g of chloroform was added to 492 mg (0.541 mmol) of Intermediate 1-1 to dissolve it, and then 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 were added and reacted at room temperature for 2 hours. The solution after the reaction was washed with 5 wt % saline and then concentrated using an evaporator. After concentration, the solution was purified using a silica gel column with ethanol / chloroform to obtain 532 mg of Compound 1.

[0193] (II) Method for Synthesizing Compound 2 <Synthesis of Intermediate 11-1> 25.0 g (189 mmol) of (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol and 19.3 g (284 mmol) of imidazole were dissolved in 251 g of dimethylformamide at room temperature. 57.3 g (208 mmol) of TBDPS-Cl was added to the resulting mixture and allowed to react at room temperature for 1 hour. Chloroform was then added to the reaction solution, which was then washed with a 5 wt% aqueous solution of sodium hydrogen phosphate and ion-exchanged water, followed by dehydration by adding sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 56.0 g of Intermediate 11-1.

[0194]

[0195] <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 aqueous NaOH solution was added to adjust the pH to 7.0. Chloroform was added to the resulting mixture, which was then washed with 20 wt % saline, and sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 44.9 g of Intermediate 11-2.

[0196]

[0197] <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 allowed to react at room temperature for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt % sodium bicarbonate water, and 20 wt % saline, followed by dehydration with the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 9.03 g of Intermediate 14-1.

[0198]

[0199] <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 a 1 M TBAF THF solution was added to the resulting mixture and reacted at room temperature for 18 hours. 94.3 g of ethyl acetate was then added to the reaction solution. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0) and 20 wt % saline, and then dehydrated by adding sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 4.90 g of Intermediate 14-2.

[0200]

[0201] <Intermediate 14> 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 at room temperature and allowed to react for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt % aqueous sodium bicarbonate, and 20 wt % saline, followed by dehydration with the addition of sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 3.16 g of Intermediate 14.

[0202]

[0203] <Synthesis of Compound 2> Using Intermediate 14 and Intermediate 1-1, 0.215 g of Compound 2 was synthesized in the same manner as in the synthesis of Compound 1 described above.

[0204] In one embodiment of the present invention, the concentration of ionic lipid (1) in the alcohol solution (i.e., lipid solution) in step a) is preferably 0.05 to 20 mM, more preferably 0.1 to 15 mM, and even more preferably 1 to 10 mM, from the viewpoint of the solubility of the lipid.

[0205] In another embodiment of the present invention, the concentration of ionic lipid (1) in the alcohol solution (i.e., lipid solution) in step a) is preferably 0.05 to 30 mM, more preferably 0.1 to 25 mM, and even more preferably 0.3 to 20 mM, from the viewpoint of the solubility of the lipid.

[0206] When two or more types of ionic lipid (1) are used, the term "ionic lipid (1) concentration" in this specification refers to the total concentration of the two or more types of ionic lipid (1). The term "concentration of other components" also has the same meaning as the term "ionic lipid (1) concentration."

[0207] The content of ionic lipid (1) in the target lipid nanoparticles is preferably 20 to 75 mol%, more preferably 30 to 70 mol%, and even more preferably 35 to 60 mol%, based on the total lipids, from the viewpoints of nucleic acid encapsulation efficiency, nucleic acid release efficiency within cells, and stability of the lipid nanoparticles.

[0208] In addition, when two or more types of ionic lipid (1) are used, the "content of ionic lipid (1)" in this specification means the total content of two or more types of ionic lipid (1). The "content of other components" has the same meaning as the "content of ionic lipid (1)". In addition, in this specification, the "content of B relative to A (mol%)" or "amount of B relative to A (mol%)" means "100 × amount of B (mol) / amount of A (mol)".

[0209] The alcohol solution in step a) preferably further contains a phospholipid. The phospholipid may be used alone or in combination of two or more kinds.

[0210] 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 lyso forms thereof.

[0211] 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-dierucoyl-sn-glycero-3-phosphocholine (DEPC), Examples include 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), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

[0212] In the above specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), the phosphocholine moiety is substituted with phosphoethanolamine, phosphoserine, phosphoglycerol, or phosphatidic acid, respectively, to give 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).

[0213] As the phospholipid, PC and PE are preferred, DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) are more preferred, DOPC, DSPC, and DEPC are further preferred, and DOPC is particularly preferred.

[0214] When a phospholipid is used, the concentration of the phospholipid in the alcohol solution (i.e., lipid solution) in step a) in one embodiment of the present invention is preferably 0.01 to 5 mM, more preferably 0.05 to 3 mM, and even more preferably 0.1 to 2 mM, from the viewpoint of the solubility of the lipid.

[0215] When a phospholipid is used, the concentration of the phospholipid in the alcohol solution (i.e., lipid solution) in step a) in another embodiment of the present invention is preferably 0.01 to 20 mM, more preferably 0.01 to 15 mM, and even more preferably 0.03 to 10 mM, from the viewpoint of the solubility of the lipid.

[0216] The content of phospholipids in the target lipid nanoparticles is preferably 0 to 35 mol%, more preferably 2.5 to 30 mol%, and even more preferably 5 to 30 mol%, based on the total lipids, from the viewpoints of nucleic acid encapsulation efficiency, nucleic acid release efficiency within cells, and lipid nanoparticle stability.

[0217] The alcohol solution (i.e., lipid solution) in step a) contains a sterol. The sterol may be used alone or in combination of two or more kinds.

[0218] Examples of sterols include cholesterol, lanosterol, phytosterol, zymosterol, zymostenol, desmosterol, stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol. Preferred sterols are cholesterol, lanosterol, and phytosterol, with cholesterol being more preferred.

[0219] In one embodiment of the present invention, the concentration of the sterol in the alcohol solution (i.e., lipid solution) in step a) is preferably 0.05 to 15 mM, more preferably 0.1 to 10 mM, and even more preferably 0.5 to 10 mM, from the viewpoint of sterol solubility.

[0220] In another embodiment of the present invention, the concentration of the sterol in the alcohol solution (i.e., lipid solution) in step a) is preferably 0.001 to 25 mM, more preferably 0.01 to 20 mM, and even more preferably 0.05 to 15 mM, from the viewpoint of sterol solubility.

[0221] The content of sterol in the target lipid nanoparticles is preferably 5 to 60 mol%, more preferably 7 to 55 mol%, and even more preferably 10 to 50 mol%, based on the total lipids, from the viewpoints of nucleic acid encapsulation efficiency, nucleic acid release efficiency within cells, and lipid nanoparticle stability.

[0222] The alcohol solution (i.e., lipid solution) in step a) contains a PEG lipid. As used herein, "PEG lipid" refers to a lipid having a polyethylene glycol (PEG) chain. Only one type of PEG lipid may be used, or two or more types may be used in combination.

[0223] PEG lipids are used to coat the surface of lipid nanoparticles with hydrophilic polyethylene glycol (PEG) to prevent aggregation of lipid nanoparticles and to prevent interactions between biological components and particles when administered to the body.

[0224] The PEG moiety in the PEG lipid can be of any molecular weight. In some embodiments, the number average molecular weight of the PEG moiety is preferably 200 to 10,000, more preferably 1,000 to 10,000. The PEG moiety can be linear or branched.

[0225] Examples of PEG lipids include PEG-phospholipids (i.e., phospholipids bound to PEG), PEG-ceramides (i.e., ceramides bound to PEG), PEG-diacylglycerols (i.e., diacylglycerols bound to PEG), and PEG-cholesterols (i.e., cholesterols bound to PEG). Of these, PEG-diacylglycerols are preferred.

[0226] As the PEG lipid, PEG-diacylglycerol (i.e., diacylglycerol bound to PEG) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000 is preferred, PEG-dimyristoylglycerol (i.e., dimyristoylglycerol bound to PEG) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000 and PEG-distearoylglycerol (i.e., distearoylglycerol bound to PEG) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000 are more preferred, and PEG-dimyristoylglycerol (e.g., DMG-PEG2000 used in the Examples described below) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000 is even more preferred.

[0227] The concentration of the PEG lipid in the alcohol solution (i.e., lipid solution) in step a) is preferably 0.001 to 1 mM, more preferably 0.005 to 0.8 mM, and even more preferably 0.01 to 0.5 mM, from the viewpoint of the solubility of the lipid.

[0228] The content of PEG lipid in the target lipid nanoparticles is preferably 0.1 to 6.0 mol%, more preferably 0.5 to 5 mol%, and even more preferably 0.7 to 3.0 mol%, based on the total moles of other lipids, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability. Here, the "PEG lipid" refers to the PEG lipid used in step a), and the "PEG lipid" does not include ligand-bound PEG lipids (a type of ligand-bound lipid) that may be used in step c). Furthermore, the "other lipid" refers to lipids other than the PEG lipid used in step a). For example, when an ionic lipid (1), a sterol, and a PEG lipid are used as lipids in step a), the "total of other lipids" refers to the sum of the ionic lipid (1) and the sterol. Furthermore, for example, when an ionic lipid (1), a phospholipid, a sterol, and a PEG lipid are used as lipids in step a), the "total of other lipids" refers to the sum of the ionic lipid (1), the phospholipid, and the sterol.

[0229] The concentration of total lipids is preferably 0.1 to 30 mM, more preferably 0.5 to 25 mM, and even more preferably 1 to 20 mM, from the viewpoint of the solubility of the total lipids and the encapsulation of nucleic acids.

[0230] The concentration of total lipids in the alcohol solution (i.e., lipid solution) in step a) is preferably 0.1 to 40 mM, more preferably 0.5 to 30 mM, and even more preferably 1 to 25 mM, from the viewpoint of the solubility of total lipids and nucleic acid encapsulation.

[0231] In step a), an acidic buffer solution having a pH of 1 to 6.5 is used. Examples of the acidic buffer solution include HCl / KCl buffer solution, p-toluenesulfonic acid / p-toluenesulfonic acid Na salt buffer solution, tartaric acid / NaOH buffer solution, citrate buffer solution, phthalic acid HK / HCl buffer solution, glycine / HCl buffer solution, trans-aconitic acid / NaOH buffer solution, formic acid / formic acid Na salt buffer solution, 3,3-dimethylglutaric acid / NaOH buffer solution, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer solution, phenylacetic acid / phenylacetic acid Na salt buffer solution, acetic acid / acetic acid Na salt buffer solution, succinic acid / NaOH buffer solution, phthalic acid HK / NaOH buffer solution, cacodylate Na / HCl buffer solution, maleic acid HNa / NaOH buffer solution, maleic acid / trishydroxymethylaminomethane (Tris) / NaOH buffer solution, phosphate buffer solution, KH ２ P.O. ４ / NaOH buffer, imidazole / HCl buffer, s-collidine (2,4,6-trimethylpyridine) / HCl buffer, triethanolamine HCl / NaOH buffer, 5,5-diethylbarbituric acid Na / HCl buffer, N-methylmorpholine / HCl buffer, pyrophosphate Na / HCl buffer, 2-morpholinoethanesulfonic acid (MES) buffer, malic acid buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, 1,4-piperazinediethanesulfonic acid (PIPES) buffer, N-(2-acetamido)-2-aminoethanesulfonic acid (ACE S) buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, Bis-Tris buffer, Bis-Tris propane buffer, anhydrous sodium carbonate buffer, glycylglycine buffer, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO) buffer, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer.

[0232] The acidic buffer solution is preferably a malic acid buffer solution, a citrate buffer solution, an acetic acid / sodium acetate buffer solution, or a 2-morpholinoethanesulfonic acid (MES) buffer solution, more preferably a malic acid buffer solution or an MES buffer solution, and even more preferably a malic acid buffer solution. The pH of the acidic buffer solution is 1 to 6.5, preferably 3 to 6.5.

[0233] The total concentration of the acid and its salt in the acidic buffer solution (hereinafter sometimes abbreviated as "buffer solution concentration") is preferably 10 to 100 mM, more preferably 10 to 80 mM, from the viewpoint of the formation of lipid nanoparticles.

[0234] <Replacement of Dispersion Medium> The dispersion medium of a suspension of lipid nanoparticles that does not contain nucleic acid, a suspension of lipid nanoparticles that encapsulate nucleic acid, a suspension of ligand-modified lipid nanoparticles that does not contain nucleic acid, or a suspension of ligand-modified lipid nanoparticles that encapsulate nucleic acid (hereinafter, these may be collectively abbreviated as "lipid nanoparticle suspension") can be replaced with another buffer solution different from the acidic buffer solution used in step a) by operations such as ultrafiltration, dialysis, dilution, etc. The other buffer solution contains water as a solvent, and the suspension in which the dispersion medium has been replaced contains water as a dispersion medium.

[0235] Other buffer solutions include, for example, tartaric acid / NaOH buffer solution, phthalic acid HK / HCl buffer solution, glycine / HCl buffer solution, trans-aconitic acid / NaOH buffer solution, formic acid / formic acid Na salt buffer solution, 3,3-dimethylglutaric acid / NaOH buffer solution, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer solution, phenylacetic acid / phenylacetic acid Na salt buffer solution, acetic acid / acetic acid Na salt buffer solution, succinic acid / NaOH buffer solution, phthalic acid HK / NaOH buffer solution, cacodylate Na / HCl buffer solution, maleic acid HNa / NaOH buffer solution, maleic acid / trishydroxymethylaminomethane (Tris) / NaOH buffer solution, phosphate buffer solution, KH ２ P.O. ４ / NaOH buffer solution, 2-morpholinoethanesulfonic acid (MES) buffer solution, malic acid buffer solution, phthalic acid buffer solution, maleic acid buffer solution, succinic acid buffer solution, tartaric acid buffer solution, citrate buffer solution, bis-tris buffer solution, glycylglycine buffer solution, etc.

[0236] The other buffer solution is preferably a malic acid buffer, a citrate buffer, an acetic acid / Na acetate buffer, a 2-morpholinoethanesulfonic acid (MES) buffer, or a maleic acid / trishydroxymethylaminomethane (Tris) / NaOH buffer, more preferably a malic acid buffer or an MES buffer, and even more preferably an MES buffer. The pH of the other buffer solution is preferably 4.5 to 6.9, more preferably 5.0 to 6.9, and even more preferably 5.5 to 6.9. The total concentration of the acid and its salt in the other buffer solution (hereinafter sometimes abbreviated as "buffer concentration") is preferably 10 to 100 mM, more preferably 20 to 50 mM, from the viewpoint of nucleic acid encapsulation and lipid nanoparticle formation.

[0237] It is preferable to exchange the dispersion medium so that the concentration of each component in the suspension after the exchange of the dispersion medium is approximately the same as the concentration of each component in the suspension before the exchange of the dispersion medium (i.e., the suspension obtained in step a)).

[0238] <Mixing of Other Components> The lipid nanoparticle suspension may be mixed with other components. Examples of the other components include sugars such as monosaccharides, sugar alcohols, disaccharides, oligosaccharides, and polysaccharides. The sugar is preferably a disaccharide, more preferably sucrose. The concentration of the sugar in the lipid nanoparticle suspension is preferably 0 to 320 mg / mL, more preferably 0 to 160 mg / mL.

[0239] <Storage of Suspension of Nucleic Acid-Free Lipid Nanoparticles> The suspension of nucleic acid-free lipid nanoparticles obtained in step a) may be frozen or lyophilized and stored before use in step b), step c'), or step d), or may be stored as a suspension. Furthermore, the suspension of lipid nanoparticles obtained in other steps may also be frozen or lyophilized and stored before use in the next step, or may be stored as a suspension. Storage may be carried out under air or in an inert atmosphere (for example, under an argon atmosphere).

[0240] The temperature at which the suspension of nucleic acid-free lipid nanoparticles is frozen is preferably −200° C. to −5° C., more preferably −85° C. to −10° C., and the freezing time is preferably 1 to 72 hours, more preferably 3 to 24 hours.

[0241] The temperature during freeze-drying of the suspension of nucleic acid-free lipid nanoparticles is preferably -80°C to 50°C, more preferably -60°C to 40°C, the pressure during freeze-drying is preferably 0 to 300 mTorr, more preferably 50 to 250 mTorr, and the time for freeze-drying is preferably 12 to 120 hours, more preferably 24 to 100 hours. It is preferable that freeze-drying is initially performed at a low temperature, and then the temperature is increased stepwise.

[0242] From the viewpoint of the stability of the components of the lipid nanoparticles, the storage temperature of a frozen or lyophilized suspension of nucleic acid-free lipid nanoparticles is preferably −80° C. to 0° C., more preferably −80° C. to −10° C., and even more preferably −80° C. to −20° C. When a suspension of nucleic acid-free lipid nanoparticles is stored as is, the storage temperature is preferably 1° C. to 50° C., more preferably 1° C. to 30° C., even more preferably 1° C. to 10° C., and most preferably 1° C. to 5° C., from the viewpoint of the stability of the components of the lipid nanoparticles.

[0243] When a frozen or lyophilized suspension of nucleic acid-free lipid nanoparticles has been stored, the resulting frozen product is thawed at 0 to 100°C to prepare a suspension before use in step b) or step c'), or water is added to the resulting lyophilized product and the resulting mixture is maintained at 0 to 100°C to prepare a suspension. It is desirable to add water to the lyophilized product so that the concentrations of the components of the suspension obtained after the addition of water are approximately the same as the concentrations of the components of the suspension before lyophilization.

[0244] From the viewpoint of the stability of the components of the lipid nanoparticles, the melting temperature of the frozen product or the temperature at which the mixture of the freeze-dried product and water is maintained is preferably 0 to 95° C., more preferably 0 to 50° C., even more preferably 0 to 30° C., and most preferably 0 to 10° C. The melting of the frozen product, the addition of water to the freeze-dried product, and the maintenance of the resulting mixture are preferably carried out under atmospheric pressure.

[0245] <Step b)> In one embodiment of the manufacturing method of the present invention, step b) is carried out after step a) and other steps that are carried out as necessary. In step b), nucleic acid-free lipid nanoparticles are mixed with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulating nucleic acid. Examples of what is mixed with the nucleic acid solution in step b) include (i) a suspension of nucleic acid-free lipid nanoparticles, and (ii) a lyophilized product of nucleic acid-free lipid nanoparticles obtained by lyophilizing the suspension. In step b), preferably, a suspension of nucleic acid-free lipid nanoparticles is mixed with a nucleic acid solution. The nucleic acid solution used in step b) contains water as a solvent, and the suspension of nucleic acid-encapsulated lipid nanoparticles obtained in step b) contains water as a dispersion medium. In addition, the suspension of nucleic acid-free lipid nanoparticles used in step b) of a preferred embodiment of the present invention contains water as a dispersion medium.

[0246] The mixing in step b) can be performed, for example, by pipetting or by using a device including a microchannel or a mixer, including, but not limited to, a vortex mixer, a T-junction mixer, and a jet mixer.

[0247] Examples of nucleic acids include those described below. The nucleic acid is preferably mRNA.

[0248] In step b), the molar ratio of the total amino groups of the ionic lipids in the nucleic acid-free lipid nanoparticles (for example, a suspension of nucleic acid-free lipid nanoparticles, or a lyophilized product of nucleic acid-free lipid nanoparticles) to the total phosphate groups of the nucleic acid in the nucleic acid solution (total amount of amino groups of the ionic lipids (mol) / total amount of phosphate groups of the nucleic acid (mol)) (sometimes referred to herein as the "N / P ratio") is preferably 5 to 280, more preferably 10 to 140, and even more preferably 15 to 70, from the viewpoint of nucleic acid encapsulation efficiency and toxicity.

[0249] The concentration of nucleic acid in the nucleic acid solution (i.e., amount of nucleic acid (μg) / volume of solution (mL)) is not particularly limited as long as it is possible to achieve the introduction of the target nucleic acid into cells, but is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 10 to 450 μg / mL.

[0250] The nucleic acid solution is preferably an aqueous solution of nucleic acid. In order to increase the rate of nucleic acid encapsulation in lipid nanoparticles, the aqueous solution of nucleic acid may contain alcohol. Examples of alcohol include methanol, ethanol, n-butanol, and tert-butanol. Only one type of alcohol may be used, or two or more types may be used in combination. The alcohol is preferably ethanol. The concentration of alcohol in the aqueous solution of nucleic acid is preferably 0 to 50 v / v%, more preferably 0 to 30 v / v%, and even more preferably 0 to 25 v / v%.

[0251] The aqueous nucleic acid solution is preferably an acidic buffer solution containing nucleic acid. The pH of the acidic buffer solution is preferably 1 to 6.5, more preferably 3 to 6.5. Examples of the acidic buffer solution are the same as the examples of the acidic buffer solution in step a). The acidic buffer solution is preferably an acetic acid / Na acetate buffer solution, a maleic acid / trishydroxymethylaminomethane (Tris) / NaOH buffer solution, a 2-morpholinoethanesulfonic acid (MES) buffer solution, a malic acid buffer solution, or a citrate buffer solution, and more preferably an MES buffer solution. The total concentration of the acid and its salt in the acidic buffer solution (i.e., the buffer solution concentration) is preferably 10 to 100 mM, more preferably 10 to 80 mM, from the viewpoint of nucleic acid stability.

[0252] The volume ratio of the suspension of nucleic acid-free lipid nanoparticles to the nucleic acid solution in step b) (volume of the suspension of nucleic acid-free lipid nanoparticles:volume of the nucleic acid solution) is not particularly limited as long as it is a volume ratio that allows nucleic acid to be encapsulated, but from the viewpoint of efficient encapsulation of nucleic acid, it is preferably 1:20 to 20:1, more preferably 1:12 to 12:1.

[0253] The concentration of nucleic acid in the suspension of nucleic acid-encapsulated lipid nanoparticles obtained in step b) (i.e., amount of nucleic acid (μg) / volume of suspension (mL)) is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 1 to 450 μg / mL. In step b) of a preferred embodiment of the present invention, nucleic acid-free lipid nanoparticles (e.g., a suspension of nucleic acid-free lipid nanoparticles or a lyophilized product of nucleic acid-free lipid nanoparticles) are mixed with a nucleic acid solution so as to obtain a suspension of the above concentration.

[0254] To enhance the nucleic acid encapsulation efficiency, a mixture of nucleic acid-free lipid nanoparticles (preferably a suspension of nucleic acid-free lipid nanoparticles) and a nucleic acid solution may be maintained at a predetermined temperature for a predetermined time. The temperature is preferably 0 to 95°C, more preferably 0 to 60°C, and even more preferably 0 to 40°C, and the time is preferably 1 minute to 2 hours, more preferably 1 to 60 minutes, and even more preferably 1 to 30 minutes. Note that the mixture in this embodiment may contain other components (e.g., lipids not bound to a ligand).

[0255] <Step c)> In one embodiment of the manufacturing method of the present invention, step c) is carried out after step b) and other steps that are carried out as necessary.In step c), a suspension of lipid nanoparticles encapsulating nucleic acid is mixed with a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.The suspension of lipid nanoparticles encapsulating nucleic acid used in step c) contains water as a dispersion medium, and the suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid obtained in step c) contains water.

[0256] The mixing in step c) can be performed, for example, by pipetting or by using a device including a microchannel or a mixer, including, but not limited to, a vortex mixer, a T-junction mixer, and a jet mixer.

[0257] Examples of the ligand include peptides (including proteins), antibodies, nucleic acids (including oligonucleotides), aptamers, sugars, lipids, etc. The ligand is preferably a peptide, antibody, or sugar that has specific affinity for a molecule expressed on the surface of a target cell, more preferably a peptide or antibody, and even more preferably an antibody. Examples of the peptide, antibody, or sugar that is the ligand include those described below.

[0258] Specific examples of ligands include transferrin, erythropoietin, EGF, VEGF, PDGF, IL-8, SDF1, thrombin, etanercept, romiplostim abatacept, HER2, transferrin receptor, glucose transporter, MDR1, muromonab, ibritumomab, tositumomab, rituximab, basiliximab, cetuximab, brentuximab, margetuximab, daclizumab, and tositumomab. rastuzumab, pertuzumab, gemtuzumab, alemtuzumab, natalizumab, tocilizumab, mogamulizumab, obinutuzumab, vedolizumab, pembrolizumab, atezolizumab, ocrelizumab, inotuzumab, benralizumab, ibalizumab, polatuzumab, crizanlizumab, sacituzumab, inebilizumab, tafasitamab, satralizumab, loncastuximab, dostallimab, panitumumab, Ipilimumab, ramucirumab, nivolumab, necitumumab, daratumumab, brodalumab, olaratumab, avelumab, durvalumab, dupilumab sarilumab, teprotunumab, amivantamab, anifrolumab, tisotumab, pavinafsp, PD-1, PD-L1, CTLA4, glutamine, leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, histidine , Fc domain-binding peptides, RGD peptides, DNA aptamers, RNA aptamers, AS1411, A10, Sgc8, HeA21, HeA23, estrone, anisamide, methotrexate, adenosine, thiamine, riboflavin, biotin, glucose, mannose, lactose, maltose, N-acetylglucosamine, sucrose, galactose, sialyl Lewis X, sialic acid, and hyaluronic acid.

[0259] The ligand is preferably a ligand having cell-specific activity. Here, "cell-specific ligand" means a ligand that has a higher affinity for one or more specific types of cells (including tissues and organs containing the cells) than for other cells. The ligand-modified lipid nanoparticles encapsulating cell-specific nucleic acids obtained by the production method of the present invention can be selectively delivered to specific cells.

[0260] An example of a cell-specific peptide is integrin α, which is highly expressed in the vascular system and some cancer cells. ｖ β ３ Examples of suitable peptides include, but are not limited to, RGD peptides containing an RGD motif (e.g., GRGDS pentapeptide) that have affinity for dystroglycan, which is highly expressed in muscle cells, and peptides derived from the laminin α2 chain that have affinity for dystroglycan. Alternatively, examples of suitable peptides include polypeptides that are natural ligands for cell surface receptors for cytokines, hormones, etc.

[0261] Examples of cell-specific antibodies include any antibody against a molecule expressed on the surface of a target cell. Specific examples of such antibodies include, but are not limited to, an anti-transferrin receptor antibody, an anti-glucose transporter antibody, an anti-insulin receptor antibody, an anti-LAT1 antibody, an anti-MDR1 antibody, an anti-BCRP antibody, an anti-SMVT1 antibody, an anti-Cluaudin5 antibody, an anti-LDL receptor antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD7 antibody, an anti-CD8 antibody, an anti-CD19 antibody, an anti-CD28 antibody, and an anti-CD147 antibody. Alternatively, an antibody used as an existing antibody drug or an antibody recognizing the same antigen as such an antibody can be used as a ligand for targeting a target cell expressing the antigen.

[0262] The antibody used in the present invention may be a complete antibody molecule, but may also be, for example, a single chain antibody (scFv), Fab, or F(ab'). ２ Any other antibody fragments such as Fab', Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc. may be preferably used. In this specification, unless otherwise specified, the term "antibody" is used to encompass antibody fragments.

[0263] Other examples of nucleic acids include ligands for Toll-like receptors, such as double-stranded RNA and CpG oligonucleotides; sugars include N-acetylgalactosamine, which has affinity for the asialoglycoprotein receptor expressed in hepatocytes; and lipids include various lipids (e.g., lysophosphatidylserine) that are natural ligands for G protein-coupled receptors.

[0264] The ligand is more preferably a brain cell-tropic ligand or a T cell-tropic ligand. Examples of brain cell-tropic ligands include anti-transferrin receptor antibodies, anti-glucose transporter antibodies, anti-insulin receptor antibodies, anti-LAT1 antibodies, anti-CD147 antibodies, anti-MDR1 antibodies, anti-BCRP antibodies, anti-SMVT1 antibodies, and anti-Cluaudin 5 antibodies. Examples of T cell-tropic ligands include anti-CD3 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD28 antibodies, and anti-LDL receptor antibodies.

[0265] From the viewpoint of achieving both selectivity for target cells and desirable particle properties, the amount of ligand in the target lipid nanoparticle is preferably 0.001 to 30 mol%, more preferably 0.001 to 10 mol%, and even more preferably 0.01 to 5 mol%, relative to the total amount of lipid in the lipid nanoparticle. It is preferable to mix a suspension of lipid nanoparticles encapsulating nucleic acid with a ligand-bound lipid so as to achieve such an amount of ligand.

[0266] As used herein, the term "ligand-conjugated lipid" refers to a lipid to which a ligand is covalently bound. The ligand-conjugated lipid is preferably a ligand-conjugated PEG lipid. As used herein, the term "ligand-conjugated PEG lipid" refers to a PEG lipid to which a ligand is covalently bound.

[0267] The method for producing the ligand-conjugated PEG lipid is not particularly limited, and the ligand-conjugated PEG lipid can be produced by known methods. For example, (i) an activated PEG lipid (i.e., a PEG lipid having a reactive group capable of binding to a ligand) as described in WO 2023 / 054243 may be reacted with a ligand to produce a ligand-conjugated PEG lipid; (ii) a compound having a reactive group (e.g., dibenzocyclooctyne-N-hydroxysuccinimidyl ester) may be reacted with a ligand to form a ligand having a reactive group, and a PEG lipid having a group (e.g., an azide group) capable of forming a covalent bond with the reactive group may be reacted with the ligand to produce a ligand-conjugated PEG lipid. Of these, the latter production method (hereinafter abbreviated as "production method (ii)") is preferred.

[0268] In production method (ii), the reaction between the compound having a reactive group and the ligand is preferably carried out in a solvent. Examples of the solvent include water and a buffer solution. The reaction temperature is preferably 0 to 95°C, more preferably 0 to 40°C, and even more preferably 0 to 25°C. The reaction time is preferably 10 minutes to 24 hours, more preferably 10 hours to 24 hours. The pH during the reaction is preferably 6 to 9, more preferably 7 to 9.

[0269] In production method (ii), the reaction between the PEG lipid having a group capable of forming a covalent bond with the reactive group and the ligand having the reactive group is preferably carried out in a solvent. Examples of the solvent include water and buffer solutions. The reaction temperature is preferably 0 to 95°C, more preferably 0 to 40°C, and even more preferably 0 to 25°C. The reaction time is preferably 1 to 24 hours, more preferably 10 to 24 hours, and even more preferably 16 to 20 hours.

[0270] The PEG moiety in the ligand-conjugated PEG lipid can be of any molecular weight. In some embodiments, the number average molecular weight of the PEG moiety is preferably 200 to 10,000, more preferably 1,000 to 10,000. The PEG moiety can be linear or branched.

[0271] Examples of the PEG lipid in the ligand-bound PEG lipid include PEG-phospholipid (i.e., phospholipid bound to PEG), PEG-ceramide (i.e., ceramide bound to PEG), PEG-diacylglycerol (i.e., diacylglycerol bound to PEG), and PEG-cholesterol (i.e., cholesterol bound to PEG). Among these, PEG-phospholipid and PEG-diacylglycerol are preferred.

[0272] The PEG lipid in the ligand-bound PEG lipid is preferably a PEG-phospholipid (i.e., a phospholipid bound to PEG) or a PEG-diacylglycerol (i.e., a diacylglycerol bound to PEG) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000, more preferably a PEG-phospholipid having a number-average molecular weight of the PEG moiety of 1,000 to 10,000, and more preferably a PEG-1,2-diacyl-sn-glycerol having a number-average molecular weight of the PEG moiety of 1,000 to 10,000. More preferred is 1,2-diacyl-sn-glycero-3-phosphoethanolamine (i.e., PEG-bound 1,2-diacyl-sn-glycero-3-phosphoethanolamine), and particularly preferred is PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (i.e., PEG-bound 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, such as PEG-DSPE used in the Examples below) in which the number-average molecular weight of the PEG moiety is 1,000 to 10,000.

[0273] The ligand-conjugated PEG lipid is preferably used in the form of an aqueous solution containing the ligand-conjugated PEG lipid. From the viewpoint of the efficiency of ligand insertion into lipid nanoparticles, the concentration of the ligand-conjugated PEG lipid in the aqueous solution is preferably 0.0001 to 0.12 mM, more preferably 0.001 to 0.06 mM.

[0274] During the mixing in step c), other components may be mixed in. Examples of other components include lipids to which no ligand is bound.

[0275] In step c), the suspension of lipid nanoparticles encapsulating nucleic acid may be mixed with a ligand-bound lipid and a lipid to which no ligand is bound.The order of mixing is not particularly limited, for example, (i) the suspension of lipid nanoparticles encapsulating nucleic acid may be mixed with a ligand-bound lipid and a lipid to which no ligand is bound at the same time; (ii) first, the suspension of lipid nanoparticles encapsulating nucleic acid may be mixed with a ligand-bound lipid, and then the resulting mixture may be mixed with a lipid to which no ligand is bound; (iii) the suspension of lipid nanoparticles encapsulating nucleic acid may be mixed with a ligand-bound lipid, and then the resulting mixture may be mixed with a suspension of lipid nanoparticles encapsulating nucleic acid; (iv) first, prepare an aqueous solution or aqueous dispersion containing a ligand-bound lipid and a lipid to which no ligand is bound, and then mix the resulting aqueous solution or aqueous dispersion with a suspension of lipid nanoparticles encapsulating nucleic acid. The aqueous solution or aqueous dispersion of (iv) above is, for example, an aqueous solution or aqueous dispersion (reaction mixture) containing a ligand-bound lipid (product) and a lipid to which no ligand is bound (unreacted reactant) obtained by a synthesis reaction of the ligand-bound lipid.

[0276] When a lipid not bound to a ligand is used in step c), from the viewpoint of achieving both desirable physical properties of the ligand-modified lipid nanoparticles and selectivity for target cells, the amount of the lipid not bound to a ligand used in step c) is preferably 0.01 to 5.0 mol%, more preferably 0.1 to 3.0 mol%, relative to the total lipid. Note that the "amount of lipid not bound to a ligand used in step c)" does not include the amount of lipid not bound to a ligand used other than step c) (for example, the ionic lipid used in step a).

[0277] The "unliganded lipid" used in step c) is preferably a PEG lipid that is not bound to a ligand. The description of the unliganded PEG lipid is the same as the description of the PEG lipid in step a).

[0278] The PEG lipid to which no ligand is bound in step c) is preferably a PEG-phospholipid (i.e., a phospholipid bound to PEG) or a PEG-diacylglycerol (i.e., a diacylglycerol bound to PEG) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000, more preferably a PEG-phospholipid having a number-average molecular weight of the PEG moiety of 1,000 to 10,000, and even more preferably a PEG-1,2-diacyl-sn-glycero-3-phosphoethanolamine (i.e., 1,2-diacyl-sn-glycero-3-phosphoethanolamine bound to PEG) having a number-average molecular weight of the PEG moiety of 1,000 to 10,000, PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine having a number average molecular weight of the PEG moiety of 1,000 to 10,000 (i.e., PEG-bound 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, such as PEG-DSPE used in the Examples below) is particularly preferred.

[0279] When using a PEG lipid to which no ligand is bound in step c), from the viewpoint of achieving both the desirable physical properties of the ligand-modified lipid nanoparticles and selectivity to target cells, the amount of the PEG lipid to which no ligand is bound used in step c) is preferably 0.01 to 5.0 mol%, more preferably 0.1 to 3.0 mol%, relative to the total lipid. Note that the "amount of the PEG lipid to which no ligand is bound used in step c") does not include the amount of the PEG lipid to which no ligand is bound used other than step c) (for example, the PEG lipid used in step a).

[0280] When using a ligand-conjugated PEG lipid and a ligand-free PEG lipid in step c), it is preferable to use an aqueous solution containing them. From the viewpoint of the efficiency of insertion into lipid nanoparticles, the concentration of the ligand-conjugated PEG lipid in the aqueous solution is preferably 0.0001 to 0.12 mM, more preferably 0.001 to 0.06 mM, and the concentration of the ligand-free PEG lipid in the aqueous solution is preferably 0.0001 to 0.12 mM, more preferably 0.001 to 0.06 mM.

[0281] For efficient modification of nucleic acid-encapsulated lipid nanoparticles with ligand-conjugated lipids, a mixture of a suspension of nucleic acid-encapsulated lipid nanoparticles and a ligand-conjugated lipid may be maintained at a predetermined temperature for a predetermined time. The temperature for the maintenance is preferably 0 to 95°C, more preferably 0 to 40°C, and even more preferably 0 to 25°C, and the maintenance time is preferably 1 minute to 5 hours, more preferably 1 minute to 2 hours, and even more preferably 1 to 60 minutes. Note that the mixture in the above embodiment may contain other components (e.g., lipids not bound to a ligand).

[0282] <Step c')> In one embodiment of the production method of the present invention, step c') is performed after step a) and other steps that are performed as necessary. In step c'), a suspension of nucleic acid-free, ligand-modified lipid nanoparticles is obtained by mixing nucleic acid-free lipid nanoparticles, water, and ligand-bound lipids. The suspension of nucleic acid-free, ligand-modified lipid nanoparticles obtained in step c') contains water as a dispersion medium.

[0283] In step c'), examples of the manner in which water and other components are mixed include (i) a suspension of nucleic acid-free lipid nanoparticles (i.e., a suspension containing nucleic acid-free lipid nanoparticles and water) and a ligand-bound lipid, (ii) a nucleic acid-free lipid nanoparticle (e.g., a lyophilized product of nucleic acid-free lipid nanoparticles) and an aqueous solution or aqueous dispersion containing a ligand-bound lipid (preferably an aqueous solution containing a ligand-bound lipid), and (iii) a nucleic acid-free lipid nanoparticle (e.g., a lyophilized product of nucleic acid-free lipid nanoparticles), water, and a ligand-bound lipid are simultaneously mixed. Among these, the above-mentioned modes (i) and (ii) are preferred, and the above-mentioned mode (i) is more preferred.

[0284] The mixing in step c') can be performed, for example, by pipetting or by using a device including a microchannel or a mixer, including, but not limited to, a vortex mixer, a T-junction mixer, and a jet mixer.

[0285] During the mixing in step c'), other components may be mixed in. Examples of other components include lipids to which no ligand is bound.

[0286] In step c'), the nucleic acid-free lipid nanoparticles, water, ligand-conjugated lipids, and lipids to which no ligand is bound may be mixed. The order of mixing is not particularly limited, and for example, (i) nucleic acid-free lipid nanoparticles, water, ligand-conjugated lipids, and non-liganded lipids may be mixed simultaneously; (ii) a suspension of nucleic acid-free lipid nanoparticles (i.e., a suspension containing nucleic acid-free lipid nanoparticles and water), ligand-conjugated lipids, and non-liganded lipids may be mixed simultaneously; (iii) a suspension of nucleic acid-free lipid nanoparticles (i.e., a suspension containing nucleic acid-free lipid nanoparticles and water) may be mixed with ligand-conjugated lipids, and then the resulting mixture may be mixed with non-liganded lipids; (iii) water, ligand-conjugated lipids, and non-liganded lipids may be mixed first, and then the resulting aqueous solution or aqueous dispersion may be mixed with non-nucleic acid-free lipid nanoparticles; or (iv) an aqueous solution or aqueous dispersion containing ligand-conjugated lipids and non-liganded lipids may be prepared first, and then the resulting aqueous solution or aqueous dispersion may be mixed with non-nucleic acid-free lipid nanoparticles. The aqueous solution or aqueous dispersion of (iv) above is, for example, an aqueous solution or aqueous dispersion (reaction mixture) containing a ligand-bound lipid (product) and a lipid to which no ligand is bound (unreacted reactant) obtained by a synthesis reaction of the ligand-bound lipid.

[0287] When a lipid not bound to a ligand is used in step c'), from the viewpoint of achieving both desirable physical properties of the ligand-modified lipid nanoparticles and selectivity for target cells, the amount of the lipid not bound to a ligand used in step c') is preferably 0.01 to 5.0 mol%, more preferably 0.1 to 3.0 mol%, relative to the total lipid. Note that the "amount of lipid not bound to a ligand used in step c')" does not include the amount of lipid not bound to a ligand used other than step c') (for example, the ionic lipid used in step a).

[0288] When using a PEG lipid to which no ligand is bound in step c'), from the viewpoint of achieving both the desirable physical properties of the ligand-modified lipid nanoparticles and selectivity to target cells, the amount of the PEG lipid to which no ligand is bound used in step c') is preferably 0.01 to 5.0 mol%, more preferably 0.1 to 3.0 mol%, relative to the total lipid. Note that the "amount of the PEG lipid to which no ligand is bound used in step c')" does not include the amount of the PEG lipid to which no ligand is bound used other than step c') (for example, the amount of the PEG lipid used in step a).

[0289] The descriptions of the ligand, the ligand-bound lipid, and the ligand-free lipid in step c') are the same as those in step c).

[0290] From the viewpoint of achieving both selectivity for target cells and desirable particle properties, the amount of ligand in the target lipid nanoparticle is preferably 0.001 to 30 mol%, more preferably 0.001 to 10 mol%, and even more preferably 0.01 to 5 mol%, relative to the total amount of lipid in the lipid nanoparticle. It is preferable to mix nucleic acid-free lipid nanoparticles, water, and ligand-bound lipids to achieve such an amount of ligand.

[0291] For efficient modification of nucleic acid-free lipid nanoparticles with ligand-conjugated lipids, a mixture of nucleic acid-free lipid nanoparticles, water, and ligand-conjugated lipids (preferably a mixture of a suspension of nucleic acid-free lipid nanoparticles and ligand-conjugated lipids) may be maintained at a predetermined temperature for a predetermined time. The temperature is preferably 0 to 95°C, more preferably 0 to 40°C, and even more preferably 0 to 25°C, and the time is preferably 1 minute to 5 hours, more preferably 1 minute to 2 hours, and even more preferably 1 to 60 minutes. Note that the mixture in this embodiment may contain other components (e.g., lipids not bound to ligands).

[0292] <Step b')> In one embodiment of the production method of the present invention, step b') is performed after step c') and other steps that are performed as necessary. In step b'), a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acids is obtained by mixing ligand-modified lipid nanoparticles that do not contain nucleic acids with a nucleic acid solution. The nucleic acid solution used in step b') contains water as a solvent, and the suspension of ligand-modified lipid nanoparticles encapsulating nucleic acids obtained in step b') contains water as a dispersion medium.

[0293] Examples of materials to be mixed with the nucleic acid solution in step b') include (i) a suspension of nucleic acid-free ligand-modified lipid nanoparticles, and (ii) a lyophilized product of nucleic acid-free ligand-modified lipid nanoparticles obtained by lyophilizing the suspension. In step b'), the suspension of nucleic acid-free ligand-modified lipid nanoparticles is preferably mixed with the nucleic acid solution. The suspension of nucleic acid-free ligand-modified lipid nanoparticles used in step b') of a preferred embodiment of the present invention contains water as a dispersion medium.

[0294] The mixing in step b') can be performed, for example, by pipetting or by using a device including a microchannel or a mixer, including, but not limited to, a vortex mixer, a T-junction mixer, and a jet mixer.

[0295] Examples of nucleic acids include those described below. The nucleic acid is preferably mRNA. The description of the nucleic acid solution in step b') (e.g., its concentration) is the same as the description of the nucleic acid solution in step b).

[0296] In step b'), the molar ratio of the total amino groups of the ionic lipids in the nucleic acid-free ligand-modified lipid nanoparticles (for example, a suspension of nucleic acid-free ligand-modified lipid nanoparticles, or a lyophilized product of nucleic acid-free ligand-modified lipid nanoparticles) to the total phosphate groups of the nucleic acid in the nucleic acid solution (total amount of amino groups of ionic lipids (mol) / total amount of phosphate groups of nucleic acid (mol)) (sometimes referred to herein as the "N / P ratio") is preferably 5 to 280, more preferably 10 to 140, and even more preferably 15 to 70, from the viewpoint of nucleic acid encapsulation efficiency and toxicity.

[0297] In step b'), the volume ratio of the suspension of ligand-modified lipid nanoparticles not containing nucleic acid to the nucleic acid solution (volume of the suspension of ligand-modified lipid nanoparticles not containing nucleic acid:volume of the nucleic acid solution) is not particularly limited as long as it is a volume ratio that allows nucleic acid to be encapsulated, but from the viewpoint of efficient encapsulation of nucleic acid, it is preferably 1:20 to 20:1, more preferably 1:12 to 12:1.

[0298] To enhance the nucleic acid encapsulation efficiency, a mixture of nucleic acid-free ligand-modified lipid nanoparticles (preferably a suspension of nucleic acid-free ligand-modified lipid nanoparticles) and a nucleic acid solution may be maintained at a predetermined temperature for a predetermined time. The temperature is preferably 0 to 95°C, more preferably 0 to 60°C, and even more preferably 0 to 40°C, and the time is preferably 1 minute to 2 hours, more preferably 1 to 60 minutes, and even more preferably 1 to 30 minutes. Note that the mixture in this embodiment may contain other components (e.g., lipids not bound to a ligand).

[0299] The concentration of nucleic acid in the suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid obtained in step b') (i.e., amount of nucleic acid (μg) / volume of suspension (mL)) is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 1 to 450 μg / mL. In step b') of a preferred embodiment of the present invention, ligand-modified lipid nanoparticles not containing nucleic acid are mixed with a nucleic acid solution so as to achieve the above-mentioned concentration.

[0300] <Step d)> In one embodiment of the manufacturing method of the present invention, step d) is carried out after step a) and other steps that are carried out as necessary.In step d), nucleic acid-free lipid nanoparticles are mixed with nucleic acid-free lipid nanoparticles, a nucleic acid solution, and a ligand-bound lipid, thereby obtaining a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.In step d), the material to be mixed with the nucleic acid solution and the ligand-bound lipid can be, for example, (i) a suspension of nucleic acid-free lipid nanoparticles, and (ii) a lyophilized product of nucleic acid-free lipid nanoparticles obtained by lyophilizing the suspension.In step d), preferably, a suspension of nucleic acid-free lipid nanoparticles is mixed with nucleic acid solution and ligand-bound lipid.

[0301] The mixing in step d) can be performed, for example, by pipetting or by using a device including a microchannel or a mixer, including, but not limited to, a vortex mixer, a T-junction mixer, and a jet mixer.

[0302] The order of mixing in step d) is not particularly limited, and for example, (i) may be simultaneously mixed with nucleic acid-free lipid nanoparticles (preferably, a suspension of nucleic acid-free lipid nanoparticles), a nucleic acid solution, and a ligand-bound lipid; (ii) may first be mixed with nucleic acid-free lipid nanoparticles (preferably, a suspension of nucleic acid-free lipid nanoparticles) and a nucleic acid solution, and then the resulting mixture may be simultaneously mixed with ligand-bound lipid; or (iii) may first be mixed with a nucleic acid solution and a ligand-bound lipid, and then the resulting solution containing nucleic acid and ligand-bound lipid may be mixed with nucleic acid-free lipid nanoparticles (preferably, a suspension of nucleic acid-free lipid nanoparticles). Of the above embodiments, embodiment (iii) is preferred.

[0303] During the mixing in step d), other components may be mixed in. Examples of other components include lipids to which no ligand is bound.

[0304] In step d), nucleic acid-free lipid nanoparticles (preferably, a suspension of nucleic acid-free lipid nanoparticles), a nucleic acid solution, a ligand-bound lipid, and a lipid to which no ligand is bound may be mixed. The order of mixing is not particularly limited, and for example, (i) nucleic acid-free lipid nanoparticles (preferably, a suspension of nucleic acid-free lipid nanoparticles), a nucleic acid solution, a ligand-bound lipid, and a lipid to which no ligand is bound may be mixed simultaneously; (ii) first, an aqueous solution or aqueous dispersion containing a ligand-bound lipid and a lipid to which no ligand is bound is prepared, and then the resulting aqueous solution or aqueous dispersion is mixed with a nucleic acid solution, and then the resulting mixture is mixed with a lipid nanoparticle to which no nucleic acid is bound (preferably, a suspension of lipid nanoparticles to which no nucleic acid is bound). The aqueous solution or aqueous dispersion in (ii) may, for example, be an aqueous solution or aqueous dispersion (reaction mixture) containing a ligand-bound lipid (product) and a lipid to which no ligand is bound (unreacted reactant) obtained in the synthesis reaction of the ligand-bound lipid.

[0305] When a lipid not bound to a ligand is used in step d), from the viewpoint of achieving both desirable physical properties of the ligand-modified lipid nanoparticles and selectivity for target cells, the amount of the lipid not bound to a ligand used in step d) is preferably 0.01 to 5.0 mol%, more preferably 0.1 to 3.0 mol%, relative to the total lipid. Note that the "amount of lipid not bound to a ligand used in step d)" does not include the amount of lipid not bound to a ligand used other than step d) (for example, the ionic lipid used in step a).

[0306] When using a PEG lipid to which no ligand is bound in step d), from the viewpoint of achieving both the desirable physical properties of the ligand-modified lipid nanoparticles and selectivity for target cells, the amount of the PEG lipid to which no ligand is bound used in step d) is preferably 0.01 to 5.0 mol%, more preferably 0.1 to 3.0 mol%, relative to the total lipid. Note that the "amount of the PEG lipid to which no ligand is bound used in step d)" does not include the amount of the PEG lipid to which no ligand is bound used other than step d) (for example, the PEG lipid used in step a).

[0307] The descriptions of the ligand, the ligand-bound lipid, and the ligand-free lipid in step d) are the same as those in step c).

[0308] From the viewpoint of achieving both selectivity for target cells and desirable particle properties, the amount of ligand in the target lipid nanoparticles is preferably 0.001 to 30 mol%, more preferably 0.001 to 10 mol%, and even more preferably 0.01 to 5 mol%, relative to the total amount of lipid in the lipid nanoparticles. It is preferable to mix nucleic acid-free lipid nanoparticles, a nucleic acid solution, and a ligand-bound lipid to achieve such an amount of ligand.

[0309] Examples of nucleic acids include those described below. The nucleic acid is preferably mRNA. The description of the nucleic acid solution in step d) (e.g., its concentration) is the same as the description of the nucleic acid solution in step b).

[0310] In step d), the molar ratio of the total amino groups of the ionic lipids in the nucleic acid-free lipid nanoparticles (for example, a suspension of nucleic acid-free lipid nanoparticles, or a lyophilized product of nucleic acid-free lipid nanoparticles) to the total phosphate groups of the nucleic acid in the nucleic acid solution (total amount of amino groups of the ionic lipids (mol) / total amount of phosphate groups of the nucleic acid (mol)) (sometimes referred to herein as the "N / P ratio") is preferably 5 to 280, more preferably 10 to 140, and even more preferably 15 to 70, from the viewpoint of nucleic acid encapsulation efficiency and toxicity.

[0311] In step d), the volume ratio of the suspension of nucleic acid-free lipid nanoparticles to the nucleic acid solution (volume ratio of the suspension of nucleic acid-free lipid nanoparticles:volume of the nucleic acid solution) is not particularly limited as long as it is a volume ratio that allows nucleic acid to be encapsulated, but from the viewpoint of efficient encapsulation of nucleic acid, it is preferably 200:1 to 1:200, more preferably 20:1 to 1:20. Note that the "nucleic acid solution" in the above volume ratio may contain components other than nucleic acid (e.g., ligand-bound lipids, lipids not bound to a ligand).

[0312] The concentration of nucleic acid in the suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid obtained in step d) (i.e., amount of nucleic acid (μg) / volume of suspension (mL)) is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 1 to 450 μg / mL. In step d) of a preferred embodiment of the present invention, nucleic acid-free lipid nanoparticles, a nucleic acid solution, and a ligand-conjugated lipid are mixed to achieve the above-mentioned concentrations.

[0313] To enhance the nucleic acid encapsulation efficiency, a mixture of nucleic acid-free lipid nanoparticles (preferably a suspension of nucleic acid-free lipid nanoparticles), a nucleic acid solution, and a ligand-conjugated lipid may be maintained at a predetermined temperature for a predetermined time. The temperature is preferably 0 to 95°C, more preferably 0 to 60°C, and even more preferably 0 to 40°C, and the time is preferably 1 minute to 2 hours, more preferably 1 to 60 minutes, and even more preferably 1 to 30 minutes. Note that the mixture in this embodiment may contain other components (e.g., lipids not conjugated to a ligand).

[0314] <Method for introducing nucleic acid into cells> The present invention also provides: (i) a method for introducing nucleic acid into cells, comprising the step of contacting cells ex vivo with ligand-modified lipid nanoparticles encapsulating nucleic acid obtained by the production method of the present invention; and (ii) a method for introducing nucleic acid into target cells in the living body, comprising the step of administering to the living body ligand-modified lipid nanoparticles encapsulating nucleic acid obtained by the production method of the present invention (hereinafter these may be collectively abbreviated as "the method of the present invention").

[0315] The method of the present invention allows any nucleic acid to be introduced into cells. Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, nucleic acids can be mono-, tri-, or tri-stranded, but are preferably single- or double-stranded. Nucleic acids may also be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, other oligomers with non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs)), or other oligomers with special linkages (provided that the oligomers contain nucleotides with configurations that allow base pairing or base attachment, such as those found in DNA or RNA). Furthermore, the nucleic acid may be, for example, a nucleic acid having a known modification added thereto, a nucleic acid labeled as known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides have been replaced with an analogue, a nucleic acid having an intramolecular nucleotide modification, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid having an intercalating compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-anomeric nucleic acid, etc.), etc.

[0316] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc., preferably plasmid DNA, cDNA, and antisense DNA, more preferably plasmid DNA. Circular DNA such as plasmid DNA can also be digested with appropriate restriction enzymes and used as linear DNA.

[0317] The type of RNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., and preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.

[0318] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0319] The ligand-modified lipid nanoparticles encapsulating nucleic acids obtained by the production method of the present invention can be administered in vivo, for example, for the purpose of preventing and / or treating diseases. Therefore, the nucleic acids used in the present invention are preferably those that have preventive and / or therapeutic activity against a specific disease (prophylactic / therapeutic nucleic acids). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0320] The particle size of the ligand-modified lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. The particle size can be measured using a particle size distribution analyzer such as Zetasizer Nano (Malvern).

[0321] The surface potential (zeta potential) of ligand-modified lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably −15 to +15 mV, more preferably −10 to +10 mV. In conventional gene transfer, particles with a positively charged surface potential have primarily been used. Particles with a positively charged surface potential are useful as a method for promoting electrostatic interaction with heparin sulfate on the negatively charged cell surface and promoting cellular uptake. However, a positive surface charge may inhibit the release of nucleic acids from the carrier due to interaction with the delivered nucleic acid within the cell, or inhibit protein synthesis due to interaction between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface charge within the above range. Surface charge can be measured using a zeta potential measuring device such as a Zetasizer Nano. The surface charge of ligand-modified lipid nanoparticles encapsulating nucleic acids can be adjusted by the composition of their constituent components.

[0322] The step of contacting cells with ligand-modified lipid nanoparticles encapsulating nucleic acids in vitro will be specifically described below.

[0323] The cells are suspended in an appropriate medium and cultured under appropriate conditions several days before contact with the nucleic acid-encapsulating ligand-modified lipid nanoparticles, and the cells may or may not be in a proliferative phase at the time of contact.

[0324] The culture medium at the time of contact may be a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 30% by weight or less, more preferably 20% by weight or less. If the medium contains excess proteins such as serum, there is a possibility that contact between the ligand-modified lipid nanoparticles encapsulating nucleic acids and the cells may be inhibited.

[0325] The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the type of cell, etc., but is usually 1×10 ４ ~1 x 10 ７ cells / mL range.

[0326] To the cells prepared in this manner, for example, a suspension of the above-mentioned ligand-modified lipid nanoparticles encapsulating nucleic acid is added. The amount of the suspension added is not particularly limited and can be set appropriately taking into account the number of cells, etc. The concentration of the ligand-modified lipid nanoparticles encapsulating nucleic acid when contacted with the cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved. However, the total lipid concentration is typically 0.0075 to 14 mM, preferably 0.05 to 10 mM, more preferably 0.075 to 7 mM, and the nucleic acid concentration is preferably 0.1 to 1000 μg / mL, more preferably 0.1 to 375 μg / mL, and even more preferably 4 to 170 μg / mL.

[0327] After adding the above-mentioned suspension to the cells, the cells are cultured. ２ The concentration and other factors are appropriately set taking into consideration the type of cells. When the cells are mammalian cells, the temperature is usually about 37°C, the humidity is about 95%, and CO ２ The concentration is about 5%. The incubation time can also be set appropriately taking into account conditions such as the type of cells used, but is usually in the range of 0.1 to 96 hours, preferably 0.2 to 72 hours, and more preferably 0.5 to 48 hours. If the incubation time is too short, the nucleic acid may not be sufficiently introduced into the cells, and if the incubation time is too long, the cells may become weak.

[0328] The nucleic acid is introduced into the cells by the above-mentioned culture, and preferably the culture medium is replaced with a fresh medium or fresh medium is added to the medium and the culture is continued. When the cells are mammalian cells, the fresh medium preferably contains serum or nutrient factors.

[0329] Furthermore, as described above, by using ligand-modified lipid nanoparticles encapsulating nucleic acids, it is possible to introduce nucleic acids into cells not only in vitro but also in vivo. That is, by administering ligand-modified lipid nanoparticles encapsulating nucleic acids to a subject, the lipid nanoparticles reach and contact target cells, and the nucleic acids encapsulated in the lipid nanoparticles are introduced into cells in vivo. The subjects to which the lipid nanoparticles can be administered are not particularly limited, and examples include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), and plants. The subjects to which the ligand-modified lipid nanoparticles encapsulating nucleic acids are administered are preferably humans or other mammals.

[0330] The type of target cell is not particularly limited, and by using ligand-modified lipid nanoparticles encapsulating nucleic acids, it is possible to introduce nucleic acids 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.).

[0331] The method of administering ligand-modified lipid nanoparticles encapsulating nucleic acids to a subject (e.g., vertebrates, invertebrates, etc.) is not particularly limited as long as it allows the lipid nanoparticles to reach and contact target cells and allows the compound introduced into the lipid nanoparticles to be introduced into the cells. Taking into consideration the type of compound to be introduced, 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 dosage of the lipid nanoparticles is not particularly limited as long as it is within a range that allows for the introduction of the compound into the cells, and can be appropriately selected taking into consideration the type of subject, administration method, type of compound to be introduced, type and site of the target cells, etc.

[0332] When the ligand-modified lipid nanoparticles encapsulating nucleic acids obtained by the production method of the present invention are used as nucleic acid transfer agents, they can be formulated according to conventional methods.

[0333] When the nucleic acid transfer agent is provided as a research reagent, the nucleic acid transfer agent may be provided (i) as a ligand-modified lipid nanoparticle encapsulating a nucleic acid, or (ii) as a mixture with, for example, water or other physiologically acceptable liquid (e.g., a water-soluble solvent (e.g., malic acid buffer solution, etc.), an organic solvent (e.g., ethanol, methanol, DMSO, tert-butanol, etc.), or a mixture of a water-soluble solvent and an organic solvent, etc.). The nucleic acid transfer agent of the present invention may optionally contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0334] Furthermore, when the nucleic acid transfer agent is provided as a pharmaceutical, the nucleic acid transfer agent can be provided as a ligand-modified lipid nanoparticle encapsulating a nucleic acid, or as a mixture with known pharmaceutically acceptable additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.). The nucleic acid transfer agent can be manufactured as an oral agent (e.g., tablets, capsules, etc.) or a parenteral agent (e.g., injections, sprays, etc.), or as a parenteral agent (more preferably, injections). The nucleic acid transfer agent can be formulated for use in adults as well as for children.

[0335] <Method for producing pharmaceutical compositions> The ligand-modified lipid nanoparticles encapsulating nucleic acids obtained by the production method of the present invention can be used as a drug delivery system for selectively delivering nucleic acids, etc. into specific cells, and are useful, for example, for DNA vaccines and gene therapy drugs for tumors that involve antigen gene transfer into dendritic cells, and nucleic acid pharmaceuticals that suppress the expression of target genes using RNA interference. Therefore, the present invention also provides a method for producing a pharmaceutical composition comprising the production method of the present invention.

[0336] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0337] In the following examples, the ionic lipids represented by formula (1) are shown by the names listed in the above table. The abbreviations used in the following examples have the following meanings:ALC-0315: [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (manufactured by Jenkem Technology USA) Azide-PEG-DSPE: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (manufactured by Avanti Polar Lipids) cRGD: Cyclo[Arg-Gly-Asp-D-Phe-Lys(Cys)] (manufactured by Peptide Institute, Inc.) DBCO-NHS: Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (manufactured by Merck-Millipore) DSPE-PEG-Mannose: DSPE-PEG-Mannose, MW 2,000 (manufactured by Biopharma PEG Scientific) DMG-PEG2000: 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (number average molecular weight of PEG chain: 2,000) (manufactured by NOF Corporation) DSPE-034GS: N-[N'-(succinimidyloxyglutaryl)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (number average molecular weight of PEG chain: 3,400) (manufactured by NOF Corporation) DSPE-050GS: N-[N'-(succinimidyloxyglutaryl)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (number average molecular weight of PEG chain: 5000) (manufactured by NOF Corporation) EDTA: ethylenediaminetetraacetic acid GS-020TS: 1,2-distearoyl-rac-glycero-3-(succinimidyloxycarbonyl)polyoxyethylene (number average molecular weight of PEG chain: 2000) (manufactured by NOF Corporation) LNP: lipid nanoparticles MC3: DLin-MC3-DMA (manufactured by Cayman Chemical) Methoxy-PEG: Methyl-PEG2000-DSPE (manufactured by Tokyo Chemical Industry Co., Ltd.) Nuclease free water: UltraPure. ＴＭDNase / RNase-Free Distilled Water (manufactured by Thermo Fisher Scientific) PBS: Phosphate-Buffered Saline SM-102: Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (manufactured by Cayman Chemical) Tris·HCl: Tris(hydroxymethyl)aminomethane hydrochloride Triton X-100: Nonionic surfactant (manufactured by Nacalai Tesque, Inc.)

[0338] [Preparation Example 1] Preparation of an aqueous solution containing a ligand-bound PEG lipid Using an Amicon Ultra-4 (MWCO: 50 kDa), the solvent of an aqueous solution of anti-transferrin receptor antibody (manufactured by BioXCell) was diluted with 200 mM NaHCO ３ The aqueous antibody solution from which the buffer solvent had been replaced was mixed with 2 equivalents of DBCO-NHS, and the resulting mixture was incubated at room temperature for 2 hours. The mixture was further incubated overnight at 4°C. Using an Amicon Ultra-4 (3 kDa), the solvent in the mixture was replaced with ultrapure water to prepare an aqueous DBCO-anti-transferrin receptor antibody solution.

[0339] 10.1 μL of the 74 μM DBCO-anti-transferrin receptor antibody aqueous solution obtained as described above, 7.47 μL of a 0.1 mM Azide-PEG-DSPE aqueous solution, and 32.5 μL of 2-morpholinoethanesulfonic acid buffer (pH: 6.0) were mixed, and the resulting mixture was incubated overnight at 4° C. to prepare an aqueous solution containing a ligand-bound PEG lipid (ligand: anti-transferrin receptor antibody).

[0340] Comparative Example 1 Preparation of LNPs Encapsulating mRNA but Not Modified with a Ligand An ethanol solution of 10 mM SS-OP, an ethanol solution of 20 mM cholesterol, and an ethanol solution of 10 mM 1,2-dioleoyl-sn-glycero-3-phosphocholine were mixed at a molar ratio of 52.5 / 40 / 7.5, and the resulting mixture was mixed with an ethanol solution of 1 mM DMG-PEG2000 to prepare a lipid solution (DMG-PEG2000 amount: 1.5 mol% relative to the total of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0341] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM, cholesterol concentration: 3.2 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 0.6 mM, DMG-PEG2000 concentration: 0.12 mM, total lipid concentration: 8.12 mM

[0342] Using the nanoparticle production device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malic acid buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 875 μL / min and lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of LNP suspension not containing nucleic acid (hereinafter referred to as "empty LNP suspension") (Step a)). The same operation was repeated to obtain 2 mL of empty LNP suspension (Step a)).

[0343] To each of the two resulting empty LNP suspensions (2 mL), 2.5 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added while stirring on a vortex mixer, and the resulting two 4.5 mL mixtures were combined. To the resulting 9.0 mL mixture, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was added, and the resulting mixture was subjected to ultrafiltration using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the resulting concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting dilution was subjected to ultrafiltration. To 100 μL of the resulting concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with a exchanged dispersion medium. The LNP suspension was stored under argon atmosphere at 4°C until use.

[0344] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire amount of the obtained mRNA solution and 15 μL of empty LNP suspension from which the dispersion medium had been replaced were mixed using a vortex mixer, and the resulting mixture was incubated at 37°C for 5 minutes. After incubation, phosphate buffer (150 μL, buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare an LNP suspension encapsulating mRNA (N / P ratio: 36, mRNA concentration: 10 μg / mL) (step b)).

[0345] Next, 7.47 μL of a 0.1 mM aqueous Azide-PEG-DSPE solution and 42.6 μL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) were mixed, and the resulting solution was incubated overnight at 4°C. The entire amount of the solution after incubation was mixed with the entire amount of the above-mentioned LNP suspension encapsulating mRNA, and the resulting mixture was incubated at 4°C for 30 minutes to prepare an Azide-PEG-DSPE-modified LNP suspension encapsulating mRNA.

[0346] Comparative Example 2 Preparation of Ligand-Modified LNPs Encapsulating mRNA An ethanol solution of 10 mM SS-OP, an ethanol solution of 10 mM cholesterol, and an ethanol solution of 5 mM 1,2-dioleoyl-sn-glycero-3-phosphocholine were mixed at a molar ratio of 52.5 / 40 / 7.5, and the resulting mixture was mixed with an ethanol solution of 1 mM DMG-PEG and an aqueous solution of 1 mM Azide-PEG-DSPE to prepare a lipid solution (DMG-PEG2000 amount: 1.5 mol%, and Azide-PEG-DSPE amount: 0.8 mol%, relative to the total of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0347] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 2.1 mM, cholesterol concentration: 1.6 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 0.3 mM, DMG-PEG2000 concentration: 0.06 mM, Azide-PEG-DSPE concentration: 0.0032 mM, total lipid concentration: 4.092 mM

[0348] Using the nanoparticle manufacturing device "iLiNP" (Lilac Pharma Co., Ltd.), a malic acid buffer solution containing mRNA encoding luciferase (mRNA concentration: 0.167 mg / mL, buffer concentration: 20 mM, pH 3.0) was mixed at a flow rate of 750 μL / min and a lipid solution at a flow rate of 250 μL / min for a total of 1 minute to prepare an LNP suspension (1 mL) encapsulating mRNA. A similar procedure was performed to prepare an LNP suspension (1 mL) encapsulating mRNA.

[0349] To each of the two resulting mRNA-encapsulated LNP suspensions (1 mL), 1.0 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added while stirring with a vortex mixer, and the resulting two 2.0 mL mixtures were combined. To the resulting 4.0 mL mixture, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 5.5) was added, and the mixture was concentrated to 0.2 mL or less using an Amicon Ultra-4 (MWCO: 100 kDa). After concentration by ultrafiltration, the resulting concentrate was diluted with phosphate buffer (4 mL, buffer concentration: 9.6 mM, pH: 7.4), and the resulting dilution was subjected to ultrafiltration. Malic acid (malic acid concentration: 20 mM, sodium chloride concentration: 30 mM, pH: 5.0) was added to bring the volume of the mixture to 4.0 mL, and the resulting mixture was again concentrated to 200 μL by ultrafiltration to prepare an LNP suspension containing mRNA with the dispersion medium exchanged.

[0350] F(ab') ２ F(ab') fragment was added in an amount of 2 equivalents of DBCO-NHS. ２ The DBCO-NHS aqueous solution was added to the aqueous fragment solution, and the mixture was incubated at room temperature for 2 hours and then at 4° C. overnight. Ultrapure water was added to bring the volume of the mixture to 0.5 mL, and the mixture was ultrafiltered using an Amicon Ultra-0.5 to remove unreacted DBCO-NHS, yielding DBCO-bound F(ab′) ２ The fragments were prepared as follows: DBCO-conjugated F(ab') ２ From the absorbance at 280 nm and 309 nm of the aqueous fragment solution, DBCO-bound F(ab') ２ The number of DBCO-introduced fragments was calculated. The number of DBCO-introduced F(ab') fragments was calculated based on the number of azide groups in the LNPs encapsulating the mRNA. ２ The DBCO-bound F(ab') fragment was added to the LNP suspension encapsulating the mRNA so that the fragments were in equimolar amounts. ２ The fragment was added and incubated overnight at 4°C to prepare a ligand-modified LNP suspension encapsulating mRNA (amount of ligand relative to total lipids: 0.8 mol%).

[0351] Example 1 Preparation of Ligand-Modified LNPs Encapsulating mRNA An ethanol solution of 10 mM SS-OP, an ethanol solution of 2 mM cholesterol, and an ethanol solution of 10 mM 1,2-dioleoyl-sn-glycero-3-phosphocholine were mixed at a molar ratio of 52.5 / 40 / 7.5, and the resulting mixture was mixed with an ethanol solution of 1 mM DMG-PEG2000 to prepare a lipid solution (DMG-PEG2000 amount relative to the total of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphocholine: 1.5 mol%).

[0352] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM, cholesterol concentration: 3.2 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 0.6 mM, DMG-PEG2000 concentration: 0.12 mM, total lipid concentration: 8.12 mM

[0353] Using the nanoparticle production device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malic acid buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 875 μL / min and lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a)). The same procedure was repeated to obtain 2 mL of empty LNP suspension (step a)).

[0354] To each of the two resulting empty LNP suspensions (2 mL), 2.5 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added while stirring on a vortex mixer, and the resulting two 4.5 mL mixtures were combined. To the resulting 9.0 mL mixture, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was added, and the resulting mixture was subjected to ultrafiltration using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the resulting concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting dilution was subjected to ultrafiltration. To 100 μL of the resulting concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with a exchanged dispersion medium. The LNP suspension was stored under argon atmosphere at 4°C until use.

[0355] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire amount of the resulting mRNA solution (135 μL) and the empty LNP suspension (15 μL) from which the dispersion medium had been replaced were mixed using a vortex mixer, and the resulting mixture was incubated at 37°C for 5 minutes. To the incubated mixture, phosphate buffer (150 μL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare an LNP suspension encapsulating mRNA (N / P ratio: 36, mRNA concentration: 10 μg / mL) (step b)).

[0356] The LNP suspension (150 μL) encapsulating mRNA and the entire amount of the aqueous solution containing the ligand-bound PEG lipid obtained in Preparation Example 1 were mixed by pipetting and incubated at 4°C for 30 minutes to prepare a ligand-modified LNP suspension encapsulating mRNA (amount of ligand relative to total lipid: 0.8 mol% (step c)).

[0357] Furthermore, ligand-modified LNP suspensions encapsulating mRNA were prepared in the same manner as above, except that the incubation temperature during preparation of the ligand-modified LNP suspensions encapsulating mRNA (hereinafter referred to as "incubation temperature during ligand modification") was changed to 37° C. or 50° C. In this way, three suspensions were prepared, each with a different incubation temperature during ligand modification.

[0358] [Test Example 1] Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA of Example 1 or Comparative Example 2 were analyzed. 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) assay. The results are shown in Table 2 below. Note that the LNP used in Example 1 was one that was incubated at 4°C during ligand modification.

[0359]

[0360] As shown in Table 2, the ligand-modified LNPs encapsulating mRNA in Example 1 had a higher mRNA encapsulation rate than the ligand-modified LNPs encapsulating mRNA in Comparative Example 2.

[0361] Comparative Example 3 Preparation of LNPs Encapsulating mRNA but Not Modified with a Ligand An Azide-PEG-DSPE-modified LNP suspension encapsulating mRNA was prepared using the same procedure as in Comparative Example 1, except that: (i) a lipid solution was prepared by mixing a 10 mM ethanol solution of SS-OP, a 20 mM ethanol solution of cholesterol, a 10 mM ethanol solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine, a 1 mM ethanol solution of DMG-PEG2000, and a 0.5 mM ethanol solution of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (a fluorescent dye) in a molar ratio of 52.5 / 40 / 7.5 / 1.5 / 0.5; and (ii) an mRNA encoding luciferase was used instead of the mRNA encoding NanoLuc (registered trademark) luciferase.

[0362] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM, cholesterol concentration: 3.2 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 0.6 mM, DMG-PEG2000 concentration: 0.12 mM, total lipid concentration: 8.12 mM

[0363] Furthermore, Azide-PEG-DSPE-modified LNP suspensions encapsulating mRNA were prepared in the same manner as above, except that the incubation temperature during preparation of the Azide-PEG-DSPE-modified LNP suspensions encapsulating mRNA (hereinafter referred to as "incubation temperature during Azide-PEG-DSPE modification") was changed to 37° C. or 50° C. In this way, three suspensions were prepared, each with a different incubation temperature during Azide-PEG-DSPE modification.

[0364] Example 2 Preparation of Ligand-Modified LNP Encapsulating mRNA A lipid solution was prepared by mixing a 10 mM ethanol solution of SS-OP, a 20 mM ethanol solution of cholesterol, a 10 mM ethanol solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine, a 1 mM ethanol solution of DMG-PEG2000, and a 0.5 mM ethanol solution of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (fluorescent dye) in a molar ratio of 52.5 / 40 / 7.5 / 1.5 / 0.5. A ligand-modified LNP suspension encapsulating mRNA was prepared using the same procedure as in Example 1, except that this lipid solution was used and that the incubation during ligand modification was performed at 4°C, 37°C, or 50°C.

[0365] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM, cholesterol concentration: 3.2 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 0.6 mM, DMG-PEG2000 concentration: 0.12 mM, total lipid concentration: 8.12 mM

[0366] Test Example 2: Evaluation of LNP particle size, PdI, mRNA encapsulation rate, etc. The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA of Example 2 were measured in the same manner as in Test Example 1. The results are shown in Table 3 below. Note that the "temperature" in Table 3 below refers to the "incubation temperature during ligand modification."

[0367]

[0368] [Test Example 3] Evaluation of LNP uptake into mouse breast cancer-derived 4T1 cells in vitro (FACS analysis) 7.5 × 10 ４ The cells were seeded onto a 12-well plate at 100 cells / well. 24 hours later, either the ligand-modified LNP suspension encapsulating mRNA obtained in Example 2 or the ligand-unmodified LNP suspension encapsulating mRNA but obtained in Comparative Example 3 was added to the 12-well plate so that the lipid concentration per well was 10 μM. The mixture in the wells was incubated for 2 hours. After incubation, the mixture in the wells was washed twice with phosphate buffer (pH 7.4) to remove LNP not incorporated into 4T1 cells. The 4T1 cells were then detached with trypsin. The 4T1 cells were centrifuged at 500 × g and 4°C for 5 minutes, and the resulting cell pellet was suspended in 1000 μL of FACS buffer (PBS containing 0.05 wt% sodium azide aqueous solution and 0.5 wt% fetal bovine serum albumin) to prepare a suspension. The resulting suspension was then centrifuged at 500 x g and 4°C for 5 minutes, and the resulting cell pellet was used as a sample to measure the fluorescence intensity derived from LNP in 4T1 cells using NovoCyte. The results are shown in Table 4 below. Note that the "temperature" described in 4 below refers to the "incubation temperature during ligand modification" in Example 2 and the "incubation temperature during Azide-PEG-DSPE modification" in Comparative Example 3.

[0369]

[0370] As shown in Table 4, regardless of the incubation temperature during ligand modification or Azide-PEG-DSPE modification, the ligand-modified LNPs encapsulating the mRNA of Example 2 had improved fluorescence intensity compared to the LNPs encapsulating the mRNA of Comparative Example 3 but not modified with a ligand, and a significant improvement in the amount of uptake into 4T1 cells was observed.

[0371] Example 3 A ligand-modified LNP suspension encapsulating mRNA was prepared in the same manner as in Example 1, except that mRNA encoding NanoLuc (registered trademark) luciferase was used instead of mRNA encoding luciferase, and the incubation temperature during ligand modification was set to 4°C.

[0372] Test Example 4 Evaluation of LNP Gene Expression Activity In Vivo C57BL / 6 mice were administered the ligand-modified LNP suspension encapsulating mRNA of Example 3, or the LNP suspension encapsulating mRNA but not modified with a ligand of Comparative Example 1, or PBS via the tail vein (mRNA amount: 1.0 μg).

[0373] Twenty-one hours after administration, the mice were euthanized and their whole brains were removed. To the whole brains, 800 μL of homogenization buffer (pH 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added, and the brains were disrupted using zirconia beads. 500 μL of the resulting supernatant was centrifuged at 13,000 rpm at 4°C for 10 minutes. 20 μL of the resulting supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate, and the luminescence derived from NanoLuc® luciferase was measured using a GloMax. The supernatant was also diluted 100-fold with ultrapure water, and the protein concentration of the resulting solution was measured using the BCA method. The luminescence intensity was normalized by the protein amount. The results are shown in Table 5 below.

[0374]

[0375] As shown in Table 5, the ligand-modified LNPs encapsulating the mRNA of Example 3 had improved luminescence intensity compared to the LNPs encapsulating the mRNA of Comparative Example 1 but not modified with a ligand, demonstrating a significant improvement in gene expression activity in the mouse brain.

[0376] Example 4 Preparation of Ligand-Modified LNPs Encapsulating mRNA A ligand-modified LNP suspension encapsulating mRNA was prepared in the same manner as in Example 1, except that instead of the 10 mM SS-OP ethanol solution, a 10 mM SM-102 ethanol solution, a 10 mM MC3 ethanol solution, a 10 mM ALC-0315 ethanol solution, a 10 mM compound 3 ethanol solution, a 10 mM compound 4 ethanol solution, a 10 mM compound 5 ethanol solution, or a 10 mM compound 6 ethanol solution was used.

[0377] Test Example 5 Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA of Example 4 were measured in the same manner as in Test Example 1. The results are shown in Table 6 below.

[0378]

[0379] [Preparation Example 2] Preparation of an aqueous solution containing ligand-bound PEG lipid and ligand-unbound PEG lipid. Using an Amicon Ultra-4 (MWCO: 50 kDa), the solvent of an aqueous solution of anti-CD3 antibody (manufactured by Invitrogen) was diluted with 100 mM NaHCO ３ The antibody aqueous solution from which the buffer solvent had been replaced was mixed with 2 equivalents of DBCO-NHS, and the resulting mixture was incubated at room temperature for 2 hours. The mixture was further incubated overnight at 4°C. Using an Amicon Ultra-4 (3 kDa), the solvent in the mixture was replaced with ultrapure water to prepare a DBCO-anti-CD3 antibody aqueous solution.

[0380] 20 μL of the 12 μM DBCO-anti-CD3 antibody aqueous solution obtained as described above, 12.0 μL of a 0.02 mM Azide-PEG-DSPE aqueous solution, 14.4 μL of a 0.1 mM Methoxy-PEG aqueous solution, and 103.6 μL of 2-morpholinoethanesulfonic acid buffer (pH: 6.0) were mixed, and the resulting mixture was incubated overnight at 4 ° C. to prepare an aqueous solution containing a ligand-bound PEG lipid (ligand: anti-CD3 antibody) and a ligand-unbound PEG lipid (Methoxy-PEG) (ligand-bound PEG lipid concentration: 0.0016 mM, ligand-unbound PEG lipid concentration: 0.0096 mM).

[0381] Example 5 Preparation of Ligand-Modified LNPs Encapsulating mRNA An ethanol solution of 10 mM SS-OP, an ethanol solution of 10 mM cholesterol, and an ethanol solution of 10 mM 1,2-dioleoyl-sn-glycero-3-phosphocholine were mixed at a molar ratio of 52.5 / 40 / 7.5, and the resulting mixture was mixed with an ethanol solution of 1 mM DMG-PEG2000 to prepare a lipid solution (DMG-PEG2000 amount relative to the total of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphocholine: 1.0 mol%).

[0382] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 10.5 mM, cholesterol concentration: 1.5 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 8 mM, DMG-PEG2000 concentration: 0.3 mM, total lipid concentration: 20.3 mM

[0383] Using the nanoparticle production device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malic acid buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 875 μL / min and lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a)). The same procedure was repeated to obtain 2 mL of empty LNP suspension (step a)).

[0384] To each of the two empty LNP suspensions (2 mL), 2.5 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added while stirring on a vortex mixer, and the resulting two 4.5 mL mixtures were combined. To the resulting 9.0 mL mixture, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was added, and the resulting mixture was subjected to ultrafiltration using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the resulting concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting dilution was subjected to ultrafiltration. To 15 μL of the resulting concentrate (lipid amount: 300 nmol), 60 μL of sucrose solution dissolved in 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) to a concentration of 320 mg / mL was added to prepare an empty LNP suspension with the dispersion medium exchanged. The LNP suspension was stored frozen at -20°C under an argon atmosphere and thawed at room temperature before use.

[0385] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding luciferase (mRNA amount: 2.5 μg) with 2-morpholinoethanesulfonic acid buffer (75 μL, buffer concentration: 40 mM, pH: 6.0). The entire amount of the obtained mRNA solution (75 μL) and the empty LNP suspension (75 μL) from which the dispersion medium had been replaced were mixed using a vortex mixer, and the resulting mixture was incubated at 75°C for 5 minutes. To the incubated mixture, phosphate buffer (150 μL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare an LNP suspension encapsulating mRNA (N / P ratio: 36, mRNA concentration: 13.3 μg / mL) (step b)).

[0386] The LNP suspension (150 μL) encapsulating mRNA and an aqueous solution (150 μL) containing ligand-bound PEG lipids and ligand-free PEG lipids obtained by the method described in Preparation Example 2 were mixed using a vortex mixer and incubated at 4°C for 30 minutes. Phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was then added to prepare a ligand-modified LNP suspension encapsulating mRNA (amount of ligand relative to total lipids: 0.08 mol%, amount of ligand-free PEG lipid relative to total lipids: 0.48 mol%) (step c)).

[0387] Comparative Example 4 Preparation of LNPs Encapsulating mRNA but Not Modified with Ligands An LNP suspension encapsulating mRNA was prepared by performing step a), exchange of dispersion medium, cryopreservation, and thawing, as well as step b), in the same manner as in Example 5. This LNP suspension encapsulating mRNA (150 μL) and 2-morpholinoethanesulfonic acid buffer (150 μL, buffer concentration: 20 mM, pH: 6.0) were mixed using a vortex mixer, and phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare an LNP suspension encapsulating mRNA (N / P ratio: 36).

[0388] Test Example 6 Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNP encapsulating mRNA of Example 5 and the LNP encapsulating mRNA but not modified with a ligand of Comparative Example 4 were measured in the same manner as Test Example 1. The results are shown in Table 7 below.

[0389]

[0390] As shown in Table 7, the ligand-modified LNPs encapsulating mRNA in Example 5, despite being ligand-modified, showed a favorable mRNA encapsulation rate, similar to the LNP suspension encapsulating mRNA in Comparative Example 4.

[0391] Preparation Example 3 Preparation of Lyophilized LNPs An ethanol solution of 10 mM SS-OP, an ethanol solution of 10 mM cholesterol, and an ethanol solution of 10 mM 1,2-dioleoyl-sn-glycero-3-phosphocholine were mixed at a molar ratio of 52.5 / 40 / 7.5, and the resulting mixture was mixed with an ethanol solution of 1 mM DMG-PEG2000 at a molar ratio of 52.5 / 40 / 7.5 / 1.5 to prepare a lipid solution.

[0392] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM, cholesterol concentration: 3.2 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 0.6 mM, DMG-PEG2000 concentration: 0.12 mM, total lipid concentration: 8.12 mM

[0393] Using a nanoparticle production device "NanoAssembler Ignite" (manufactured by Precision NanoSystems), malic acid buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 875 μL / min and lipid solution at a flow rate of 125 μL / min at 25° C. for 2 minutes to obtain 2 mL of empty LNP suspension (step a)). A similar operation was performed to obtain 2 mL of empty LNP suspension (step a)).

[0394] To each of the two empty LNP suspensions (2 mL), 2.5 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added while stirring on a vortex mixer, and the resulting two 4.5 mL mixtures were combined. To the resulting 9.0 mL mixture, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was added, and the resulting mixture was subjected to ultrafiltration using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the resulting concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting dilution was subjected to ultrafiltration. To 10 μL of the obtained concentrate (lipid amount: 400 nmol), 100 μL of sucrose solution dissolved in 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) to a concentration of 320 mg / mL was added to prepare an empty LNP suspension with the dispersion medium exchanged.

[0395] Lyophilization was performed by first freezing the empty LNP suspension after exchanging the dispersion medium at atmospheric pressure until its temperature reached -40°C. The pressure was then reduced to 200 mTorr, and the temperature was then increased as described below. Specifically, the frozen product was allowed to stand at -40°C for 1 hour under 200 mTorr pressure, followed by heating, then at -30°C for 6 hours, then at -20°C for 6 hours, then at -10°C for 6 hours, then at 0°C for 6 hours, then at 10°C for 3 hours, then at 20°C for 3 hours, then at 30°C for 3 hours, and then the pressure was returned to atmospheric pressure to recover the lyophilized LNP. The resulting lyophilized LNP was stored at -80°C under an argon atmosphere until use.

[0396] Example 6 Preparation of Ligand-Modified LNP Encapsulating mRNA An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding luciferase (mRNA amount: 2.0 μg) with nuclease-free water (200 μL). The total amount of the obtained mRNA solution (200 μL) and the lyophilized LNP obtained in Preparation Example 3 were mixed by pipetting, and the resulting mixture was incubated at 37 ° C. or 75 ° C. for 5 minutes. After incubation, phosphate buffer (200 μL, buffer concentration: 9.6 mM, pH 7.4) was added to the mixture to prepare an LNP suspension encapsulating mRNA (N / P ratio: 69, mRNA concentration: 5 μg / mL) (step b)).

[0397] The mRNA-encapsulated LNP suspension (100 μL) and the entire amount of aqueous solution containing the ligand-bound PEG lipid and the ligand-unbound PEG lipid obtained by the method described in Preparation Example 2 were mixed by pipetting and incubated for 30 minutes at 4° C. After incubation, phosphate buffer (200 μL, buffer concentration: 9.6 mM, pH 7.4) was added to the mixture to prepare a ligand-modified LNP suspension encapsulating mRNA (amount of ligand relative to total lipid: 0.08 mol%) (step c)).

[0398] Example 7 Preparation of Ligand-Modified LNP Encapsulating mRNA The lyophilized LNP obtained in Preparation Example 3 and the entire amount of aqueous solution containing ligand-bound PEG lipids and ligand-free PEG lipids obtained by the method described in Preparation Example 2 were mixed by pipetting and incubated at 4°C for 30 minutes to prepare a ligand-modified LNP suspension that did not contain mRNA (amount of ligand relative to total lipids: 0.8 mol% (step c')).

[0399] An mRNA solution was prepared by mixing an mRNA aqueous solution (mRNA amount: 2.0 μg) encoding NanoLuc® luciferase with nuclease-free water (200 μL). The total amount of the obtained mRNA solution (200 μL) and a ligand-modified LNP suspension (100 μL) not containing mRNA were mixed using a vortex mixer, and the resulting mixture was incubated at 75 ° C. for 5 minutes. After incubation, phosphate buffer (200 μL, buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare a ligand-modified LNP suspension encapsulating mRNA (N / P ratio: 69, mRNA concentration: 4 μg / mL) (step b').

[0400] Test Example 7 Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA of Examples 6 and 7 were measured in the same manner as Test Example 1. The results are shown in Table 8 below. Note that the "temperature" in Table 8 below refers to the "incubation temperature during encapsulation of nucleic acid."

[0401]

[0402] As shown in Table 8, the ligand-modified LNPs encapsulating mRNA in Examples 6 and 7 both showed favorable mRNA encapsulation rates.

[0403] [Preparation Example 4] Preparation of an aqueous solution containing a ligand-bound PEG lipid An aqueous solution containing a ligand-bound PEG lipid was prepared in the same manner as in Preparation Example 1, except that the ligand used was changed to cRGD and the lipid used was changed to GS-020TS, DSPE-034GS, or DSPE-050GS. The ligand-bound PEG lipids obtained using GS-020TS, DSPE-034GS, or DSPE-050GS are hereinafter referred to as "DSPE-034-RGD," "DSPE-050-RGD," or "GS-020-RGD," respectively.

[0404] Preparation Example 5 Preparation of Empty LNP Suspension A 10 mM ethanol solution of SS-OP, a 2 mM ethanol solution of cholesterol, and a 10 mM ethanol solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine were mixed at a molar ratio of 52.5 / 40 / 7.5, and the resulting mixture was mixed with a 1 mM ethanol solution of DMG-PEG2000 to prepare a lipid solution (DMG-PEG2000 amount relative to the total of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphocholine: 1.5 mol%).

[0405] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 10.5 mM, cholesterol concentration: 1.5 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 8 mM, DMG-PEG2000 concentration: 0.3 mM, total lipid concentration: 20.3 mM

[0406] Using the nanoparticle production device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malic acid buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 875 μL / min and lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a)). The same procedure was repeated to obtain 2 mL of empty LNP suspension (step a)).

[0407] To each of the two resulting empty LNP suspensions (2 mL), 2.5 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added while stirring on a vortex mixer, and the resulting two 4.5 mL mixtures were combined. To the resulting 9.0 mL mixture, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was added, and the resulting mixture was subjected to ultrafiltration using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the resulting concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting dilution was subjected to ultrafiltration. To 100 μL of the resulting concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with a exchanged dispersion medium. The LNP suspension was stored under argon atmosphere at 4°C until use.

[0408] Example 8 Preparation of Ligand-Modified LNPs Encapsulating mRNA An mRNA-encapsulated LNP suspension was prepared in the same manner as in Example 5, except that the empty LNP suspension obtained in Preparation Example 5, in which the dispersion medium had been exchanged, was used and that incubation during nucleic acid encapsulation was carried out at 37°C (N / P ratio: 36, mRNA concentration: 8.3 μg / mL) (step b).

[0409] The mRNA-encapsulated LNP suspension (75 μL) and an aqueous solution containing the ligand-conjugated PEG lipid obtained in Preparation Example 4 (i.e., DSPE-034-RGD, DSPE-050-RGD, or GS-020-RGD) or an aqueous solution containing 0.032 mg / mL DSPE-PEG-Mannose (75 μL) were mixed by pipetting and incubated at 4° C. for 30 minutes to prepare a ligand-modified LNP suspension encapsulating mRNA (amount of ligand relative to total lipid: 0.8 mol% (step c)).

[0410] Example 9 Preparation of Ligand-Modified LNP Encapsulating mRNA The empty LNP suspension (15 μL) from which the dispersion medium had been exchanged, obtained in Preparation Example 5, and the aqueous solution containing DSPE-034-RGD or the aqueous solution containing DSPE-PEG-Mannose (150 μL) obtained in Preparation Example 4 were mixed by pipetting and incubated at 4° C. for 30 minutes to prepare a ligand-modified LNP suspension not containing mRNA (amount of ligand relative to total lipids: 0.8 mol % (step c′)).

[0411] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding luciferase (mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132 μL, buffer concentration: 20 mM, pH: 6.0). The entire amount of the obtained mRNA solution (135 μL) and an mRNA-free ligand-modified LNP suspension (165 μL) were mixed using a vortex mixer, and the resulting mixture was incubated at 37°C or 75°C for 5 minutes. To the incubated mixture, phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare an LNP suspension encapsulating mRNA (N / P ratio: 69, mRNA concentration: 5 μg / mL) (step b').

[0412] Example 10 Preparation of Ligand-Modified LNPs Encapsulating mRNA An aqueous solution containing the ligand-conjugated PEG lipid obtained in Preparation Example 4 (i.e., DSPE-034-RGD, DSPE-050-RGD, or GS-020-RGD) or an aqueous solution containing DSPE-PEG-Mannose (150 μL) was mixed with an mRNA solution encoding luciferase (mRNA amount: 3 μg, 2-morpholinoethanesulfonic acid buffer: 132 μL, buffer concentration: 20 mM, pH: 6.0) by pipetting to obtain an aqueous solution containing the ligand-conjugated PEG lipid and mRNA.

[0413] The total amount (285 μL) of the resulting aqueous solution containing the ligand-PEG lipid and mRNA and the empty LNP suspension (15 μL) obtained in Preparation Example 5, in which the dispersion medium had been exchanged, were mixed using a vortex mixer, and the resulting mixtures were incubated at 37° C. for 5 minutes and then at 4° C. for 30 minutes. To the incubated mixture, phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare a ligand-modified LNP suspension encapsulating mRNA (N / P ratio: 69) (step d)).

[0414] Test Example 8 Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA in Examples 8 to 10 were measured in the same manner as in Test Example 1. The results are shown in Table 9 below. Table 9 also lists the ligand-bound PEG lipids used in Examples 8 to 10.

[0415]

[0416] As shown in Table 9, the ligand-modified LNPs encapsulating mRNA in Examples 8 to 10 showed favorable mRNA encapsulation rates regardless of the type of ligand PEG-conjugated lipid.

[0417] Example 11 Preparation of Ligand-Modified LNP Encapsulating mRNA A lipid solution was prepared by mixing a 10 mM ethanol solution of SS-OP, a 20 mM ethanol solution of cholesterol, a 10 mM ethanol solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine, and a 1 mM ethanol solution of DMG-PEG2000 in a molar ratio of 52.5 / 40 / 7.5 / 1.5. A ligand-modified LNP suspension encapsulating mRNA was prepared using the same procedure as in Example 1, except that this lipid solution was used, and the incubation temperature during nucleic acid encapsulation in step b) was 4°C, 37°C, 60°C, or 95°C, and the incubation time during nucleic acid encapsulation was 1 minute, 5 minutes, 30 minutes, 60 minutes, or 120 minutes.

[0418] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 10.5 mM, cholesterol concentration: 1.5 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 8 mM, DMG-PEG2000 concentration: 0.3 mM, total lipid concentration: 20.3 mM

[0419] Test Example 9 Evaluation of LNP particle size, PdI, mRNA encapsulation rate, etc. The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA of Example 11 were measured in the same manner as Test Example 1. The results are shown in Table 10 below. Note that the "temperature" in Table 10 below refers to the "incubation temperature during encapsulation of nucleic acid," and the "time" refers to the "incubation time during encapsulation of nucleic acid."

[0420]

[0421] Example 12 Preparation of Ligand-Modified LNP Encapsulating mRNA A lipid solution was prepared by mixing a 10 mM ethanol solution of SS-OP, a 20 mM ethanol solution of cholesterol, a 10 mM ethanol solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine, and a 1 mM ethanol solution of DMG-PEG2000 in a molar ratio of 52.5 / 40 / 7.5 / 1.5. A ligand-modified LNP suspension encapsulating mRNA was prepared using the same procedure as in Example 1, except that this lipid solution was used and that the incubation during ligand modification in step c) was carried out at the temperature and for the time shown in Table 11 below.

[0422] The concentrations of each lipid and total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 10.5 mM, cholesterol concentration: 1.5 mM, 1,2-dioleoyl-sn-glycero-3-phosphocholine concentration: 8 mM, DMG-PEG2000 concentration: 0.3 mM, total lipid concentration: 20.3 mM.

[0423] Test Example 10: Evaluation of LNP particle size, PdI, mRNA encapsulation rate, etc. The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNPs encapsulating mRNA of Example 12 were measured in the same manner as Test Example 1. The results are shown in Table 11 below. Note that "temperature" in Table 11 below refers to the "incubation temperature during ligand modification," and "time" refers to the "incubation time during ligand modification."

[0424]

[0425] Preparation Example 6 Preparation of an aqueous solution containing a ligand-conjugated PEG lipid, and an aqueous solution containing a ligand-conjugated PEG lipid and a PEG lipid to which no ligand is bound. An aqueous solution containing a ligand-conjugated PEG lipid, and an aqueous solution containing a ligand-conjugated PEG lipid and a PEG lipid to which no ligand is bound (hereinafter sometimes referred to as "aqueous solution 6-1" to "aqueous solution 6-7") were prepared in the same manner as in Preparation Example 2, except that the concentrations of the ligand-conjugated PEG lipid (ligand: anti-CD3 antibody) and the PEG lipid to which no ligand is bound (Methoxy-PEG) in the aqueous solution were changed as shown in Table 12.

[0426]

[0427] [Example 13] A 0.5 mM ethanol solution of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (fluorescent dye) was added to the lipid solution of Example 1 to prepare a lipid solution containing a fluorescent dye in which the molar ratio of SS-OP / cholesterol / 1,2-dioleoyl-sn-glycero-3-phosphocholine / DMG-PEG2000 / 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate was 52.5 / 40 / 7.5 / 1.5 / 0.5; The lipid solution containing the fluorescent dye was used instead of the lipid solution of Example 1; and In place of the aqueous solution containing the ligand-bound PEG lipid obtained in Preparation Example 1, any of Aqueous Solutions 6-1 to 6-7 obtained in Preparation Example 6 was used in step c). Other than that, in the same manner as in Example 1, suspensions of ligand-modified LNPs encapsulating mRNA (hereinafter, sometimes referred to as "Suspension 13-1" to "Suspension 13-7") were prepared.

[0428] Test Example 11: Evaluation of LNP particle size, PdI, mRNA encapsulation rate, etc. The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNP encapsulating mRNA of Example 13 were measured in the same manner as Test Example 1. The results are shown in Table 13 below. Table 13 below also lists the ligand-modified LNP suspensions encapsulating mRNA of Example 13 (Suspensions 13-1 to 13-7) and the aqueous solutions of Preparation Example 6 used (Aqueous Solutions 6-1 to 6-7). In addition, the "Amount of Ligand" and "Amount of Ligand-Unbound PEG Lipid" columns in Table 13 below also list the "Amount of Ligand (Anti-CD3 Antibody) Relative to Total Lipid" and the "Amount of Ligand-Unbound PEG Lipid (Methoxy-PEG) Used in Step c) Relative to Total Lipid, respectively."

[0429]

[0430] Test Example 12 Evaluation of LNP Gene Expression Activity In Vivo Ligand-modified LNP suspensions No. 13-1 to No. 13-3 encapsulating mRNA obtained in Example 13, or an LNP suspension encapsulating mRNA of Comparative Example 3 but not modified with a ligand, were administered to C57BL / 6 mice via the tail vein (mRNA amount: 1.0 μg).

[0431] Twenty-one hours after administration, the mice were euthanized and the spleens were removed. The resulting spleens were collected in a petri dish containing 3 mL of RPMI-1640 with L-Gln liquid (Nacalai Tesque, Inc.) (1% by weight fetal bovine serum (Sigma)). Spleen cells were strained using tweezers, and the cells were suspended in spleen cell medium using a 5 mL syringe. The resulting cell suspension was passed through a 70 μL cell strainer and collected in a 15 mL tube. The collected cells were centrifuged at 4°C and 500 g for 4 minutes, and the supernatant was removed to obtain a cell pellet. The cell pellet was suspended in 1 mL of Red Blood Cell Lysing Buffer, and the cell suspension was incubated at room temperature for 5 minutes to remove red blood cells. The cell suspension after removal of red blood cells was diluted 5-fold with FACS buffer (PBS containing 0.05% by weight of sodium azide aqueous solution and 0.5% by weight of fetal bovine serum albumin). The resulting dilution was centrifuged, and the supernatant was removed. 5 mL of FACS buffer was added to the cell pellet, and the mixture was centrifuged and the supernatant was removed. The resulting cell pellet was suspended in 3 mL of FACS buffer. The resulting cell suspension was diluted 10-fold with FACS buffer. 18 μL of the diluted cell suspension was mixed with 2 μL of acridine orange, and the resulting mixture was used for cell counting using a LUNA-FL (Logos Biosystems). When the concentration of the suspended cells was 1 x 10 ６FACS buffer was added to the cell suspension to a concentration of 1000 cells / mL. The cell suspension was then centrifuged at 4°C and 500xg for 4 minutes, and the supernatant was removed to obtain a cell pellet. To prevent nonspecific staining, a blocking solution was prepared by mixing 2 μL of CD16 / 32 with 98 μL of FACS buffer. 50 μL of blocking solution was added to the cell pellet and the mixture was stirred to obtain a cell suspension. The resulting cell suspension was then incubated at 4°C for 10 minutes in the dark. BV605-labeled anti-CD45 antibody, BV421-labeled anti-CD3ε antibody, BV510-labeled anti-CD19 antibody, PE-labeled anti-CD4 antibody, and AF488-labeled anti-CD8a antibody were diluted with blocking solution to the manufacturer's recommended concentrations to obtain an antibody cocktail for staining. 54 μL of the antibody cocktail for staining was added to 50 μL of the cell suspension, and the resulting mixture was incubated at 4°C for 30 minutes in the dark. Five minutes before observation, 5 μL of 7-AAD Viability Staining Solution (BioLegend) was added to each mixture, and dead cell staining was performed. 1 mL of FACS buffer was added to the cell suspension containing the antibody cocktail and 7-AAD, and the centrifugation and supernatant removal procedures were repeated twice. The resulting cell pellet was suspended in 500 μL of FACS buffer. The resulting suspension (sample) was analyzed using Novocyte, and the percentage of DiD-positive T cells (%) (= 100 × number of T cells exhibiting DiD fluorescence / total number of T cells) was calculated. Here, T cells exhibiting DiD fluorescence were T cells that had taken up LNP. Specifically, the group of live cells in the cell suspension that were stained with BV421-labeled anti-CD3ε antibody was defined as T cells, and the percentage of T cells that exhibited DiD fluorescence (i.e., T cells that had taken up LNP) relative to the total number of T cells was calculated. The results are shown in Table 14 below.

[0432]

[0433] As shown in Table 14, the ligand-modified LNPs encapsulating the mRNA of Example 13 showed an improved percentage of DiD-positive T cells (%) compared to the LNPs encapsulating the mRNA of Comparative Example 3 but not modified with a ligand.

[0434] Example 14 Preparation of Ligand-Modified LNP Encapsulating mRNA Step a) and exchange of the dispersion medium were carried out in the same manner as in Preparation Example 5, except that 1 mM 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (fluorescent dye) was added to the lipid solution of Preparation Example 5 to prepare a lipid solution containing a fluorescent dye in which the molar ratio of SS-OP / cholesterol / 1,2-dioleoyl-sn-glycero-3-phosphocholine / DMG-PEG2000 / 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate was 52.5 / 40 / 7.5 / 1.5 / 0.5, and that the lipid solution containing the fluorescent dye was used instead of the lipid solution of Preparation Example 5, and an empty LNP suspension with the exchanged dispersion medium was prepared.

[0435] Using a nanoparticle manufacturing device "NanoAssembler Ignite" (manufactured by Precision NanoSystems), the empty LNP suspension after the dispersion medium exchange was mixed with the luciferase-encoding mRNA solution at a flow rate of 5 mL / min for 55 seconds at 25°C to obtain a 9.2 mL suspension (9200 μL, mRNA amount: 184 μg). This solution was divided into 4.6 mL aliquots, which were either left unincubated or incubated at 37°C for 5 minutes to prepare mRNA-encapsulated LNP suspensions (N / P ratio: 36, mRNA concentration: 20 μg / mL) (step b)).

[0436] Using a nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems), the obtained mRNA-encapsulated LNP suspension was mixed at a flow rate of 0.5 mL / min with an aqueous solution containing the ligand-bound lipid PEG and ligand-free PEG lipid obtained in Preparation Example 2 at a flow rate of 0.5 mL / min at 25 ° C. for 60 seconds, or the obtained mRNA-encapsulated LNP suspension was mixed at a flow rate of 5 mL / min with an aqueous solution containing the ligand-bound lipid PEG and ligand-free PEG lipid obtained in Preparation Example 2 at a flow rate of 5 mL / min at 25 ° C. for 6 seconds to obtain a 1 mL suspension. This suspension was divided into 0.5 mL aliquots and incubated either unincubated or at 4 ° C. for 30 minutes. To this solution, phosphate buffer (0.5 mL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare a ligand-modified LNP suspension encapsulating mRNA (amount of ligand relative to total lipids: 0.1 mol%, 0.2 mol%, or 0.4 mol%) (step c)).

[0437] Test Example 13: Evaluation of LNP particle size, PdI, mRNA encapsulation rate, etc. The particle size, PdI, and mRNA encapsulation rate of the ligand-modified LNP encapsulating mRNA of Example 14 were measured in the same manner as Test Example 1. The results are shown in Table 15 below. Table 15 also lists the incubation treatments in steps b) and c), the flow rate of the LNP suspension encapsulating mRNA in step c), and the flow rate of the aqueous solution containing the ligand-bound lipid PEG and the PEG lipid to which no ligand is bound. The two flow rates are listed together in the "Flow rate in step c)" column in Table 15 below. The "Amount of ligand (anti-CD3 antibody) relative to total lipid" is also listed in the "Ligand amount" column in Table 15 below.

[0438]

[0439] The lipid nanoparticles encapsulating nucleic acids obtained by the production method of the present invention are useful for nucleic acid medicines, gene therapy, biochemical experiments, and the like.

[0440] This application is based on Japanese Patent Application No. 2023-159114, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing ligand-modified lipid nanoparticles encapsulating nucleic acid, comprising: a) mixing an alcohol solution containing an ionic lipid, a sterol, and a PEG lipid with an acidic buffer solution having a pH of 1 to 6.5 to obtain a suspension of lipid nanoparticles containing no nucleic acid; and b) mixing the lipid nanoparticles containing no nucleic acid with a nucleic acid solution to obtain a suspension of lipid nanoparticles containing nucleic acid; and c) mixing the suspension of lipid nanoparticles containing nucleic acid with a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles containing nucleic acid; or c') mixing the lipid nanoparticles containing no nucleic acid with water and a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles containing no nucleic acid; and b') mixing the ligand-modified lipid nanoparticles containing no nucleic acid with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles containing nucleic acid; or d) mixing the lipid nanoparticles containing no nucleic acid with a nucleic acid solution and a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles containing nucleic acid.

2. The method according to claim 1, wherein in step b), a suspension of lipid nanoparticles not containing nucleic acid is mixed with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulating nucleic acid; or in step c'), a suspension of lipid nanoparticles not containing nucleic acid is mixed with a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles not containing nucleic acid, and in step b'), a suspension of ligand-modified lipid nanoparticles not containing nucleic acid is mixed with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid; or in step d), a suspension of lipid nanoparticles not containing nucleic acid is mixed with a nucleic acid solution and a ligand-bound lipid to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.

3. The method according to claim 2, comprising steps a), b) and c), or comprising steps a), c') and b').

4. The method according to any one of claims 1 to 3, comprising maintaining a mixture of the nucleic acid-free lipid nanoparticles of step b) and a nucleic acid solution, or a mixture of the nucleic acid-free ligand-modified lipid nanoparticles of step b') and a nucleic acid solution at 0 to 95°C for 1 minute to 2 hours.

5. The method according to any one of claims 1 to 3, comprising maintaining a mixture of the suspension of lipid nanoparticles encapsulating nucleic acid (step c) and ligand-conjugated lipid, or a mixture of lipid nanoparticles not containing nucleic acid (step c'), water and ligand-conjugated lipid, at 0 to 95°C for 1 minute to 5 hours.

6. The method according to claim 1 or 2, comprising maintaining the mixture of the nucleic acid-free lipid nanoparticles, the nucleic acid solution and the ligand-bound lipids in step d) at 0 to 95°C for 1 minute to 2 hours.

7. The method according to claim 1 or 2, wherein in step c), a suspension of lipid nanoparticles encapsulating nucleic acid is mixed with a ligand-bound lipid and a lipid to which no ligand is bound to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid; or in step c'), lipid nanoparticles not containing nucleic acid are mixed with water, a ligand-bound lipid and a lipid to which no ligand is bound to obtain a suspension of ligand-modified lipid nanoparticles not containing nucleic acid; or in step d), lipid nanoparticles not containing nucleic acid are mixed with a nucleic acid solution, a ligand-bound lipid and a lipid to which no ligand is bound to obtain a suspension of ligand-modified lipid nanoparticles encapsulating nucleic acid.

8. The method according to any one of claims 1 to 3, wherein the alcoholic solution in step a) further comprises a phospholipid.

9. The ionic lipid has the formula (1): (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms; ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate 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, at least one aromatic ring, and optionally having a heteroatom; ａ and n ｂ are each independently 0 or 1; ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, ９ -O-CO-(CH ２ ) ａ -* (3) (In formula (3), * represents a bond position, R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10. (In formula (4), * represents a 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 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), * represents a 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 alkynediyl 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; R １２ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; １３ At least one ethylene group or at least one trimethylene group in may be replaced with 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 each independently 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, and X ４ represents an oxygen atom, NH, or a sulfur atom.) represents a group having 50 or less carbon atoms and represented by the following formula: (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate. The method according to any one of claims 1 to 3, comprising at least one selected from the group consisting of a compound represented by the following formula:

10. The method of claim 9, wherein the ionic lipid comprises a compound represented by formula (1).

11. The method according to any one of claims 1 to 3, wherein the ligand in the ligand-binding lipid is a ligand having brain cell tropism or a ligand having T cell tropism.

12. A method for introducing a nucleic acid into a cell, comprising a step of contacting the cell ex vivo with the ligand-modified lipid nanoparticles encapsulating the nucleic acid obtained by the method according to any one of claims 1 to 3.

13. A method for introducing a nucleic acid into a target cell in a living organism, comprising the step of administering to the living organism ligand-modified lipid nanoparticles encapsulating the nucleic acid obtained by the method according to any one of claims 1 to 3.

14. A method for producing a pharmaceutical composition comprising the method according to any one of claims 1 to 3.

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