Lipid nanoparticles for delivering nucleic acid to peripheral blood mononuclear cells, and method for delivering nucleic acid to peripheral blood mononuclear cells using same

Abstract

The present invention provides lipid nanoparticles for delivering nucleic acid to peripheral blood mononuclear cells, and a method for delivering nucleic acid to peripheral blood mononuclear cells using the same, with which the efficiency of delivering nucleic acid to peripheral blood mononuclear cells can be improved. The present invention provides lipid nanoparticles used for delivering a nucleic acid to peripheral blood mononuclear cells, said nucleic acid containing: an ionic lipid represented by formula (1); the phospholipid 1,2-diacyl-sn-glycero-3-phosphocholine, in which the acyl groups have 20 or more carbons and at least one of the acyl groups is an alkenoyl group; cholesterol; and dimyristoylglycerol PEG represented by the formula CH2(OR6)–CH(OR7)–CH2(OR8) (the symbols in the formula are as defined in the specification). The present invention further provides a method for delivering nucleic acid to peripheral blood mononuclear cells using said lipid nanoparticles.

Description

Lipid nanoparticles for delivering nucleic acids to peripheral blood mononuclear cells and a method for delivering nucleic acids to peripheral blood mononuclear cells using the same

[0001] The present invention relates to lipid nanoparticles used for delivering nucleic acids to peripheral blood mononuclear cells, and methods for using the same to deliver nucleic acids to peripheral blood mononuclear cells.

[0002] Effective and safe nucleic acid delivery carriers are required for the practical application of 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.

[0003] In addition to viral vectors, electroporation has been developed as a method for in vitro nucleic acid delivery. Because electroporation involves physically introducing nucleic acids by creating holes in cells, it poses challenges in terms of cellular stress and cytotoxicity. Meanwhile, lipid nanoparticles using ionic lipids have been developed as low-toxicity nucleic acid delivery carriers that can be used both in vitro and in vivo.

[0004] Ionic lipids are broadly composed of an amine moiety and a lipid moiety. For example, the amine moiety, which protonates under acidic conditions, interacts electrostatically with nucleic acids, which are polyanions, to form lipid nanoparticles, which promote cellular uptake and deliver nucleic acids into cells.

[0005] A well-known and widely used ionic lipid is 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP). It is known that by combining such a known ionic lipid with a phospholipid, cholesterol, and a PEG lipid, lipid nanoparticles can be formed, and nucleic acids can be delivered into cells (see Non-Patent Document 1).

[0006] For example, 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 connected by a biodegradable disulfide bond. Patent Document 1 shows that the ionic lipid improves in vivo kinetics such as blood stability and tumor targeting. Furthermore, 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 within cells, and it has also been shown that the cleavage of disulfide bonds within cells has the effect of dissociating nucleic acids from the lipid membrane structure. In fact, compared to the known ionic lipid DODAP, it exhibits a higher nucleic acid delivery efficiency, revealing that the ionic lipid can improve intracellular kinetics, such as improving the efficiency of delivery of nucleic acids into the cytoplasm.

[0007] For example, 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 delivery to the cytoplasm.

[0008] As mentioned above, ionic lipids with improved intracellular dynamics have been developed by increasing endosomal escape efficiency and membrane fusion ability, and it has been shown that they can efficiently deliver nucleic acids into established cell lines and living organisms.

[0009] US Patent Application Publication No. 2014 / 0335157 WO 2019 / 188867 WO 2020 / 039631

[0010] Molecular Therapy, 25(7): 1467-1475 (2017)Molecular Therapy, 26(6): 1509-1519 (2018)Adv. Funct. Mater., 30: 1910575 (2020)Gene Therapy, 23: 699-707 (2016)Nanomedicine: NBM, 13: 1377-1387 (2017)

[0011] Efficient nucleic acid delivery to blood cells such as peripheral blood mononuclear cells (PBMCs) is important in the fields of cancer immunotherapy, such as CAR-T therapy, and regenerative medicine, such as the generation and differentiation of iPS cells. However, because the efficiency of nucleic acid delivery to peripheral blood mononuclear cells, such as T cells extracted from the body, is insufficient using lipid nanoparticles made of ionic lipids, development using viral vectors and electroporation methods remains ahead of the curve.

[0012] Therefore, in order to more efficiently introduce nucleic acids into lipid nanoparticles, further improvement in the efficiency of nucleic acid delivery to peripheral blood mononuclear cells is required. For example, Patent Document 3 shows that the efficiency of nucleic acid delivery to peripheral blood mononuclear cells can be increased by making the surface of lipid nanoparticles cationic under physiological pH conditions.

[0013] However, since compounds that are cationic under physiological pH conditions generally have high cytotoxicity, it is desirable to use neutral lipid nanoparticles with low cytotoxicity for practical use.

[0014] In this regard, Non-Patent Documents 2, 3, and 4 each disclose the use of ionic lipids with different structural features for lipid nanoparticles for liver delivery. In these documents, different phospholipids are used as components of lipid nanoparticles, and the optimal combination with the phospholipid varies depending on the structure of the ionic lipid.

[0015] On the other hand, Non-Patent Document 5 shows an example in which the appropriate phospholipid varies depending on the cell type to be transfected, even when lipid nanoparticles use the same ionic lipid.

[0016] As mentioned above, there are examples of using lipid nanoparticles to improve the efficiency of nucleic acid transfer into peripheral blood mononuclear cells. However, because the appropriate phospholipid varies depending on the structure of the ionic lipid, this is not fully satisfactory for practical use, and further improvements in nucleic acid delivery efficiency are required.

[0017] In view of the above problems, the present invention aims to provide lipid nanoparticles for delivering nucleic acids to peripheral blood mononuclear cells, which can improve the efficiency of nucleic acid delivery to peripheral blood mononuclear cells, and a method for delivering nucleic acids to peripheral blood mononuclear cells using the same.

[0018] As mentioned above, the appropriate combination of ionic lipids and phospholipids in lipid nanoparticles varies depending on the cell type, and efficient nucleic acid delivery requires optimizing the combination of ionic lipids and phospholipids.

[0019] In view of the above problems, the inventors conducted extensive research and found that neutral lipid nanoparticles produced using an ionic lipid that has a pKa suitable for endosomal escape and that decomposes specifically in the reductive environment within a cell, and a specific 1,-diacyl-sn-glycero-3-phosphocholine as a phospholipid, can improve the efficiency of nucleic acid delivery to peripheral blood mononuclear cells. The present invention, based on this finding, is as follows.

[0020] [1] Formula (1):

[0021]

[0022] (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 nｂ 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, or a group represented by the formula (4): R ９ —O—CO—(CH ２ )a- (4) (In formula (4), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10. ) represents a group represented by the formula (2): ) an ionic lipid represented by the formula (2): a phospholipid having an acyl group of 20 or more carbon atoms and at least one of the acyl groups being 1,2-diacyl-sn-glycero-3-phosphocholine which is an alkenoyl group; cholesterol; and ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (2) (In formula (2), R ６ , R ７ and R ８ wherein any two of the groups represent myristoyl groups, and the remaining group represents an alkyl group having 1 to 6 carbon atoms linked via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000.

[0023] [2] The lipid nanoparticles according to [1], wherein the phospholipid is at least one selected from the group consisting of 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), and 1,2-dinervoyl-sn-glycero-3-phosphocholine (DNPC).

[0024] [3] The ionic lipid represented by formula (1) is represented by the following formula:

[0025]

[0026] The lipid nanoparticle according to [1] or [2], wherein the lipid nanoparticle is an ionic lipid represented by the formula:

[0027] [4] The ionic lipid is 20 to 60 mol%, the phospholipid is 5 to 20 mol%, the cholesterol is 30 to 70 mol%, and the dimyristoylglycerol PEG lipid is 0.5 to 1.5 mol% relative to the total of the ionic lipid, the phospholipid, and the cholesterol. [1] to [3] Any one of the lipid nanoparticles described.

[0028] [5] A method for delivering nucleic acid to peripheral blood mononuclear cells, comprising contacting the lipid nanoparticles according to any one of [1] to [4], which contain nucleic acid, with peripheral blood mononuclear cells.

[0029] [6] Formula (1):

[0030]

[0031] (wherein each symbol is as defined in [1]), an ionic lipid represented by the formula (1): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (2) (In formula (2), each symbol is as defined in [1].) Use of lipid nanoparticles containing dimyristoylglycerol PEG represented by the following formula for producing a pharmaceutical used for delivering nucleic acids to peripheral blood mononuclear cells.

[0032] [7] The use according to [6], wherein the phospholipid is at least one selected from the group consisting of 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), and 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC).

[0033] [8] The ionic lipid represented by formula (1) is represented by the following formula:

[0034]

[0035] The use according to [6] or [7], wherein the ionic lipid is represented by the formula:

[0036] [9] The use according to any one of [6] to [8], wherein the ionic lipid is 20 to 60 mol%, the phospholipid is 5 to 20 mol%, the cholesterol is 30 to 70 mol%, and the dimyristoylglycerol PEG lipid is 0.5 to 1.5 mol% relative to the total of the ionic lipid, the phospholipid, and the cholesterol.

[0037] The lipid nanoparticles of the present invention can efficiently deliver nucleic acids to peripheral blood mononuclear cells by optimizing the combination of ionic lipids and phospholipids and the lipid composition.

[0038] FIG. 1 shows the gene expression activity in mouse primary T cells of the lipid nanoparticles of Example 1, Comparative Example 1, and Comparative Example 2. FIG. 2 shows the amount of uptake into mouse primary T cells of the lipid nanoparticles of Example 2, Comparative Example 3, and Comparative Example 4. FIG. 3 shows the gene expression activity in mouse primary T cells of the lipid nanoparticles of Examples 1 and 3 to 8, and Comparative Example 1. FIG. 4 shows the gene expression activity in mouse primary T cells of the lipid nanoparticles of Examples 6, 7, and 9 to 14, and Comparative Example 1. FIG. 5 shows the gene expression activity in mouse primary T cells of the lipid nanoparticles of Examples 6, 15 to 18, and Comparative Example 1. FIG. 6 shows the gene expression activity in human primary T cells of the lipid nanoparticles of Example 6, Comparative Example 5, and Comparative Example 6.

[0039] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention relates to lipid nanoparticles comprising an ionic lipid represented by formula (1) (i.e., an ionic lipid having a tertiary amino group, a lipid moiety, and a disulfide bond as a biodegradable group), a phospholipid which is 1,2-diacyl-sn-glycero-3-phosphocholine in which the acyl group has 20 or more carbon atoms and at least one of the acyl groups is an alkenoyl group, cholesterol, and dimyristoylglycerol PEG represented by formula (2), and a method for delivering nucleic acids to peripheral blood mononuclear cells using the same.

[0040] Lipid Nanoparticles In this specification, "lipid nanoparticles" (sometimes abbreviated as "LNPs" in this specification) refer to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are aligned toward the aqueous phase side of the interface, and having a particle diameter of less than 1 μm, and "amphipathic lipid" refers to a lipid having both a hydrophilic group and a hydrophobic group.

[0041] The particle size of the lipid nanoparticles of the present invention 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). The particle size of the lipid nanoparticles can be appropriately adjusted depending on the method for producing the lipid nanoparticles. In this specification, "particle size" refers to the average particle size (zeta mean) measured by dynamic light scattering.

[0042] Examples of amphipathic lipids include ionic lipids, phospholipids, and PEG lipids. In this specification, "PEG" refers to polyethylene glycol, "PEG lipid" refers to a lipid modified with PEG, and "Y modified with X" (e.g., X:PEG, Y:lipid) refers to Y bound to X. In other words, "PEG lipid" refers to a lipid bound to PEG.

[0043] The lipid nanoparticles of the present invention may contain lipids other than the ionic lipid represented by formula (1), phospholipids, cholesterol, and dimyristoylglycerol PEG represented by formula (2) (hereinafter referred to as "other lipids"). Examples of other lipids include sterols other than cholesterol and PEG lipids other than dimyristoylglycerol PEG represented by formula (2).

[0044] The amount of other lipids in the lipid nanoparticles of the present invention is preferably 0 to 50 mol%, more preferably 0 to 30 mol%, and even more preferably 0 to 10 mol% relative to the total amount of lipids in the lipid nanoparticles. Here, "total amount of lipids in lipid nanoparticles" refers to, for example, when the lipid nanoparticles contain an ionic lipid represented by formula (1), a phospholipid, cholesterol, and a dimyristoylglycerol PEG represented by formula (2) and other lipids as constituent components, the total amount of the ionic lipid represented by formula (1), the phospholipid, cholesterol, and the dimyristoylglycerol PEG represented by formula (2) and other lipids. Furthermore, in this specification, "the amount of B relative to A (mol%)" means "100 × the amount of B (mol) / the amount of A (mol)." For example, "the amount of other lipids relative to the total amount of lipids (mol%)" means "100 × the amount of other lipids (mol) / the total amount of lipids (mol)."

[0045] It is most preferable that no other lipids are used in the present invention, i.e., the lipids constituting the lipid nanoparticles of the present invention consist of an ionic lipid represented by formula (1), a phospholipid, cholesterol, and dimyristoylglycerol PEG represented by formula (2).

[0046] Ionic Lipid The ionic lipid used in the present invention is an ionic lipid represented by the following formula (1) (sometimes abbreviated as "ionic lipid (1)" in this specification). Only one type of ionic lipid (1) may be used, or two or more types may be used in combination.

[0047]

[0048] (In formula (1), R １ａ and R １ｂeach 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 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, or a group represented by the formula (4): R ９ —O—CO—(CH ２ )a- (4) (In formula (4), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.

[0049] R １ａ and R １ｂare 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.

[0050] 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

[0051] X 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, and preferably each independently represents a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups.

[0052] 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.

[0053] 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 as follows.

[0054]

[0055] X １ R ５ represents 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.

[0056] 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 an aziridylene group, an azetidylene group, a pyrrolidine group, a piperidylene group, an imidazolidylene group, and a piperaziylene group, preferably a pyrrolidine group, a piperidylene group, and a piperaziylene group, and most preferably a piperidylene group.

[0057] 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 as follows.

[0058]

[0059] X ２ In the formula, p is 1 or 2. When p is 1, X ２ is a pyrrolidine group, and when p is 2, X ２ is a piperidylene group. Preferably, p is 2.

[0060] 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 as follows.

[0061]

[0062] X ３ The w in is 1 or 2. When w is 1, X ３ is an imidazolidylene group, and when w is 2, X ３ is a piperazylene group.

[0063] 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

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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

[0068] 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 limited. ａ and Y ｂ is an ester bond, preferably -Z ａ -CO-O-R ２ａ - and -Z ｂ -CO-O-R ２ｂ - structure.

[0069] 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

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Z ａ and Z ｂ Preferred specific structures of Z １ Examples include:

[0074]

[0075] 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.

[0076] 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.

[0077] Z １ R ４is 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.

[0078] 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

[0079] n ａ and n ｂ are each independently 0 or 1.

[0080] n ａ is n ｂ may be the same as or different from, but preferably, n ａ is n ｂ is the same as

[0081] R ３ａand 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, or a group represented by the formula (4): R ９ —O—CO—(CH ２ )a- (4) (In formula (4), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.) Preferably, each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and most preferably, each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0082] The residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride is a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with a hydroxyl group of *-O-CO-CH ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure in which the hydroxyl group of a sterol derivative is replaced by *-O-CO-CH. * represents the bonding position with a fat-soluble vitamin. The residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride is a residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride. 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.

[0083] 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.

[0084] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and preferably cholesterol or cholestanol.

[0085] 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 even more 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, Tetradecenyl 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, i Examples include a cosadienyl group, a henicosadienyl group, a docosadienyl group, an octadecatrienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, a docosahexaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl 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.

[0086] 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.

[0087] R ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the formula (I) 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 in the alkyl group, 3 to 40, does not include the number of carbon atoms in the cyclopropane ring. The number of cyclopropane rings in the alkyl group is preferably one. ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the formula (5):

[0088]

[0089] (In formula (5), b and c are each independently 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 (5) include 7-(2-octylcyclopropyl)heptyl.

[0090] In formula (4), R９The aliphatic hydrocarbon group having 2 to 20 carbon atoms represented by the formula (I) 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 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 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 dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, and a heptadecenyl group. Examples of the alkyl group 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.

[0091] In formula (4), 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.

[0092] 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

[0093] In one embodiment of the present invention, R １ａ is R １ｂ is the same as X ａ is X ｂ is the same as R ２ａ is R ２ｂ is the same as 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

[0094] Suitable examples of the ionic lipid (1) include the following ionic lipids: [Ionic lipid (1-1)] 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 an acyclic 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., a piperidylene group); R ２ａ and R ２ｂare 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; 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 hetero atom (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; 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).

[0095] [Ionic lipid (1-2)] 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 an acyclic 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., a piperidylene 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, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; 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; 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).

[0096] [Ionic lipid (1-3)] 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 are each independently, X １ :

[0097]

[0098] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0099]

[0100] (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; ａ and Z ｂ are each independently, Z １ :

[0101]

[0102] (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; 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).

[0103] Specific examples of the ionic lipid (1) include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, and TS-P4C2.

[0104] Specific examples of the ionic lipid (1) include Lipid 1 to Lipid 20 described in WO 2021 / 195529 A2, and in particular, the following Lipid 1, Lipid 5, and Lipid 8.

[0105]

[0106]

[0107]

[0108] Among the specific examples of the ionic lipid (1), SS-OP is preferred. That is, the ionic lipid (1) is preferably an ionic lipid represented by the following formula:

[0109]

[0110] The amount of ionic lipid (1) in the lipid nanoparticles of the present invention is preferably 20 to 60 mol%, more preferably 25 to 55 mol%, and even more preferably 30 to 50 mol%, based on the total amount of ionic lipid (1), phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0111] The ionic lipid (1) can be produced by known methods (for example, the methods described in WO 2019 / 188867 A1, US 9708628 B2, and WO 2021 / 195529 A2).

[0112] The lipid nanoparticles of the present invention contain 1,2-diacyl-sn-glycero-3-phosphocholine (PC) as a phospholipid, in which the acyl group has 20 or more carbon atoms and at least one of the acyl groups is an alkenoyl group. Furthermore, the phospholipid may be the (PC) alone, or may be used in combination with the (PC) or other phospholipids. In this case, the ratio of the other phospholipids to the (PC) is not particularly limited as long as it is within a range in which the effects of the present invention can be exhibited.

[0113] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholines in which the acyl group has 20 or more carbon atoms and at least one of the acyl groups has an alkenoyl group include 1-arachidoyl-2-eicosenoyl-sn-glycero-3-phosphocholine (AEiPC), 1-eicosenoyl-2-arachidoyl-sn-glycero-3-phosphocholine (EiAPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC), 1-behenoyl-2-erucoyl-sn-glycero-3-phosphocholine (BEPC), 1-erucoyl-2-behenoyl-sn-glycero-3-phosphocholine (EBPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), Examples of such phospholipids include 1-lignoceroyl-2-nervonoyl-sn-glycero-3-phosphocholine (LiNPC), 1-nervonoyl-2-lignoceroyl-sn-glycero-3-phosphocholine (NLiPC), and 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC). Phospholipids may be referred to by their abbreviations herein. For example, 1,2-diacyl-sn-glycero-3-phosphocholine may be referred to as PC, and 1,2-dierucoyl-sn-glycero-3-phosphocholine may be referred to as DEPC. Furthermore, phospholipids may be referred to as C22:1PC, for example, based on the number of carbon atoms and degree of unsaturation of the fatty acid.

[0114] The phospholipid is preferably 1,2-diacyl-sn-glycero-3-phosphocholine, in which the acyl group has 20 or more carbon atoms and at least one of the acyl groups has one unsaturated bond; more preferably 1,2-diacyl-sn-glycero-3-phosphocholine, in which the acyl group has 20 or more carbon atoms and both acyl groups have one unsaturated bond; even more preferably at least one selected from the group consisting of DEiPC, DEPC, and DNPC, and most preferably DEPC. These phospholipids are thought to exhibit similar effects because they have similar acyl chain structures. The number of carbon atoms in the acyl group is the number of carbon atoms including the carbonyl carbon. The number of carbon atoms in the acyl group is preferably 20 to 26, more preferably 20 to 24.

[0115] The amount of phospholipid in the lipid nanoparticles of the present invention is preferably 5 to 20 mol%, more preferably 10 to 20 mol%, and even more preferably 10 to 15 mol%, based on the total amount of ionic lipid (1), phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0116] Cholesterol The lipid nanoparticles of the present invention contain cholesterol. The amount of cholesterol in the lipid nanoparticles of the present invention is preferably 30 to 70 mol %, more preferably 35 to 65 mol %, and even more preferably 40 to 60 mol %, based on the total amount of ionic lipid (1), phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0117] Dimyristoylglycerol PEG The lipid nanoparticles of the present invention are represented by the formula (2): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (2) (In formula (2), R ６ , R ７ and R ８wherein any two of the formulas represent myristoyl groups, and the remaining formula represents an alkyl group having 1 to 6 carbon atoms linked via a polyethylene glycol (PEG) chain having a number-average molecular weight of 1,000 to 3,000.

[0118] The number average molecular weight of the PEG chain in formula (2) is 1,000 to 3,000, and preferably 1,500 to 2,500. The number average molecular weight of the PEG used to form the PEG chain can be measured by gel permeation chromatography (GPC).

[0119] The alkyl group having 1 to 6 carbon atoms may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include 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 is preferred.

[0120] The amount of dimyristoylglycerol PEG (2) in the lipid nanoparticles of the present invention is preferably 0.25 to 2 mol%, more preferably 0.5 to 1.5 mol%, and even more preferably 0.5 to 1.0 mol%, based on the total amount of ionic lipid (1), phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0121] Optimal Composition The optimal molar ratio of ionic lipid (1):phospholipid:cholesterol:dimyristoylglycerol PEG (2) in the lipid nanoparticles of the present invention is 40:10:50:0.75.

[0122] Method for Producing Lipid Nanoparticles The lipid nanoparticles of the present invention can be produced by dispersing a lipid raw material containing an ionic lipid (1), a phospholipid, cholesterol, and dimyristoylglycerol PEG (2) in an appropriate dispersion medium (e.g., an aqueous dispersion medium or an alcoholic dispersion medium), and then performing an operation to induce organization as necessary.

[0123] Examples of the "operation to induce organization" for producing the lipid nanoparticles of the present invention include the ethanol dilution method using a microchannel or a vortex, simple hydration, ultrasonic treatment, heating, vortexing, ether injection, French press method, cholic acid method, Ca ２＋ Examples of known methods include fusion, freeze-thaw, and reverse-phase evaporation. The ethanol dilution method using a microchannel or vortex is preferred, and the ethanol dilution method using a microchannel is even more preferred. In the ethanol dilution method using a microchannel, a dispersion containing lipid nanoparticles can be produced by mixing an acidic buffer solution containing nucleic acid with an ethanol solution of lipids using, for example, NanoAssemblr (registered trademark) (Precision NanoSystems). The dispersion produced by this method contains lipid nanoparticles and a dispersion medium (an acidic buffer solution and ethanol). The dispersion medium (particularly ethanol) can be removed or replaced by ultrafiltration, dialysis, dilution, or other procedures.

[0124] The present invention also provides a method for delivering nucleic acid to peripheral blood mononuclear cells, which comprises contacting the lipid nanoparticles of the present invention containing nucleic acid with peripheral blood mononuclear cells of a subject. In this method, it is preferable to transfect the target cells with the lipid nanoparticles containing nucleic acid.

[0125] Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, any one of mono-, tri-, and tri-stranded nucleic acids can be used, but single- or double-stranded nucleic acids are preferred. Nucleic acids may be, for example, nucleotides having N-glycosides of purine or pyrimidine bases, oligomers having non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs)), or oligomers having special linkages (provided that the oligomers contain nucleotides having a configuration that allows base pairing or base attachment as found in DNA or RNA).

[0126] 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 naturally occurring nucleotides are replaced with an analogue, a nucleic acid having a modified nucleotide, 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 containing an intercurrent 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.

[0127] 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 of DNA include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc., and preferably plasmid DNA, cDNA, and antisense DNA, and more preferably plasmid DNA. Circular DNA such as plasmid DNA can also be digested with appropriate restriction enzymes and used as linear DNA.

[0128] 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 of RNA 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.

[0129] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0130] The nucleic acid used in the present invention is a nucleic acid used for in vitro / ex vivo gene transfer, specifically, a nucleic acid for gene transfer into primary cells extracted from a living body for use in CAR-T therapy, etc. More specifically, the nucleic acid of the present invention is preferably a nucleic acid having preventive and / or therapeutic activity in patients with a specific disease (a so-called prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy and cell therapy.

[0131] Examples of nucleic acids used in cell therapy include nucleic acids encoding chimeric antigen receptors (CARs) and T cell receptors (TCRs). Nucleic acids encoding CARs used in cell therapy include an antigen-binding domain of an antibody capable of specifically recognizing a surface antigen to be recognized by a target immune cell, an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain. Nucleic acids encoding TCRs used in cell therapy are nucleic acids encoding the α chain and β chain of a TCR capable of specifically recognizing a surface antigen to be recognized by a target T cell. Types of nucleic acids encoding CARs and TCRs include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids.

[0132] The particle size of the lipid nanoparticles encapsulating nucleic acid is not particularly limited, but is preferably 10 nm to 500 nm, and more preferably 30 nm to 300 nm. Measurement of the particle size can be performed using a particle size distribution analyzer such as Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles encapsulating nucleic acid can be adjusted appropriately depending on the manufacturing method.

[0133] The surface potential (zeta potential) of lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably −15 to +15 mV, more preferably −10 to +10 mV. Previous gene transfer techniques primarily used positively charged particles. This is useful as a method for promoting electrostatic interactions with negatively charged heparin sulfate on cell surfaces and facilitating cellular uptake. However, a positive surface potential can result in (a) inhibition of nucleic acid release from the carrier due to intracellular interaction with the delivered nucleic acid, and (b) inhibition of protein synthesis due to interaction between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface potential (zeta potential) within the above range. Measurement of the surface potential (zeta potential) can be performed using a zeta potential measuring device such as a Zetasizer Nano. The surface potential (zeta potential) of lipid nanoparticles can be adjusted by the composition of the lipid nanoparticle's components.

[0134] By contacting peripheral blood mononuclear cells with the lipid nanoparticles of the present invention encapsulating nucleic acids, the lipid nanoparticles are introduced into the peripheral blood mononuclear cells, and the nucleic acids encapsulated in the lipid nanoparticles are delivered to the peripheral blood mononuclear cells. Peripheral blood mononuclear cells are a general term for mononuclear cells isolated from peripheral blood. The peripheral blood mononuclear cells to which the lipid nanoparticles are introduced are not particularly limited as long as they are contained in peripheral blood, and examples thereof include T cells, B cells, NK cells, dendritic cells, macrophages, and monocytes. The target cells are also not particularly limited, and examples include cells from mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), and fish (e.g., zebrafish, medaka, etc.). The target cells to which the lipid nanoparticles are introduced are preferably human or other mammalian cells.

[0135] The method for introducing nucleic acid-encapsulated lipid nanoparticles into target cells is not particularly limited as long as the lipid nanoparticles can deliver nucleic acid into the cells, and any known method (e.g., transfection, reverse transfection, etc.) can be appropriately selected. Transfection is preferred as a method for introducing nucleic acid into target cells using the lipid nanoparticles of the present invention. The amount of lipid nanoparticles used can be appropriately selected taking into account the type of target cells, the introduction method, etc.

[0136] The step of contacting lipid nanoparticles encapsulating nucleic acids with peripheral blood mononuclear cells in vitro (in vitro / ex vivo) will be specifically described below.

[0137] Peripheral blood mononuclear cells are suspended in an appropriate medium several days before contact with the lipid nanoparticles and cultured under appropriate conditions. Activating factors (e.g., anti-CD3 antibody / anti-CD28 antibody in the case of T cells) and growth factors (e.g., IL-2) may or may not be added during culture. The cells may or may not be in a proliferation phase when contacted with the lipid nanoparticles.

[0138] 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 excessive proteins such as serum, there is a possibility that contact between the lipid nanoparticles and the cells may be inhibited.

[0139] The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the type of peripheral blood mononuclear cells, etc., but is usually 1 x 10 ４ ~1 x 10 ７ cells / mL range.

[0140] To the cells prepared in this manner, for example, a suspension of lipid nanoparticles encapsulating the nucleic acid described above 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 lipid nanoparticles 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, but the lipid concentration is usually 1 to 300 nmol / mL, preferably 10 to 200 nmol / mL, and the nucleic acid concentration is usually 0.01 to 100 μg / mL, preferably 0.05 to 10 μg / mL.

[0141] The above suspension is added to peripheral blood mononuclear cells, and the cells are then cultured. ２ The concentration and other factors are appropriately determined taking into consideration the type of peripheral blood mononuclear cells. When the cells are mammalian-derived cells, the temperature is usually about 37°C, the humidity is about 95%, and the CO ２ The concentration is about 5%. The incubation time can also be appropriately set taking into consideration conditions such as the type of peripheral blood mononuclear cells used, but is usually in the range of 0.1 to 120 hours, preferably 0.2 to 96 hours, and more preferably 0.5 to 72 hours. If the incubation time is too short, the nucleic acid may not be sufficiently introduced into the cells, whereas if the incubation time is too long, the cells may become weak.

[0142] The nucleic acid is introduced into the peripheral blood mononuclear 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 derived from a mammal, the fresh medium preferably contains serum or nutrient factors.

[0143] The lipid nanoparticles of the present invention can be prepared as is or in admixture with a pharmaceutically acceptable carrier.

[0144] As the pharmaceutically acceptable carrier, those commonly used as pharmaceutical ingredients can be used, such as excipients, lubricants, solvents, solubilizers, suspending agents, isotonicity agents, buffers, etc. Furthermore, pharmaceutical additives such as preservatives, antioxidants, coloring agents, sweeteners, etc. can also be used as needed.

[0145] 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 in any way.

[0146] In the following examples, the ionic lipid (1) is shown by the name shown in the above table. The abbreviations used in the following examples have the following meanings: Chol: cholesterol; DEPC: 1,2-dierucoyl-sn-glycero-3-phosphocholine; DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; DiD: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate; DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (number average molecular weight (Mn) of PEG: 2000); MES: 2-morpholinoethanesulfonic acid; PBS: phosphate buffered saline; DDW: deionized distilled water. The mol % of the lipid composition indicates the mol % relative to the total of the ionic lipid (1), phospholipid, and cholesterol.

[0147] [Manufacturing Example 1] Preparation of mRNA-encapsulated LNPs (1) Preparation of lipid ethanol solution The lipid ethanol solution was prepared by mixing 10 mM SS-OP, 5 mM phospholipid, and 10 mM Chol to a final concentration of 800 nmol, adding 1 mM DMG-PEG2000 ethanol solution to the desired ratio, and adding ethanol to bring the total volume to 360 μL.

[0148] (2) Preparation of Acidic Buffer Solution of Nucleic Acid The acidic buffer solution of nucleic acid was prepared by mixing mRNA encoding the luciferase gene with 20 mM acidic malic acid buffer (pH 3.0) containing 30 mM NaCl to give a concentration of 0.0067 μg / μL.

[0149] (3) Preparation of LNP by Ethanol Dilution Method Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), 1000 μL of nucleic acid acidic buffer solution and 360 μL of lipid ethanol solution were mixed at flow rates of 3 mL / min and 1 mL / min, respectively, to obtain 0.8 mL of LNP solution. The obtained LNP was diluted with 3 mL of 20 mM MES buffer (pH 6.5) and transferred to an Amicon Ultra 4 (Millipore). The transferred LNP solution was subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, approximately 10 minutes) and concentrated to approximately 500 μL. The resulting concentrate was diluted to 4 mL with PBS and then concentrated to approximately 250 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, approximately 10 minutes). Finally, the concentrate was diluted to a volume of 10 μg / mL with PBS.

[0150] [Test Example 1] Lipid composition of various mRNA-encapsulated LNPs Using the method described in Production Example 1, mRNA-encapsulated LNPs with the lipid composition shown in Table 2 below were prepared.

[0151]

[0152] Test Example 2: Evaluation of gene expression activity in mouse primary T cells (1) Isolation of mouse primary T cells The abdominal cavity of a C57BL / 6JJcl mouse (Japan SLC) was incised to collect the spleen, which was then crushed by rubbing a slide glass on PBS to separate the spleen cells. The cell suspension was passed through a 100 μm filter (MACS SmartStrainers (100 μm); Miltenyi Biotec) and a 30 μm filter (MACS SmartStrainers (30 μm); Miltenyi Biotec), and centrifuged at 4°C at 400 g for 3 minutes to produce a cell pellet. The supernatant was removed, and the cells were suspended in 90 μL of MACS buffer (0.5% BSA, 2 mM EDTA in PBS). 10 μL of CD90.2 MicroBeads (Miltenyi Biotec) were added, and the cells were mixed by gentle tapping and allowed to stand on ice for 15 minutes. Then, 5 mL of MACS buffer was added, and the cells were centrifuged (4°C, 400 g, 3 minutes) to form a cell pellet. The supernatant was removed, and the cells were suspended in 500 μL of degassed MACS buffer. 500 μL of degassed MACS buffer was passed through a column (MS Columns; Miltenyi Biotec) in contact with a magnet, and the column was left standing until no more liquid was dripping. A cell suspension suspended in degassed MACS buffer was then added to the column and left standing. 500 μL of degassed MACS buffer was then passed through the column and left standing. This process was repeated two more times. The column was removed from the magnet, and 1 mL of degassed MACS buffer was added. Mouse primary T cells were recovered by pushing the suspension out with a piston. A cell pellet was prepared by centrifugation (4°C, 400 g, 3 minutes), and after removing the supernatant, the cells were suspended in 1 mL of RPMI 1640 (10% FBS, 1 mM pyruvic acid, 10 mM HEPES, 100 U / mL penicillin, 100 mg / mL streptomycin, 4.5 g / L glucose, 55 μM 2-mercaptoethanol, non-essential amino acids).

[0153] (2) Culture of mouse primary T cells 2 mL of PBS was added to a 6-well plate (ThermoFisher) and incubated with anti-CD3 antibody (Ultra-LEAF ＴＭPurified anti-mouse CD3ε antibody (BioLegend), anti-CD28 antibody (Ultra-LEAF ＴＭ Purified anti-mouse CD28 Antibody (BioLegend) was added to the cells at 10 μg / mL and 0.5 μg / mL, respectively, and the cells were left to stand for 3 hours. The supernatant was then removed, and 2 mL of PBS was added. The same procedure was repeated once more, and after removing the supernatant, 1.5 × 10 mouse primary T cells isolated in (1) were added. ６ Cells were seeded at a concentration of 1000 cells / mL in 2 mL of medium, and 50 U / mL IL-2 (IL-2, Human Recombinant; PeproTech) was added. After 72 hours, 2 mL of medium containing 50 U / mL IL-2 was added. After a further 48 hours, the cells were harvested and used in various experiments.

[0154] (3) Measurement of gene expression activity Cultured mouse primary T cells were transfected with the LNPs of Example 1, Comparative Example 1, and Comparative Example 2 so that the amount of nucleic acid was 0.4 μg. Furthermore, a D-luciferin potassium aqueous solution (prepared with DDW) was added so that the final luciferin concentration was 0.1 mM. After transfection, luminescence was measured for 100 hours using an incubation luminometer (Kronos Dio; ATTO), and the total luminescence was calculated as the gene expression level using AUC. The results are shown in Figure 1. The LNP of Example 1, which used DEPC as the phospholipid, showed improved gene expression activity in mouse primary T cells compared to Comparative Examples 1 and 2.

[0155] [Test Example 3] Evaluation of LNP uptake into mouse primary T cells (1) Preparation of fluorescently labeled LNP A lipid ethanol solution containing a fluorescent dye was prepared by adding an ethanol solution of DiD to the lipid ethanol solution described in Production Example 1 so that the amount was 0.2 mol% relative to the total lipid amount. Fluorescently labeled LNP was obtained by preparing LNP using this lipid ethanol solution according to the procedure described in Production Example 1.

[0156] (2) Evaluation of LNP uptake into mouse primary T cells Cultured mouse primary T cells were transfected with the LNPs of Example 2, Comparative Example 3, and Comparative Example 4, and the cells were collected 24, 48, and 72 hours later. The amount of LNP uptake was then evaluated using a flow cytometer. The results are shown in Figure 2. The LNP with the composition of Example 2 using DEPC showed increased uptake into primary T cells at all time points compared to the LNPs with the compositions of Comparative Examples 3 and 4.

[0157] Test Example 4: Effect of phospholipid composition ratio on gene expression activity in mouse primary T cells Cultured mouse primary T cells were transfected with the LNPs of Examples 1, 3-8, and Comparative Example 1 prepared in Production Example 1 so that the amount of nucleic acid was 0.4 μg. Furthermore, an aqueous solution of D-luciferin potassium was added so that the final luciferin concentration was 0.1 mM. After transfection, luminescence was measured for 100 hours using an incubation luminometer (Kronos Dio; ATTO), and the total luminescence was calculated as the gene expression level using AUC. The results are shown in Figure 3. The compositions of Examples 6 and 7, which used 10 or 15 mol% DEPC, showed high gene expression activity.

[0158] Test Example 5: Effect of Cholesterol Composition Ratio on Gene Expression Activity in Mouse Primary T Cells Cultured mouse primary T cells were transfected with the LNPs of Examples 6, 7, 9-14 and Comparative Example 1 prepared in Production Example 1 so that the amount of nucleic acid was 0.4 μg. Furthermore, an aqueous solution of D-luciferin potassium was added so that the final luciferin concentration was 0.1 mM. After transfection, luminescence was measured for 100 hours using an incubation luminometer (Kronos Dio; ATTO), and the total luminescence was calculated as the gene expression level using AUC. The results are shown in Figure 4. The composition of Example 6, which used 50 mol% cholesterol, showed the highest gene expression activity.

[0159] Test Example 6: Effect of DMG-PEG2000 Composition Ratio on Gene Expression Activity in Mouse Primary T Cells Cultured mouse primary T cells were transfected with the LNPs of Examples 6, 15-18, and Comparative Example 1 prepared in Production Example 1 so that the amount of nucleic acid was 0.4 μg. Furthermore, an aqueous solution of D-luciferin potassium was added so that the final luciferin concentration was 0.1 mM. After transfection, luminescence was measured for 100 hours using an incubation luminometer (Kronos Dio; ATTO), and the total luminescence was calculated as the gene expression level using AUC. The results are shown in Figure 5. The highest gene expression activity was observed under the conditions of Example 6, in which 0.75 mol% DMG-PEG2000 was used.

[0160] Test Example 7: Evaluation of gene expression activity in human primary T cells (1) Culture of human primary T cells Human peripheral blood CD3+ Pan T cells (purchased from Lonza) were thawed at 37°C. 9 mL of RPMI 1640 (10% FBS, 1 mM pyruvate, 10 mM HEPES, 100 U / mL penicillin, 100 mg / mL streptomycin, 4.5 g / L glucose, 55 μM 2-mercaptoethanol, non-essential amino acids) was added, and the cells were centrifuged under centrifugation conditions (4°C, 300 g, 10 minutes). The supernatant was removed, the cells were suspended in 10 mL of medium, and 1 mL of the solution was seeded into a 12-well plate (ThermoFisher) at a concentration of 1 x 106 cells / mL. Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) beads were added at a bead:T cell ratio of 1:2, and 30 U / mL IL-2 (PeproTech) was added. After 72 hours, the cells were split to halve the cell concentration, and after another 24 hours, 1 mL of medium containing 30 U / mL IL-2 was added. After 72 hours, the cells were collected, the Dynabeads were removed, and 2 × 10 ５ The cells were seeded at 1000 cells / mL and used in the experiment.

[0161] (2) Measurement of Gene Expression Activity Cultured human primary T cells were transfected with the LNPs of Example 6, Comparative Example 5, and Comparative Example 6 prepared in Production Example 1 so that the amount of nucleic acid was 0.4 μg. Furthermore, an aqueous solution of D-luciferin potassium was added so that the final luciferin concentration was 0.1 mM. After transfection, luminescence was measured for 40 hours using an incubation luminometer (Kronos Dio; ATTO), and the total luminescence was calculated as the gene expression level using AUC. The results are shown in Figure 6. In human primary T cells, the LNPs with the composition of Example 6 using DEPC also showed higher gene expression activity than Comparative Examples 5 and 6.

[0162] The lipid nanoparticles of the present invention are useful for delivering nucleic acids to peripheral blood mononuclear cells.

[0163] This application is based on patent application No. 2022-051919 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. Formula (1): 【Chemistry 1】 (In formula (1), R １ａ and R １ｂ Each of these independently represents an alkylene group with 1 to 6 carbon atoms. X ａ and X ｂ Each of these independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. R ２ａ and R ２ｂ Each of these independently represents an alkylene group or oxydialkylene group having 8 or fewer carbon atoms. Y ａ and Y ｂ Each of these independently represents an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom. n ａ and n ｂ Each of these is independently either 0 or 1, R ３ａ and R ３ｂ Each of these is independently a residue derived from a reaction product of a lipid-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 having a cyclopropane ring, or formula (4): R ９ -O-CO-(CH ２ )a- (4) (In formula (4), R ９ This represents an aliphatic hydrocarbon group with 2 to 20 carbon atoms. (where 'a' represents an integer between 2 and 10.) (This represents the base represented by [this symbol].) Ionic lipids represented by A phospholipid having 20 or more carbon atoms in the acyl group, and at least one of the acyl groups being an alkenoyl group 1,2-diacyl-sn-glycero-3-phosphocholine. Cholesterol, and Formula (2): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (2) (In formula (2), R ６ , R ７ and R ８ Two of these represent myristoyl groups, and the remaining one represents a C1-C6 alkyl group linked via a polyethylene glycol chain with a number-average molecular weight of 1,000-3,000. Dimyristoylglycerol PEG Lipid nanoparticles used to deliver nucleic acids containing these substances to peripheral blood mononuclear cells.

2. The lipid nanoparticle according to claim 1, wherein the phospholipid is at least one selected from the group consisting of 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC), 1,2-dielicoyl-sn-glycero-3-phosphocholine (DEPC), and 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC).

3. The ionic lipid represented by formula (1) is given by the following formula: 【Chemistry 2】 Lipid nanoparticles according to claim 1 or 2, which are ionic lipids represented by .

4. Lipid nanoparticles according to claim 1 or 2, wherein, relative to the total of the ionic lipid, phospholipid and cholesterol, the ionic lipid is 20 to 60 mol%, the phospholipid is 5 to 20 mol%, the cholesterol is 30 to 70 mol%, and the dimyristoylglycerol PEG lipid is 0.5 to 1.5 mol%.

5. A method for delivering nucleic acids to peripheral blood mononuclear cells, comprising contacting peripheral blood mononuclear cells with lipid nanoparticles according to claim 1 or 2 that contain nucleic acids.

6. Formula (1): 【Transformation 3】 (In formula (1), R １ａ and R １ｂ Each of these independently represents an alkylene group with 1 to 6 carbon atoms. X ａ and X ｂ Each of these independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. R ２ａ and R ２ｂ Each of these independently represents an alkylene group or oxydialkylene group having 8 or fewer carbon atoms. Y ａ and Y ｂ Each of these independently represents an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z ａ and Z ｂ Each of these independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, possessing at least one aromatic ring, and possibly having a heteroatom. n ａ and n ｂ Each of these is independently either 0 or 1, R ３ａ and R ３ｂ Each of these is independently a residue derived from a reaction product of a lipid-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 having a cyclopropane ring, or formula (4): R ９ -O-CO-(CH ２ )a- (4) (In formula (4), R ９ This represents an aliphatic hydrocarbon group with 2 to 20 carbon atoms. (where 'a' represents an integer between 2 and 10.) (This represents the base represented by [this symbol].) Ionic lipids represented by A phospholipid having 20 or more carbon atoms in the acyl group, and at least one of the acyl groups being an alkenoyl group 1,2-diacyl-sn-glycero-3-phosphocholine. Cholesterol, and Formula (2): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (2) (In formula (2), R ６ , R ７ and R ８ Two of these represent myristoyl groups, and the remaining one represents a C1-C6 alkyl group linked via a polyethylene glycol chain with a number-average molecular weight of 1,000-3,000. Dimyristoylglycerol PEG Use of lipid nanoparticles containing nucleic acids for the manufacture of pharmaceuticals used to deliver nucleic acids to peripheral blood mononuclear cells.

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