Asymmetric structured lipid

Abstract

The purpose of the present invention is to provide: lipid nanoparticles useful as a nucleic acid delivery carrier; and a novel pH-sensitive cationic lipid for producing the lipid nanoparticles. The problem is solved by: a pH-sensitive cationic lipid comprising an asymmetric hydrocarbon chain; and lipid nanoparticles comprising the pH-sensitive cationic lipid as a constituent lipid.

Description

Asymmetric lipid structure

[0001] The present invention relates to lipid nanoparticles useful as nucleic acid delivery carriers and pH-sensitive cationic lipids for producing such lipid nanoparticles.

[0002] Lipid nanoparticles (LNPs) are used as carriers for encapsulating lipid-soluble drugs, siRNA (short interfering RNA), mRNA, and other nucleic acids and delivering them to target cells. For example, lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids have been reported as lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids such as siRNA into target cells (Patent Document 1 and Non-Patent Document 1).

[0003] As a pH-sensitive cationic lipid, for example, Jayaraman et al. developed DLin-MC3-DMA and demonstrated that it had an EDTA effect in factor 7 (F7) knockdown in mouse liver. ５０ The present inventors have also developed their own pH-sensitive cationic lipids YSK05 and YSK13-C3, and have achieved EDTA of 0.005 mg siRNA / kg in F7 knockdown. ５０ In addition, Maier et al. have developed L319, which is a biodegradable version of MC3-DMA, and have achieved EDTA of 0.06 and 0.015 mg siRNA / kg, respectively (Non-Patent Documents 3 to 5). ５０ It has been reported that the bioavailability of lipid nanoparticles containing pH-sensitive cationic lipids is as high as 0.01 mg siRNA / kg, while maintaining high safety (Non-Patent Documents 6 to 8). However, it has been revealed that the endosomal escape efficiency of lipid nanoparticles containing these pH-sensitive cationic lipids is still only about several percent (Non-Patent Document 9), and the development of a technology that can further improve bioavailability is desired.

[0004] Furthermore, Dong et al. discovered a unique lipid-like substance, cKK-E12, through high-throughput screening and found that it inhibited ED in F7 knockdown. ５０Achieved 0.002 mg siRNA / kg at 1000 kJ / min (Non-Patent Document 10). This technology is the most excellent in terms of activity in the literature, but there is no information on safety aspects such as toxicity at high doses or biodegradability of lipids. Therefore, lipid nanoparticles useful as nucleic acid delivery carriers have been developed (Patent Documents 3 and 4).

[0005] International Publication No. WO 2018 / 230710 International Publication No. WO 2018 / 190423 International Publication No. WO 2022 / 071582 International Publication No. WO 2023 / 191050

[0006] Sato et al., Journal of Controlled Release, 2019, vol.295, p.140-152.Jayaraman et al., Angewandte Chemie International Edition, 2012, vol.51, p.8529-8533.Watanabe et al., Scientific Reports, 2014, 4:4750, DOI: 10.1038 / srep04750.Yamamoto et al., Journal of Hepatology, 2016, vol.64, p.547-555.Sato et al., Molecular Therapy, 2016, vol.24, p.788-795.Maier et al., Molecular Therapy, 2013, vol.21(8), p.1570-1578.Wittrup et al., Nature Biotechnology, 2015, vol.33(8), p.870-876. 2014, vol.111(11), p.3955-3960.Leung et al.,Journal of Physical Chemistry C Nanomater Interfaces,2012,vol.116(34),p.18440-18450.

[0007] An object of the present invention is to provide lipid nanoparticles that are further useful as nucleic acid delivery carriers and novel pH-sensitive cationic lipids for producing such lipid nanoparticles.

[0008] The present inventors have discovered that lipid nanoparticles containing, as constituent lipids, pH-sensitive cationic lipids with asymmetric hydrocarbon chains are useful as nucleic acid delivery carriers that can selectively deliver genes to the liver and spleen and can express encapsulated genes and enable functional nucleic acids to function in the liver and spleen, thereby completing the present invention.

[0009] That is, the present invention relates to the following: [1] A compound represented by the following formula (I) (R １ ), (R ２ )C(OH),(CH ２ )a-(O-CO)b-X (I) where, R １ is the following formula (A) X ０ -COO-(CH ２ )p-(A) is a group represented by R ２ is represented by the following formula (B): ０ -COO-(CH ２ )q-(B) is a group represented by the formula: ０ and Y ０ is different, X ０ is represented by the following formula (C): -(CH ２ )r-CH(R １ａ ), (R ２ａ ), (C), r is an integer of 0 to 6, R １ａ and R ２ａ are each independently C １－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, a is an integer of 3 to 5, b is 0 or 1, and X is selected from any one of the following formulae (X-a) to (X-d): In the formula, d is an integer of 0 to 3; ３ａ and R ４ａ are each independently hydrogen or C １－５ is an alkyl group, where R ３ａ and R ４ａ do not bond to each other to form a ring, and Z １ , CH, CR ５ａ or N; Z ２ ~Z ７are, when present, each independently NH, NR ５ａ , C.R. ６ａ R ６ｂ or O; R ５ａ is C １－５ is an alkyl group, R ６ａ and R ６ｂ are each independently hydrogen or C １－５ is an alkyl group, provided that in formula (X-b), Z １ ~Z ５ At least one of the groups contains a nitrogen atom, and in (X-c), Z １ ~Z ６ At least one of (X-d) contains a nitrogen atom, and Z １ ~Z ７ at least one of the groups contains a nitrogen atom, and does not form an N-N bond, an N-O bond, or an O-O bond in formulae (X-b) to (X-d), p is an integer of 4 to 12, and q is an integer of 4 to 12, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

[0010] [2] Y ０ [3] The compound according to [1], or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein R is a linear saturated or unsaturated hydrocarbon group. １ａ and R ２ａ At least one of ９－１６ [4] The compound according to [1] or [2], or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein R is an alkyl group. １ａ and R ２ａ However, both are C ９－１６ The compound according to any one of [1] to [3], which is an alkyl group, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

[0011] [5] The compound according to any one of [1] to [4], or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein r is 0. [6] X is formula (X-a), d is 0, and R ３ａ and R ４ａ are each independently hydrogen or C １－５or X is a group represented by formula (X-c), d is 0, and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , and Z ６ is CH ２ and Z ４ NR ５ａ and R ５ａ is C １－５ The compound according to any one of [1] to [5], or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein R is an alkyl group.

[0012] [7] X is formula (X-a), and R ３ａ and R ４ａ are each independently hydrogen or C １－５ or X is a group represented by formula (X-c) and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , Z ６ is CH ２ and Z ４ NR ５ａ and R ５ａ is C １－５ [8] The compound according to any one of [1] to [6], or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein X is a group represented by formula (X-a) and R ３ａ and R ４ａ are each independently hydrogen or C １－５ or X is a group represented by formula (X-c) and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , Z ６ is CH ２ and Z ４ NR ５ａ and R ５ａ is C １－５ is an alkyl group, ０ is a linear saturated or unsaturated hydrocarbon group, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, according to any one of [1] to [7].

[0013] [9] Y ０ But linear C ９－２１

[10] The compound according to any one of [1] to [8], or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein Y is a saturated or unsaturated hydrocarbon group. ０ But linear C １４－１８ The compound according to any one of [1] to [9], which is a saturated or unsaturated hydrocarbon group, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

[0014]

[11] Formula (II) or (III) below In the formula, ０ is -(CH ２ )r-CH(R １ａ ), (R ２ａ ) is a group represented by the formula: r is an integer of 0 to 6; １ａ and R ２ａ are each independently C １－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, ０ and Y ０ or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein

[0015]

[12] Formula (II) or (III) below In the formula, ０ is -(CH ２ )r-CH(R １ａ ), (R ２ａ ) is a group represented by the formula: r is an integer of 0 to 6; １ａ and R ２ａ are each independently C ９－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, ０ and Y ０ or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein

[0016]

[13] The compound is The compound according to any one of [1] to

[12] , or a stereoisomer thereof, or a salt thereof, or a mixture thereof, selected from the following:

[0017]

[14] A lipid nanoparticle comprising the compound according to any one of [1] to

[13] or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

[15] The lipid nanoparticle according to

[14] , which contains a nucleic acid.

[16] The lipid nanoparticle according to

[15] , wherein the nucleic acid is siRNA.

[17] The lipid nanoparticle according to

[15] , wherein the nucleic acid is mRNA.

[0018]

[18] The lipid nanoparticles according to

[17] , wherein the nucleic acid is a gene to be expressed in liver or spleen cells.

[19] A pharmaceutical composition comprising the lipid nanoparticles according to

[14] as an active ingredient.

[20] A method for expressing a foreign gene, comprising administering the lipid nanoparticles according to

[14] , which encapsulate a foreign gene to be expressed in liver or spleen cells, to a subject animal (excluding humans), and expressing the foreign gene in the liver or spleen of the subject animal.

[0019] The lipid nanoparticles of the present invention can selectively deliver encapsulated genes or functional nucleic acids such as inhibitory nucleic acids to the liver and spleen, and can also express the encapsulated genes or activate the functional nucleic acids in the liver or spleen. Therefore, the lipid nanoparticles are useful as liver-specific or spleen-specific nucleic acid delivery carriers for gene therapy. Furthermore, the lipid nanoparticles of the present invention are less likely to induce inflammatory reactions in the body, resulting in improved safety.

[0020] Figure 1 shows the relative FLuc luminescence intensity in cells transfected with each FLuc mRNA-loaded lipid nanoparticle. Figure 1(A) shows the results of transfection into HepG2 cells, and Figure 1(B) shows the results of transfection into CHO-K1 cells. NT: no transfection, 1: CL4F12-10 / Ste, 2: CL4F12-10 / Ole, 3: CL4F12-10 / Lin, 4: CL4F12-10 / Myr, 5: CL4F12-10 / Myristole, 6: CL4F12-10 / Dec, 7: CL4F12-10. Error bars indicate standard deviation (n=3).

[0021] Figure 2 shows the FLuc luminescence intensity per unit area in the organs of mice administered with each FLuc mRNA-loaded lipid nanoparticle. Error bars indicate standard deviation (n = 3). Figure 2(A) shows the results for the liver, and Figure 2(B) shows the results for the spleen.

[0022] Figure 3 shows the ratio of Atp5f1 mRNA to F7 mRNA (F7 / Atp5f1) in the livers of mice administered siRNA-loaded lipid nanoparticles. Error bars indicate standard deviation (n = 4).

[0023] Figure 4 shows the liver damage markers and chemokine production levels 24 hours after administration of a high dose of LNP. Figure 4(A) shows the ALT values, Figure 4(B) shows the AST values, and Figure 4(C) shows the MCP-1 values. HEPES is the control group, and only HEPES buffer was administered. Error bars indicate the standard deviation (n=4).

[0024] The present invention will be described in detail below based on the embodiments of the present invention. １ ), (R ２ )C(OH),(CH ２ )a-(O-CO)b-X (I) where, R １ is the following formula (A) X ０ -COO-(CH ２ )p-(A) is a group represented by R ２ is represented by the following formula (B): ０ -COO-(CH ２)q-(B) is a group represented by the formula: ０ and Y ０ is different, X ０ is represented by the following formula (C): -(CH ２ )r-CH(R １ａ ), (R ２ａ ), (C), r is an integer of 0 to 6, R １ａ and R ２ａ are each independently C １－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, a is an integer of 3 to 5, b is 0 or 1, and X is selected from any one of the following formulae (X-a) to (X-d): In the formula, d is an integer of 0 to 3; ３ａ and R ４ａ are each independently hydrogen or C １－５ is an alkyl group, where R ３ａ and R ４ａ do not bond to each other to form a ring, and Z １ , CH, CR ５ａ or N; Z ２ ~Z ７ are, when present, each independently NH, NR ５ａ , C.R. ６ａ R ６ｂ or O; R ５ａ is C １－５ is an alkyl group, R ６ａ and R ６ｂ are each independently hydrogen or C １－５ is an alkyl group, provided that in formula (X-b), Z １ ~Z ５ At least one of the groups contains a nitrogen atom, and in (X-c), Z １ ~Z ６ At least one of (X-d) contains a nitrogen atom, and Z １ ~Z ７at least one of the groups contains a nitrogen atom, and does not form an N-N bond, an N-O bond, or an O-O bond in formulae (X-b) to (X-d), p is an integer of 4 to 12, and q is an integer of 4 to 12, or a compound represented by the formula (X-b) to (X-d), a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof.

[0025] In the present invention, R １ is the following formula (A) X ０ -COO-(CH ２ )p-(A) In the present invention, p is an integer of 4 to 12, preferably 5 to 10, particularly preferably 5 to 7, and most preferably 6. In the present invention, R ２ is represented by the following formula (B): ０ -COO-(CH ２ )q-(B) In the present invention, q is an integer of 4 to 12, preferably 5 to 10, particularly preferably 5 to 7, and most preferably 6.

[0026] In the present invention, X ０ is represented by the following formula (C): -(CH ２ )r-CH(R １ａ ), (R ２ａ In the present invention, r is an integer of 0 to 6, preferably 0 to 4, particularly preferably 0 to 2, and most preferably 0.

[0027] In the present invention, R １ａ and R ２ａ are each independently C １－１６ is an alkyl group, preferably R １ａ and R ２ａ At least one of ９－１６ alkyl group, and particularly preferably R １ａ and R ２ａ All of these are C ９－１６ It is an alkyl group.

[0028] Linear or branched chain C １－１６Examples of alkyl groups include: a methyl group, an n-ethyl group; an n-propyl group, a 1-methylethyl group; an n-butyl group, a 1-methylpropyl group, a 2-methylpropyl group, a 1,1-dimethylethyl group; an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1-ethylpropyl group, a 1,1-dimethylpropyl group, a 2,2-dimethylpropyl group; an n-hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 4-methylpentyl group, a 1-ethylbutyl group, a 1,1-dimethylbutyl group, a 2,2-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1-methyl-2,2-dimethylbutyl group; n-heptyl group, 1-methylhexyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl group, 1-ethylpentyl group, 1,1-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 4,4-dimethylpentyl group, 1-methyl-3,3-dimethylbutyl group, 2-methyl-3,3-dimethylbutyl group; n-octyl group, 1-methylheptyl group, 2-methylheptyl group, 3-methylheptyl group, 4-methylheptyl group, 5-methylheptyl group, 6-methylheptyl group, 1-ethylhexyl group, 1,1-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 5,5-dimethylhexyl group, 1-methyl-4,4-dimethylpentyl group, 2-methyl-4,4-dimethylpentyl group, 3-methyl-4,4-dimethylpentyl group;

[0029] n-nonyl group, 1-methyloctyl group, 2-methyloctyl group, 3-methyloctyl group, 4-methyloctyl group, 5-methyloctyl group, 6-methyloctyl group, 7-methyloctyl group, 1-ethylheptyl group, 1,1-dimethylheptyl group, 2,2-dimethylheptyl group, 3,3-dimethylheptyl group, 4,4-dimethylheptyl group, 5,5-dimethylheptyl group, 6,6-dimethylheptyl group, 1-methyl-5,5-dimethylhexyl group, 2-methyl-5,5-dimethylhexyl group, 3-methyl-5,5-dimethylhexyl group, 4-methyl-5,5-dimethylhexyl group; n-decyl group, 1-methylnonyl group, 2-methylnonyl group, 3-methylnonyl group, 4-methylnonyl group, 5-methylnonyl group, 6-methylnonyl group, 7-methylnonyl group, 8-methylnonyl group, 1-ethyloctyl group, 1,1-dimethyloctyl group, 2,2-dimethyloctyl group, 3,3-dimethyloctyl group, 4,4-dimethyloctyl group, 5,5-dimethyloctyl group, 6,6-dimethyloctyl group, 7,7-dimethyloctyl group, 1-methyl-6,6-dimethylheptyl group, 2-methyl-6,6-dimethylheptyl group, 3-methyl-6,6-dimethylheptyl group, 4-methyl-6,6-dimethylheptyl group, 5-methyl-6,6-dimethylheptyl group;

[0030] n-undecyl group, 1-methyldecyl group, 2-methyldecyl group, 3-methyldecyl group, 4-methyldecyl group, 5-methyldecyl group, 6-methyldecyl group, 7-methyldecyl group, 8-methyldecyl group, 9-methyldecyl group, 1-ethylnonyl group, 1,1-dimethylnonyl group, 2,2-dimethylnonyl group, 3,3-dimethylnonyl group, 4,4-dimethylnonyl group, 5,5-dimethylnonyl group, 6,6-dimethylnonyl group, 7,7-dimethylnonyl group, 8,8-dimethylnonyl group, 1-methyl-7,7-dimethyloctyl group, 2-methyl-7,7-dimethyloctyl group, 3-methyl-7,7-dimethyloctyl group, 4-methyl-7,7-dimethyloctyl group, 5-methyl-7,7-dimethyloctyl group, 6-methyl-7,7-dimethyloctyl group; n-dodecyl group, 1-methylundecyl group, 2-methylundecyl group, 3-methylundecyl group, 4-methylundecyl group, 5-methylundecyl group, 6-methylundecyl group, 7-methylundecyl group, 8-methylundecyl group, 9-methylundecyl group, 10-methylundecyl group, 1-ethyldecyl group, 1,1-dimethyldecyl group, 2,2-dimethyldecyl group, 3,3-dimethyldecyl group, 4,4-dimethyldecyl group, 5,5 -dimethyldecyl group, 6,6-dimethyldecyl group, 7,7-dimethyldecyl group, 8,8-dimethyldecyl group, 9,9-dimethyldecyl group, 1-methyl-8,8-dimethylnonyl group, 2-methyl-8,8-dimethylnonyl group, 3-methyl-8,8-dimethylnonyl group, 4-methyl-8,8-dimethylnonyl group, 5-methyl-8,8-dimethylnonyl group, 6-methyl-8,8-dimethylnonyl group, 7-methyl-8,8-dimethylnonyl group;

[0031] n-Tridecyl group, 1-methyldodecyl group, 2-methyldodecyl group, 3-methyldodecyl group, 4-methyldodecyl group, 5-methyldodecyl group, 6-methyldodecyl group, 7-methyldodecyl group, 8-methyldodecyl group, 9-methyldodecyl group, 10-methyldodecyl group, 11-methyldodecyl group, 1-ethylundecyl group, 1,1-dimethylundecyl group, 2,2-dimethylundecyl group, 3,3-dimethylundecyl group, 4,4-dimethylundecyl group, 5,5-dimethylundecyl group, 6,6-dimethylundecyl group, Methylundecyl group, 7,7-dimethylundecyl group, 8,8-dimethylundecyl group, 9,9-dimethylundecyl group, 10,10-dimethylundecyl group, 1-methyl-9,9-dimethyldecyl group, 2-methyl-9,9-dimethyldecyl group, 3-methyl-9,9-dimethyldecyl group, 4-methyl-9,9-dimethyldecyl group, 5-methyl-9,9-dimethyldecyl group, 6-methyl-9,9-dimethyldecyl group, 7-methyl-9,9-dimethyldecyl group, 8-methyl-9,9-dimethyldecyl group; n-tetradecyl group, 1-methyltridecyl group, 2-methyltridecyl group, 3-methyltridecyl group, 4-methyltridecyl group, 5-methyltridecyl group, 6-methyltridecyl group, 7-methyltridecyl group, 8-methyltridecyl group, 9-methyltridecyl group, 10-methyltridecyl group, 11-methyltridecyl group, 12-methyltridecyl group, 1-ethyldodecyl group, 1,1-dimethyldodecyl group, 2,2-dimethyldodecyl group, 3,3-dimethyldodecyl group, 4,4-dimethyldodecyl group, 5,5-dimethyldodecyl group, 6,6-dimethyldodecyl group, 7,7-dimethyldodecyl group, 8,8-dimethyldodecyl group, 9,9-dimethyldodecyl group, 10,10-dimethyldodecyl group, 11,11-dimethyldodecyl group, 1-methyl-10,10-dimethylundecyl group, 2-methyl-10,10-dimethylundecyl group, 3-methyl-10,10-dimethylundecyl group, 4-methyl-10,10-dimethylundecyl group, 5-methyl-10,10-dimethylundecyl group, 6-methyl-10,10-dimethylundecyl group, 7-methyl-10,10-dimethylundecyl group, 8-methyl-10,10-dimethylundecyl group, 9-methyl-10,10-dimethylundecyl group;

[0032] n-pentadecyl group, 1-methyltetradecyl group, 2-methyltetradecyl group, 3-methyltetradecyl group, 4-methyltetradecyl group, 5-methyltetradecyl group, 6-methyltetradecyl group, 7-methyltetradecyl group, 8-methyltetradecyl group, 9-methyltetradecyl group, 10-methyltetradecyl group, 11-methyltetradecyl group, 12-methyltetradecyl group, 13-methyltetradecyl group, 1-ethyltridecyl group, 1,1-dimethyltridecyl group, 2,2-dimethyltridecyl group, 3,3-dimethyltridecyl group, 4,4-dimethyltridecyl group, 5,5-dimethyltridecyl group, 6,6-dimethyltridecyl group, 7,7-dimethyltridecyl group, 8 ,8-dimethyltridecyl group, 9,9-dimethyltridecyl group, 10,10-dimethyltridecyl group, 11,11-dimethyltridecyl group, 12,12-dimethyltridecyl group, 1-methyl-11,11-dimethyldodecyl group, 2-methyl-11,11-dimethyldodecyl group, 3-methyl-11,11-dimethyldodecyl group, 4-methyl-11,11-dimethyldodecyl group, 5-methyl-11,11-dimethyldodecyl group, 6-methyl-11,11-dimethyldodecyl group, 7-methyl-11,11-dimethyldodecyl group, 8-methyl-11,11-dimethyldodecyl group, 9-methyl-11,11-dimethyldodecyl group, 10-methyl-11,11-dimethyldodecyl group,

[0033] n-Hexadecyl, 1-methylpentadecyl, 2-methylpentadecyl, 3-methylpentadecyl, 4-methylpentadecyl, 5-methylpentadecyl, 6-methylpentadecyl, 7-methylpentadecyl, 8-methylpentadecyl, 9-methylpentadecyl, 10-methylpentadecyl, 11-methylpentadecyl, 12-methylpentadecyl, 13-methylpentadecyl, 14-methylpentadecyl, 2,2-dimethyltetradecyl, 3,3-dimethyltetradecyl, 4,4-dimethyltetradecyl, 5,5-dimethyltetradecyl, 6,6-dimethyltetradecyl, 2-ethylpentadecyl, 3-ethylpentadecyl, 4-ethylpentadecyl, 5-ethylpentadecyl, 6-ethylpentadecyl, 2-propylpentadecyl, 3-propylpentadecyl, 4-propylpentadecyl, 5-propylpentadecyl, 2-butylpentadecyl, 3-butylpentadecyl, 4-butylpentadecyl, 2-pentylpentadecyl, 3-pentylpentadecyl, 4-pentylpentadecyl, 2-hexylpentadecyl, 3-hexylpentadecyl, 4-hexyl Cylpentadecyl, 2-heptylpentadecyl, 3-heptylpentadecyl, 2-octylpentadecyl, 2-nonylpentadecyl, 2-decylpentadecyl, 2-undecylpentadecyl, 2-dodecylpentadecyl, 2-tridecylpentadecyl, 2-tetradecylpentadecyl, 2-pentadecylpentadecyl, 2-methylhexadecyl, 3-methylhexadecyl, 4-methylhexadecyl, 5-methylhexadecyl, 6-methylhexadecyl, 7-methylhexadecyl, 2-ethylhexadecyl, 3-ethylhexadecyl, ethylhexadecyl, 4-ethylhexadecyl, 5-ethylhexadecyl, 6-ethylhexadecyl, 2-propylhexadecyl, 3-propylhexadecyl, 4-propylhexadecyl, 5-propylhexadecyl, 2-butylhexadecyl, 3-butylhexadecyl, 4-butylhexadecyl, 2-pentylhexadecyl, 3-pentylhexadecyl, 4-pentylhexadecyl, 2-hexylhexadecyl, 3-hexylhexadecyl, 2-heptylhexadecyl, 2-octylhexadecyl, 2-nonylhexadecyl hexadecyl, 2-decylhexadecyl, 2-undecylhexadecyl, 2-dodecylhexadecyl, 2-tridecylhexadecyl, 2-tetradecylhexadecyl, 2-pentadecylhexadecyl, 2,3-dimethylpentadecyl, 2,4-dimethylpentadecyl, 2,5-dimethylpentadecyl, 3,4-dimethylpentadecyl, 3,5-dimethylpentadecyl, 4,5-dimethylpentadecyl, 2,6-dimethylpentadecyl, 3,6-dimethylpentadecyl, 4,6-dimethylpentadecyl, and the like.

[0034] In the present invention, Y ０is a linear or branched, saturated or unsaturated hydrocarbon group, preferably a linear, saturated or unsaturated hydrocarbon group, more preferably a linear C ９－２１ A saturated or unsaturated hydrocarbon group, and even more preferably a linear C １４－１８ It is a saturated or unsaturated hydrocarbon group.

[0035] In the present invention, Y ０ is a linear saturated or unsaturated hydrocarbon group, Y ０ -COOH is butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), pentadecanoic acid, hexadecanoic acid (palmitic acid (Pam)), 9-hexadecenoic acid, heptadecanoic acid, octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)) , 11-octadecenoic acid, cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)), 9,12,15-octadecanetrienoic acid ((9,12,15)-linolenic acid (αLnn), 6,9,12-octadecanetrienoic acid, 9,11,13-octadecanetrienoic acid, eicosanoic acid (arachidic acid (Ach)), 8,11-eicosadienoic acid, 5,8,11-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, docosanoic acid (behenic acid (Beh)), etc. ０ is preferred, and Y ０ -COOH is decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), pentadecanoic acid, hexadecanoic acid (palmitic acid (Pam)), octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)), cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)), 9,12,15-octadecanetrienoic acid ((9,12,15)-linolenic acid (αLnn), eicosanoic acid (arachidic acid (Ach)), docosanoic acid (behenic acid (Beh)) ０More preferably, Y ０ -COOH is decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)), cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)) ０ is particularly preferred.

[0036] Y ０ is a branched saturated hydrocarbon group, Y ０ In one embodiment, the formula (D) —(CH ２ )r'-CH(R １ａ’ ), (R ２ａ’ ), (D), r' is an integer of 0 to 6, R １ａ’ and R ２ａ’ are each independently C １－１６ In the present invention, X is an alkyl group. ０ and Y ０ In the present invention, a is an integer of 3 to 5, preferably 4.

[0037] In the present invention, X is selected from any one of the following formulae (X-a) to (X-d): d is an integer of 0 to 3, and preferably d is 0. ３ａ and R ４ａ are each independently hydrogen or C １－５ alkyl group, preferably both C ３ is an alkyl group, where R ３ａ and R ４ａ do not bond to each other to form a ring, and Z １ , CH, CR ５ａ or N, preferably CH; Z ２ ~Z ７ are, when present, each independently NH, NR ５ａ , C.R. ６ａ R ６ｂ or O, preferably any one of which is NR５ａ and R ５ａ is C １－５ is an alkyl group, preferably C １ is an alkyl group, R ６ａ and R ６ｂ are each independently hydrogen or C １－５ is an alkyl group, and preferably hydrogen. １ ~Z ５ At least one of the groups contains a nitrogen atom, and in (X-c), Z １ ~Z ６ At least one of (X-d) contains a nitrogen atom, and Z １ ~Z ７ At least one of R contains a nitrogen atom, and does not form an N-N bond, an N-O bond, or an O-O bond in formulas (X-b) to (X-d). ３ａ , R ４ａ , R ５ａ , R ６ａ and R ６ｂ In C １－５ Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a tert-butyl group.

[0038] In one embodiment of the invention, X is of formula (X-a), d is 0, and R ３ａ and R ４ａ are each independently hydrogen or C １－５ is an alkyl group, preferably, X is of formula (X-a), d is 0, and R ３ａ and R ４ａ are each independently C １－５ alkyl groups, particularly preferably X is of formula (X-a), d is 0, and R ３ａ and R ４ａ is C ３ In one embodiment of the present invention, X is of formula (X-c), d is 0, and Z １ is CH, and Z ２ , Z ３ , Z ５ , and Z ６ is CH ２ and Z ４is NR ５ａ and R ５ａ is C １－５ Preferably, X is a group represented by the formula (X-c), d is 0, and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , and Z ６ is CH ２ and Z ４ is NR ５ａ and R ５ａ is C １ It is an alkyl group.

[0039] In one aspect, the present invention provides a compound represented by the following formula (II) or (III): In the formula, ０ is -(CH ２ )r-CH(R １ａ ), (R ２ａ ) is a group represented by the formula: r is an integer of 0 to 6; １ａ and R ２ａ are each independently C １－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, ０ and Y ０ are different from each other, a stereoisomer thereof, a mixture of stereoisomers, or a salt thereof.

[0040] In the present invention, r is an integer of 0 to 6, preferably 0 to 4, particularly preferably 0 to 2, and most preferably 0. １ａ and R ２ａ are each independently C １－１６ is an alkyl group, preferably R １ａ and R ２ａ At least one of ９－１６ alkyl group, and particularly preferably R １ａ and R ２ａ All of these are C ９－１６ In the present invention, Y is an alkyl group. ０is a linear or branched, saturated or unsaturated hydrocarbon group, preferably a linear, saturated or unsaturated hydrocarbon group, more preferably a linear C ９－２１ A saturated or unsaturated hydrocarbon group, and even more preferably a linear C １４－１８ It is a saturated or unsaturated hydrocarbon group.

[0041] In the present invention, Y ０ is a linear saturated or unsaturated hydrocarbon group, Y ０ -COOH is butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), pentadecanoic acid, hexadecanoic acid (palmitic acid (Pam)), 9-hexadecenoic acid, heptadecanoic acid, octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)) , 11-octadecenoic acid, cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)), 9,12,15-octadecanetrienoic acid ((9,12,15)-linolenic acid (αLnn), 6,9,12-octadecanetrienoic acid, 9,11,13-octadecanetrienoic acid, eicosanoic acid (arachidic acid (Ach)), 8,11-eicosadienoic acid, 5,8,11-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, docosanoic acid (behenic acid (Beh)), etc. ０ is preferred, and Y ０ -COOH is decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), pentadecanoic acid, hexadecanoic acid (palmitic acid (Pam)), octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)), cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)), 9,12,15-octadecanetrienoic acid ((9,12,15)-linolenic acid (αLnn), eicosanoic acid (arachidic acid (Ach)), docosanoic acid (behenic acid (Beh)) ０More preferably, Y ０ -COOH is decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)), cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)) ０ is particularly preferred.

[0042] Y ０ is a branched saturated hydrocarbon group, Y ０ In one embodiment, the formula (D) —(CH ２ )r'-CH(R １ａ’ ), (R ２ａ’ ), (D), r' is an integer of 0 to 6, R １ａ’ and R ２ａ’ are each independently C １－１６ It is an alkyl group.

[0043] In one aspect, the present invention provides a compound represented by the following formula (II) or (III): In the formula, ０ is -(CH ２ )r-CH(R １ａ ), (R ２ａ ) is a group represented by the formula: r is an integer of 0 to 6; １ａ and R ２ａ are each independently C ９－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, ０ and Y ０ In the present invention, r is an integer of 0 to 6, preferably 0 to 4, particularly preferably 0 to 2, and most preferably 0. In the present invention, R １ａ and R ２ａ are each independently C １－１６ is an alkyl group, preferably R １ａ and R ２ａ At least one of ９－１６ alkyl group, and particularly preferably R１ａ and R ２ａ All of these are C ９－１６ It is an alkyl group.

[0044] In the present invention, Y ０ is a linear or branched, saturated or unsaturated hydrocarbon group, preferably a linear, saturated or unsaturated hydrocarbon group, more preferably a linear C ９－２１ A saturated or unsaturated hydrocarbon group, and even more preferably a linear C １４－１８ It is a saturated or unsaturated hydrocarbon group. ０ is a linear saturated or unsaturated hydrocarbon group, Y ０ -COOH is butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), pentadecanoic acid, hexadecanoic acid (palmitic acid (Pam)), 9-hexadecenoic acid, heptadecanoic acid, octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)) , 11-octadecenoic acid, cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)), 9,12,15-octadecanetrienoic acid ((9,12,15)-linolenic acid (αLnn), 6,9,12-octadecanetrienoic acid, 9,11,13-octadecanetrienoic acid, eicosanoic acid (arachidic acid (Ach)), 8,11-eicosadienoic acid, 5,8,11-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, docosanoic acid (behenic acid (Beh)), etc. ０ is preferred, and Y ０-COOH is decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), pentadecanoic acid, hexadecanoic acid (palmitic acid (Pam)), octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)), cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)), 9,12,15-octadecanetrienoic acid ((9,12,15)-linolenic acid (αLnn), eicosanoic acid (arachidic acid (Ach)), docosanoic acid (behenic acid (Beh)) ０ More preferably, Y ０ -COOH is decanoic acid (capric acid (Dec)), dodecanoic acid (lauric acid (Lau)), tetradecanoic acid (myristic acid (Myr)), 9-tetradecenoic acid (mistoleic acid (Myristole)), octadecanoic acid (stearic acid (Ste)), cis-9-octadecenoic acid (oleic acid (Ole)), cis,cis-9,12-octadecadienoic acid (linoleic acid (Lin)) ０ is particularly preferred.

[0045] Y ０ is a branched saturated hydrocarbon group, Y ０ In one embodiment, the formula (D) —(CH ２ )r'-CH(R １ａ’ ), (R ２ａ’ ), (D), r' is an integer of 0 to 6, R １ａ’ and R ２ａ’ are each independently C １－１６ It is an alkyl group.

[0046] In one aspect, the present invention relates to the following compounds:

[0047] The pKa of the pH-sensitive cationic lipid represented by general formula (I) is not particularly limited, but can be selected, for example, from about 4.0 to 9.0, preferably from about 4.5 to 8.5, and it is preferable to select the type of each substituent so as to give a pKa within this range.

[0048] The pH-sensitive cationic lipid represented by general formula (I) can be easily produced, for example, by the method specifically shown in the Examples of this specification. By referring to this production method and appropriately selecting raw material compounds, reagents, reaction conditions, etc., a person skilled in the art can easily produce any lipid within the scope of general formula (I).

[0049] The pH-sensitive cationic lipid represented by general formula (I) is ０ and Y ０ These hydrocarbon chains form a hydrophobic scaffold embedded in the lipid membrane of the lipid nanoparticle. Lipid nanoparticles containing the pH-sensitive cationic lipid of the present invention, which has an asymmetric hydrophobic scaffold structure, are more likely to be delivered to the liver or spleen, and can more effectively express encapsulated genes and function functional nucleic acids than lipid nanoparticles containing lipids with a symmetric hydrophobic scaffold structure. Furthermore, the lipid nanoparticles of the present invention are less likely to cause inflammatory reactions in the body and have improved safety compared to lipid nanoparticles using conventional lipids.

[0050] The pH-sensitive cationic lipid of the present invention constituting the lipid nanoparticles of the present invention may be one type only, or may be two or more types. When the pH-sensitive cationic lipid of the present invention constituting the lipid nanoparticles of the present invention is two or more types, the amount of the pH-sensitive cationic lipid of the present invention means the total amount of lipid molecules corresponding to the pH-sensitive cationic lipid of the present invention among the lipid molecules constituting the lipid nanoparticles.

[0051] The higher the ratio of the pH-sensitive cationic lipid of the present invention to the lipid molecules constituting the lipid nanoparticles, the higher the efficiency of uptake of the lipid nanoparticles into target cells. Therefore, in the lipid nanoparticles of the present invention, the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles ([amount (mol) of the pH-sensitive cationic lipid of the present invention] / ([total amount (mol) of lipids constituting the lipid nanoparticles]) × 100%) is preferably 20 mol% or more. On the other hand, if the ratio of the pH-sensitive cationic lipid to the lipid molecules constituting the lipid nanoparticles is too high, it may be difficult to sufficiently reduce the particle size. Since lipid nanoparticles have sufficient uptake efficiency into target cells and lipid nanoparticles with a sufficiently small particle size can be obtained, the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles in the lipid nanoparticles of the present invention is more preferably 30 mol% or more, even more preferably 30 to 70 mol%, and even more preferably 40 to 60 mol%.

[0052] Among the constituent lipids of the lipid nanoparticles of the present invention, lipids other than the pH-sensitive cationic lipid of the present invention can be lipids generally used in forming liposomes. Examples of such lipids include phospholipids, sterols or sterol derivatives, glycolipids, saturated or unsaturated fatty acids, etc. These can be used alone or in combination of two or more.

[0053] Examples of phospholipids include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphalidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, and phosphatidic acid; and sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, and ceramide phosphorylethanolamine. Phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used. The fatty acid residues in the glycerophospholipids and sphingophospholipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms, with saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms being preferred. Specific examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, lignoceric acid, etc. When these glycerolipids or sphingolipids have two or more fatty acid residues, all of the fatty acid residues may be the same group or may be different groups.Examples of phospholipids are diphytanoyl phosphatidyl ethanolamine (DPhPE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC). These include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0054] Examples of sterols or sterol derivatives include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, β-sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as zymosterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; and sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside. Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0055] The constituent lipids of the lipid nanoparticles of the present invention preferably contain a neutral lipid in addition to the pH-sensitive cationic lipid of the present invention, more preferably a phospholipid or a sterol, even more preferably a sterol, and even more preferably cholesterol.

[0056] The lipid nanoparticles according to the present invention preferably contain a polyalkylene glycol-modified lipid as a lipid component. Polyalkylene glycol is a hydrophilic polymer, and by constructing lipid nanoparticles using a polyalkylene glycol-modified lipid as a lipid membrane-constituting lipid, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may improve the stability of the lipid nanoparticles, such as their blood retention.

[0057] Examples of polyalkylene glycols that can be used include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The molecular weight of the polyalkylene glycol is, for example, about 200 to 10,000, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000. In one embodiment of the present invention, the molecular weight of the polyalkylene glycol is about 200, 300, 350, 400, 500, 550, 750, 1,000, 1,500, 2,000, 3,000, 3,500, 4,000, 5,000, or 10,000 Da.

[0058] For example, stearylated polyethylene glycol (e.g., PEG 45 stearate (STR-PEG45)) can be used to modify lipids with polyethylene glycol. Other examples include N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), and N-[carbonyl-methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG). glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol), (DMG-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine glycol]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), (DMPE-PEG), and other polyethylene glycol derivatives may be used.For example, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000), N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG5000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol polyethylene glycol-750 (DMG-PEG750), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG750), Polyethylene glycol derivatives such as N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000) and N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG5000) can also be used, but the polyalkylene glycolated lipid is not limited to these.

[0059] The ratio of polyalkylene glycol-modified lipid to the total amount of lipid constituting the lipid nanoparticles of the present invention is not particularly limited, as long as it is an amount that does not impair the liver selectivity or spleen selectivity of the pH-sensitive cationic lipid of the present invention, specifically, the gene expression activity or functional activity in the liver or spleen when the lipid nanoparticles of the present invention are used as nucleic acid delivery carriers. For example, the ratio of polyalkylene glycol-modified lipid to the total amount of lipid constituting the lipid nanoparticles is preferably 0.5 to 3 mol%. The lipid nanoparticles of the present invention may contain any one of DSPC, DOPC, and DOPE as a phospholipid, and may also contain DMG-PEG as a polyalkylene glycol-modified lipid. For example, the lipid nanoparticles according to the present invention may have a lipid content of 40 to 60 (e.g., 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5) based on the total lipid content of the lipid nanoparticles. , 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, or 60) mol % pH-sensitive cationic lipid; 30-50 (e.g., 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37) mol % pH-sensitive cationic lipid relative to the total lipid content of the lipid nanoparticle; , 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50) mol % sterol (e.g., cholesterol); 5-15 (e.g., 5) mol % of the total lipid content of the lipid nanoparticle , 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15) mol% of any one of DSPC, DOPC and DOPE phospholipids; and 1-5 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4,In the lipid nanoparticles according to the present invention, the molar ratio of the pH-sensitive cationic lipid, the sterol, the phospholipid selected from DSPC, DOPC, and DOPE, and the DMG-PEG (pH-sensitive cationic lipid / sterol / phospholipid / DMG-PEG) is not particularly limited, but includes, for example, the following: (60 / 31 / 8 / 1), (60 / 31 / 7.5 / 1.5), (60 / 31 / 7 / 2), (60 / 31 / 6.5 / 2.5), (60 / 31 / 6 / 3), (60 / 31 / 5.5 / 3.5), (60 / 31 / 5 / 4), (60 / 31 / 5 / 5), (60 / 31 / 6.5 / 5), (60 / 31 / 5.5 / 5.5), (60 / 31 / ... 1 / 4.5 / 4.5), (60 / 31 / 4 / 5), (60 / 31.5 / 7.5 / 1), (60 / 30.5 / 7.5 / 2), (60 / 30 / 7.5 / 2.5), (60 / 29.5 / 7.5 / 3), (60 / 29 / 7.5 / 3.5), (60 / 28.5 / 7.5 / 4), (60 / 28 / 7.5 / 4.5), (60 / 27.5 / 7.5 / 5), (59 / 32 / 7.5 / 1.5), (58 / 33 / 7.5 / 1.5), (57 / 34 / 7.5 / 1.5), (56 / 35 / 7.5 / 1.5), (55 / 36 / 7.5 / 1.5), (60 / 29 / 10 / 1), (60 / 28.5 / 10 / 1.5), (60 / 28 / 10 / 2), (60 / 27.5 / 10 / 2.5), (60 / 27 / 10 / 3), (60 / 26.5 / 10 / 3.5), (60 / 26 / 10 / 4), (60 / 25.5 / 10 / 4.5), (60 / 25 / 10 / 5), (60 / 27 / 12 / 1), (60 / 27 / 11.5 / 1.5), (60 / 27 / 11 / 2), (60 / 27 / 10.5 / 2.5), (60 / 27 / 9.5 / 3.5), (60 / 27 / 9 / 4), (60 / 27 / 8.5 / 4.5), (60 / 27 / 8 / 5), (59 / 28 / 10 / 3), (58 / 29 / 10 / 3), (57 / 30 / 10 / 3), (56 / 31 / 10 / 3), (55 / 32 / 10 / 3), (50 / 39 / 10 / 1), (50 / 38.5 / 10 / 1.5), (50 / 38 / 10 / 2), (50 / 37.5 / 10 / 2.5), (50 / 37 / 10 / 3), (50 / 36.5 / 10 / 3.5), (50 / 36 / 10 / 4), (50 / 35.5 / 10 / 4.5), (50 / 35 / 10 / 5), (50 / 38.5 / 10.5 / 1), (50 / 38.5 / 9.5 / 2),(50 / 38.5 / 9 / 2.5), (50 / 38.5 / 8.5 / 3), (50 / 38.5 / 8 / 3.5), (50 / 38.5 / 7.5 / 4), (50 / 38.5 / 7 / 4.5), (50 / 38.5 / 6.5 / 5), (51 / 37.5 / 10 / 1.5), (52 / 36.5 / 10 / 1.5), (53 / 35.5 / 10 / 1.5), (54 / 34.5 / 10 / 1.5), (55 / 33.5 / 10 / 1.5), (49 / 39.5 / 10 / 1.5), (48 / 40.5 / 10 / 1.5), (47 / 41.5 / 10 / 1.5), (46 / 42.5 / 10 / 1.5), (45 / 43.5 / 10 / 1.5), (51 / 34 / 10 / 5), (52 / 3 3 / 10 / 5), (53 / 32 / 10 / 5), (54 / 31 / 10 / 5), (55 / 30 / 10 / 5), (40 / 44 / 15 / 1), (40 / 43.5 / 15 / 1.5), (40 / 43 / 15 / 2), (40 / 42.5 / 15 / 2.5), (40 / 42 / 15 / 3), (40 / 41.5 / 15 / 3.5), (40 / 4 1 / 15 / 4), (40 / 40.5 / 15 / 4.5), (40 / 40 / 15 / 5), (41 / 39 / 15 / 5), (42 / 38 / 15 / 5), (43 / 37 / 15 / 5), (44 / 36 / 15 / 5), (45 / 35 / 15 / 5), (41 / 41 / 14 / 4), (42 / 42 / 13 / 3), (43 / 43 / 12 / 2), and (44 / 44 / 11 / 1).

[0060] The lipid nanoparticles according to the present invention can be subjected to appropriate surface modification, etc., as necessary. The lipid nanoparticles according to the present invention can have improved blood retention by modifying the surface with a hydrophilic polymer or the like. Surface modification can sometimes be achieved by using lipids modified with these modifying groups as constituent lipids of the lipid nanoparticles.

[0061] In producing the lipid nanoparticles according to the present invention, examples of lipid derivatives that can be used to enhance blood retention include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, etc. Furthermore, in addition to polyalkylene glycols, hydrophilic polymers that can be used for surface modification to enhance blood retention include dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, and carrageenan.

[0062] Furthermore, to promote nuclear translocation of the lipid nanoparticles according to the present invention, the surface of the lipid nanoparticles can be modified with, for example, an oligosaccharide compound of three or more sugars. The type of oligosaccharide compound of three or more sugars is not particularly limited, but for example, an oligosaccharide compound having about 3 to 10 sugar units bonded thereto can be used, and preferably an oligosaccharide compound having about 3 to 6 sugar units bonded thereto can be used. Among these, oligosaccharide compounds that are glucose trimers or hexamers are preferred, and oligosaccharide compounds that are glucose trimers or tetramers are even more preferred. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose are suitable for use, and among these, maltotriose, maltotetraose, maltopentaose, or maltohexaose in which glucose is α1-4-linked are even more preferred. Maltotriose or maltotetraose are particularly preferred, and maltotriose is the most preferred. The amount of surface modification of lipid nanoparticles with an oligosaccharide compound is not particularly limited, but is, for example, about 1 to 30 mol %, preferably about 2 to 20 mol %, and more preferably about 5 to 10 mol % of the total lipid amount.

[0063] The method for surface-modifying lipid nanoparticles with oligosaccharide compounds is not particularly limited, but for example, liposomes in which the surface of lipid nanoparticles is modified with monosaccharides such as galactose or mannose (WO 2007 / 102481) are known, and the surface modification method described in this publication can be adopted. The disclosure of the above publication is incorporated herein by reference in its entirety.

[0064] Furthermore, the lipid nanoparticles according to the present invention can be imparted with one or more of the following functions: temperature-sensitive function, membrane-permeable function, gene expression function, pH-sensitive function, etc. By appropriately imparting these functions, the retention of the lipid nanoparticles in the blood can be improved, and the lipid nanoparticles can be efficiently released from endosomes after endocytosis in target cells, allowing the encapsulated nucleic acid to be more efficiently expressed in or within liver cells.

[0065] The lipid nanoparticles of the present invention may contain one or more substances selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides. Examples of charged substances that impart a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine. Examples of charged substances that impart a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Examples of membrane polypeptides include membrane surface polypeptides and membrane integral polypeptides. The amounts of these substances to be incorporated are not particularly limited and can be selected appropriately depending on the purpose.

[0066] The size of the lipid nanoparticles according to the present invention is preferably an average particle diameter of 400 nm or less, more preferably an average particle diameter of 300 nm or less, even more preferably an average particle diameter of 200 nm or less, and even more preferably an average particle diameter of 150 nm or less, since this facilitates high delivery efficiency to liver cells or spleen cells in vivo. The average particle diameter of the lipid nanoparticles refers to the Z-average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be carried out by conventional methods using a commercially available DLS device or the like.

[0067] The polydispersity index (PDI) of the lipid nanoparticles according to the present invention is approximately 0.01 to 0.7, preferably approximately 0.01 to 0.6, and more preferably approximately 0.03 to 0.3. The zeta potential at pH 7.4 can be in the range of -50 mV to 5 mV, preferably -45 mV to 5 mV. The form of the lipid nanoparticles according to the present invention is not particularly limited, but examples of forms dispersed in an aqueous solvent include unilamellar liposomes, multilamellar liposomes, spherical micelles, and amorphous layered structures. The lipid nanoparticles according to the present invention are preferably unilamellar liposomes or multilamellar liposomes.

[0068] The lipid nanoparticles according to the present invention preferably contain a target component to be delivered into target cells within the lipid membrane-covered particles. The component contained within the lipid nanoparticles according to the present invention is not particularly limited as long as it has a size that allows it to be contained. The lipid nanoparticles according to the present invention can contain any substance, such as nucleic acids, sugars, peptides, low-molecular-weight compounds, and metal compounds.

[0069] Nucleic acids are preferred as components to be encapsulated in the lipid nanoparticles of the present invention. The nucleic acid may be DNA, RNA, or an analog or derivative thereof (e.g., peptide nucleic acid (PNA) or phosphorothioate DNA). The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention may be a single-stranded nucleic acid, a double-stranded nucleic acid, linear, or cyclic. In one aspect of the present invention, the lipid nanoparticles of the present invention comprise the pH-sensitive cationic lipid of the present invention, a stereoisomer or a mixture of stereoisomers thereof, and a nucleic acid.

[0070] The nucleic acid encapsulated in the lipid nanoparticles of the present invention preferably contains a foreign gene for expression in target cells, and more preferably is a nucleic acid that functions to express the foreign gene in the cells by being incorporated into the cells. The foreign gene may be a gene that is originally contained in the genomic DNA of the target cells (preferably liver cells and spleen cells), or it may be a gene that is not contained in the genomic DNA. Examples of such nucleic acids include gene expression vectors containing a nucleic acid consisting of a base sequence encoding the target gene to be expressed. The gene expression vector may exist as an extrachromosomal gene in the introduced cells, or it may be incorporated into the genomic DNA by homologous recombination.

[0071] The gene expression vector to be encapsulated in the lipid nanoparticles of the present invention is not particularly limited, and vectors commonly used in gene therapy, etc. can be used. The gene expression vector to be encapsulated in the lipid nanoparticles of the present invention is preferably a nucleic acid vector such as a plasmid vector. The plasmid vector may remain circular, or may be encapsulated in the lipid nanoparticles of the present invention after being pre-cleaved into a linear form. The gene expression vector can be designed by standard methods using commonly used molecular biology tools based on the base sequence information of the gene to be expressed, and can be produced by various known methods.

[0072] The nucleic acid encapsulated in the lipid nanoparticles of the present invention is preferably a functional nucleic acid that controls the expression of a target gene present in a target cell. Examples of such functional nucleic acids include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, and mRNA. Alternatively, the nucleic acid may be a plasmid DNA (pDNA) that serves as an siRNA expression vector for expressing siRNA in cells. The siRNA expression vector can be prepared from a commercially available siRNA expression vector, which may also be modified as appropriate. The nucleic acid encapsulated in the lipid nanoparticles of the present invention is preferably mRNA or pDNA, as it has particularly good selectivity for the liver or spleen. In one aspect of the present invention, the lipid nanoparticles of the present invention comprise the pH-sensitive cationic lipid of the present invention, a stereoisomer or a mixture of stereoisomers thereof, and a nucleic acid, wherein the nucleic acid is mRNA or plasmid DNA.

[0073] The method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be employed. For example, all lipid components are dissolved in an organic solvent such as chloroform, and a lipid membrane is formed by drying under reduced pressure using an evaporator or spray drying using a spray dryer. An aqueous solvent containing the components to be encapsulated in the lipid nanoparticles, such as nucleic acids, is then added to the dried mixture, and the resulting mixture is emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure injection emulsifier. Liposomes can also be produced by well-known methods for producing liposomes, such as reverse-phase evaporation. To control the size of lipid nanoparticles, extrusion filtration can be performed under high pressure using a membrane filter with a uniform pore size.

[0074] The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include buffer solutions such as phosphate buffer, citrate buffer, and phosphate-buffered saline, physiological saline, cell culture media, etc. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles, but may also contain sugars (aqueous solutions) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melezinose, polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, and polyhydric alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ethers, diethylene glycol monoalkyl ethers, and 1,3-butylene glycol. To stably store lipid nanoparticles dispersed in this aqueous solvent for a long period of time, it is desirable to eliminate electrolytes from the aqueous solvent as much as possible from the viewpoint of physical stability such as suppressing aggregation. Also, from the viewpoint of chemical stability of lipids, it is desirable to set the pH of the aqueous solvent to a weakly acidic to near-neutral range (pH 3.0 to 8.0) and / or remove dissolved oxygen by nitrogen bubbling or the like.

[0075] The lipid nanoparticles of the present invention can also be produced by an alcohol dilution method using a flow channel. This method involves introducing a solution of lipid components dissolved in an alcohol solvent and a solution of water-soluble components to be incorporated into the lipid nanoparticles into an aqueous solvent through separate flow channels and then merging them to produce lipid nanoparticles. By using a microchannel with a built-in three-dimensional micromixer, which can achieve instantaneous mixing of the two liquids, lipid nanoparticles with a diameter of approximately 30 nm can be reproducibly produced (Non-Patent Document 11). As the flow channel used for production, a simple two-dimensional flow channel structure, such as that described in Patent Document 2, in which a micro-sized flow channel for flowing a raw material solution is provided with baffles (baffles) of a fixed width arranged alternately on both sides of the flow channel, can be used, as this allows for the formation of a nano-sized lipid particle formation system with high particle size controllability. The aqueous solvent used in the alcohol dilution method can be the same as described above.

[0076] When the resulting aqueous dispersion of lipid nanoparticles is freeze-dried or spray-dried, stability may be improved by using a sugar (aqueous solution) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melezinose; polysaccharides such as cyclodextrin; or sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol. Furthermore, when the aqueous dispersion is frozen, stability may be improved by using a polyhydric alcohol (aqueous solution) such as the above-mentioned sugars or glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ethers, diethylene glycol monoalkyl ethers, and 1,3-butylene glycol. In one aspect of the present invention, the lipid nanoparticles according to the present invention are freeze-dried.

[0077] In one aspect, the present invention relates to a lipid nanoparticle formulation comprising the pH-sensitive cationic lipid of the present invention, its stereoisomer, or a mixture of stereoisomers. In another aspect, the present invention relates to a lipid nanoparticle formulation comprising (i) a sterol or a sterol derivative, (ii) a polyalkylene glycol-modified lipid, (iii) a nucleic acid, (iv) a buffer, (v) a disaccharide, and (vi) the pH-sensitive cationic lipid of the present invention, its stereoisomer, or a mixture of stereoisomers. Examples of sterols or sterol derivatives include cholesterol, sitosterol, etc., preferably cholesterol. Examples of polyalkylene glycol-modified lipids include polyethylene glycol-modified lipids, polypropylene glycol-modified lipids, etc., preferably polyethylene glycol-modified lipids. Examples of nucleic acids include siRNA, pDNA, mRNA, etc., preferably mRNA. Examples of buffers include HEPES buffer, phosphate buffer, Tris buffer, etc. Examples of disaccharides include lactose, sucrose, cellobiose, trehalose, and maltose, with sucrose being preferred. The concentration of the disaccharide in the lipid nanoparticle formulation is, for example, 1% to 20% by weight, preferably 5% to 15% by weight. The molar ratio of the sterol or sterol derivative to the pH-sensitive cationic lipid, its stereoisomer, or a mixture of stereoisomers is, for example, 68.5:20 to 28.5:60.

[0078] In the present invention, the lipid nanoparticle formulation may be prepared by suspending lipid nanoparticles in an aqueous solution. The pH of the lipid nanoparticle formulation of the present invention is, for example, 5.5 to 8.5, preferably 6.8 to 8.0, at 25° C. In one aspect, the present invention relates to a resuspended formulation in which the lipid nanoparticle formulation is resuspended by adding water or an aqueous solution.

[0079] The lipid nanoparticles of the present invention have excellent stability, for example, being stable for one week or more when stored at −80° C., and / or being stable for one week, two weeks, three weeks, four weeks, five weeks, or more when stored at 5° C., and / or being stable for one week, two weeks, three weeks, four weeks, five weeks, or more when stored at 25° C., and / or being stable for three days, one week, two weeks, three weeks, four weeks, five weeks, or more when stored at 40° C.

[0080] When lipid nanoparticles according to the present invention encapsulating a gene expression vector are administered to an animal, the gene expression vector encapsulated in the lipid nanoparticles is preferentially expressed in the liver or spleen over other organs. Similarly, when lipid nanoparticles according to the present invention encapsulating an siRNA expression vector are administered to an animal, the siRNA expression vector encapsulated in the lipid nanoparticles is preferentially expressed in the liver or spleen over other organs, thereby suppressing the expression of the gene targeted by the expression vector. For example, when lipid nanoparticles according to the present invention encapsulating a foreign gene to be expressed in liver cells or spleen cells are administered to a test animal, the foreign gene can be expressed in the liver or spleen of the test animal.

[0081] Due to this highly selective gene expression activity in the liver or spleen, the lipid nanoparticles of the present invention function as gene expression carriers targeting the liver or spleen. By encapsulating a foreign gene to be expressed in liver cells or spleen cells in the lipid nanoparticles of the present invention and then administering them to a subject animal, the foreign gene is expressed in the liver or spleen of the subject animal. Therefore, the lipid nanoparticles of the present invention are useful as an active ingredient in pharmaceutical compositions used in gene therapy, and are particularly useful as an active ingredient in pharmaceutical compositions used in gene therapy targeting the liver or spleen. In one aspect, the present invention relates to a pharmaceutical composition for liver delivery containing the pH-sensitive cationic lipid of the present invention, a stereoisomer thereof, or a mixture of stereoisomers. In another aspect, the present invention relates to a pharmaceutical composition for spleen delivery containing the pH-sensitive cationic lipid of the present invention, a stereoisomer thereof, or a mixture of stereoisomers.

[0082] The animals to which the lipid nanoparticles of the present invention are administered are not particularly limited and may be humans or non-human animals, including mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quails, and ducks.

[0083] I. Synthesis of pH-Sensitive Cationic Lipids 1. Synthesis of CL4F12-10 [Synthesis Example 1] Synthesis of CL4F12-10 CL4F12-10 was synthesized as follows. 7-(4-(dipropylamino)butyl)tridecane-1,7,13-triol (1.0 mmol) was dissolved in 4 mL of dichloromethane, followed by the addition of 2-decyldodecanoic acid (2.40 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol), and the reaction was carried out at room temperature overnight. The solvent was removed using a rotary evaporator, and the residue was suspended in ethyl acetate, followed by filtration to remove insoluble matter. The filtrate was washed with 0.5 N aqueous sodium hydroxide and saturated saline, and the organic layer was dehydrated by adding anhydrous sodium sulfate. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product. The crude product was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] and silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to obtain 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl bis(2-decyldodecanoate) (CL4F12-10).

[0084] 2. Synthesis of CL4F12-10 Derivatives [Synthesis Example 2] Synthesis of CL4F12-10 Derivatives The CL4F12-10 derivatives, CL4F12-10 / Ste, CL4F12-10 / Ole, CL4F12-10 / Lin, CL4F12-10 / Myr, CL4F12-10 / Myristole, and CL4F12-10 / Dec, were synthesized as follows. 7-(4-(dipropylamino)butyl)tridecane-1,7,13-triol (1.0 mmol) was dissolved in 4 mL of dichloromethane, followed by the addition of 2-decyldodecanoic acid (1.0 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol), and the reaction was carried out at room temperature overnight. After the solvent was removed using a rotary evaporator, the residue was suspended in ethyl acetate and then washed with 0.5 N aqueous sodium hydroxide and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product. The crude product was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] to give 7-(4-(dipropylamino)butyl)-1,7-hydroxytridecane-13-(2-decyldodecanoate).

[0085] Next, 7-(4-(dipropylamino)butyl)-1,7-hydroxytridecane-13-(2-decyl dodecanoate) (1.0 mmol) was dissolved in 4 mL of dichloromethane, and linear fatty acids (stearic acid, oleic acid, linoleic acid, myristic acid, myristoleic acid, capric acid) (1.2 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol) were added, followed by overnight reaction at room temperature. After the solvent was removed using a rotary evaporator, the resulting mixture was suspended in ethyl acetate and washed with 0.5 N aqueous sodium hydroxide and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product. The crude product was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] and silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to give CL4F12-10 / Ste, CL4F12-10 / Ole, CL4F12-10 / Lin, CL4F12-10 / Myr, CL4F12-10 / Myristole, and CL4F12-10 / Dec.

[0086] 3. Synthesis of CL15F12-4 Derivative [Synthesis Example 3] Synthesis of CL4F12-4 / Ole The synthesis of CL15F12-4 / Ole, a CL15F12-4 derivative, was carried out as follows. 5,11-Dihydroxy-5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate (1.0 mmol) was dissolved in 4 mL of dichloromethane, followed by the addition of 2-butyldodecanoic acid (1.0 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol), and the reaction was carried out at room temperature overnight. After the solvent was removed using a rotary evaporator, the mixture was suspended in ethyl acetate and then washed with 0.5 N aqueous sodium hydroxide and saturated saline. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product, which was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] to obtain 1,7-dihydroxy-7-(4-(1-methylpiperidine)-4-carbonyl)oxy)butyl)tridecane-13-(2-butyldodecanoate).

[0087] Subsequently, 1,7-dihydroxy-7-(4-(1-methylpiperidine)-4-carbonyl)oxy)butyl)tridecane-13-(2-butyldodecanoate) (1.0 mmol) was dissolved in 4 mL of dichloromethane, and the linear fatty acid oleic acid (1.2 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol) were added, followed by a reaction at room temperature overnight. After the solvent was removed using a rotary evaporator, the residue was suspended in ethyl acetate and then washed with 0.5 N aqueous sodium hydroxide and saturated saline, followed by separation and washing. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product. The crude product was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] and silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to obtain CL15F12-4 / Ole.

[0088] 4. Synthesis of CL15F11-9 Derivative [Synthesis Example 4] Synthesis of CL15F11-9 / Ole CL15F11-9 / Ole, a CL15F11-9 derivative, was synthesized as follows. 5,11-Dihydroxy-5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate (1.0 mmol) was dissolved in 4 mL of dichloromethane, followed by the addition of 2-nonylundecanoic acid (1.0 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol), and the reaction was carried out at room temperature overnight. After the solvent was removed using a rotary evaporator, the mixture was suspended in ethyl acetate and then washed with 0.5 N aqueous sodium hydroxide and saturated saline. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product, which was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] to obtain 1,7-dihydroxy-7-(4-(1-methylpiperidine)-4-carbonyl)oxy)butyl)tridecane-13-(2-nonylundecanoate).

[0089] Next, 1,7-dihydroxy-7-(4-(1-methylpiperidine)-4-carbonyl)oxy)butyl)tridecane-13-(2-nonylundecanoate) (1.0 mmol) was dissolved in 4 mL of dichloromethane, and the linear fatty acid oleic acid (1.2 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.10 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol) were added, followed by a reaction at room temperature overnight. After the solvent was removed using a rotary evaporator, the residue was suspended in ethyl acetate and then washed with 0.5 N aqueous sodium hydroxide and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was removed using a rotary evaporator to obtain a crude product. The crude product was purified by ODS-modified silica gel chromatography [eluent: water (0.1% TFA):acetonitrile (0.1% TFA), (continuous gradient)] and silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to obtain CL15F11-9 / Ole.

[0090] II. Preparation and Evaluation of Lipid Nanoparticles 1. Preparation of Buffer Solution (1) Preparation of Citrate Buffer Solution 6.90 g of citric acid (Fujifilm Wako Pure Chemical Industries, Ltd., #030-05525) and 4.15 g of sodium citrate dihydrate (Aldrich, #W302600) were added to ultrapure water to prepare 1 L of citrate buffer solution (pH 3.5). (2) Preparation of HEPES Buffer Solution 90.0 g of sucrose (Fujifilm Wako Pure Chemical Industries, Ltd., #196-00015) and 20 ml of HEPES buffer solution (1 mol / L, Nacalai Tesque, #17557-94) were added to ultrapure water to prepare 1 L of HEPES buffer solution (pH 7.45). (3) Preparation of TE buffer solution TE buffer solution (pH 7.5) was prepared by diluting 20x RNase-free TE buffer solution (20x) (Invitrogen, #T11493) with DEPC-Treated Water (Nippon Gene, #314-90205) 20 times.

[0091] 2. Preparation of Lipid Nanoparticles Lipid nanoparticles were prepared by the alcohol dilution method using a microfluidic device. Specifically, the procedure was as follows. The pH-sensitive cationic lipids used were CL4F12-10, CL4F12-10 / Ste, CL4F12-10 / Ole, CL4F12-10 / Lin, CL4F12-10 / Myr, CL4F12-10 / Myristole, or CL4F12-10 / Dec. The pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG2000 (NOF Corp., #GM-020) were mixed in a molar ratio of 50:38.5:10:1.5 and diluted with ethanol to a total lipid concentration of 8 mM to prepare a lipid solution. The nucleic acids used were mRNA encoding firefly luciferase (Fluc) (TriLink BioTechnologies, #L-7202) or siRNA (siF7) directed against the mouse factor 7 gene (F7). The sequences of the siRNA directed against the mouse F7 gene are shown in Table 1. Nucleic acid solutions were prepared by diluting mRNA and siRNA with citrate buffer to 44.1 μg / ml and 128 μg / ml, respectively. Using a NanoAssemblr Benchtop (PRECISION NANOSYSTEMS), the prepared lipid and nucleic acid solutions were mixed in a microfluidic device at a flow rate ratio of 1:3 and a total flow rate of 12 ml / min, and then diluted 2-fold with HEPES buffer. The resulting lipid nanoparticle solution was dialyzed overnight at 5°C against HEPES buffer using a dialysis membrane with a molecular weight cutoff of 12-14 kD (REPLIGEN, Spectra / Por 4). The dialyzed lipid nanoparticle solution was concentrated using an ultrafiltration unit (Amicon Ultra-15) and stored at -80°C.

[0092] 3. Measurement of Nucleic Acid Encapsulation Rate in Lipid Nanoparticles The nucleic acid encapsulation rate of lipid nanoparticles was measured using Ribogreen reagent (Invitrogen, #R114491). Specifically, a solution for measuring the surface nucleic acid concentration of lipid nanoparticles (measurement solution A) was prepared by diluting the lipid nanoparticle solution with TE buffer. A solution for measuring the total nucleic acid concentration of lipid nanoparticles (measurement solution B) was prepared by diluting the lipid nanoparticle solution with TE buffer containing 1% (w / w) Triton-X100 (Sigma-Aldrich, #T8787). 100 μl of each measurement solution and 100 μl of Ribogreen reagent were mixed on a 96-well microplate, incubated at room temperature for 5 minutes, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm. The nucleic acid concentration was calculated using a calibration curve prepared over a nucleic acid concentration range of 0 to 2.5 μg / ml. The encapsulation rate of lipid nanoparticles was calculated using the following formula: Encapsulation rate %=(nucleic acid concentration in measurement solution B (μg / ml)−nucleic acid concentration in measurement solution A (μg / ml))÷nucleic acid concentration in measurement solution B (μg / ml)×100 The results are shown in Tables 2 and 3.

[0093] 4. Measurement of mean particle size and polydispersity index of lipid nanoparticles The lipid nanoparticle solution was diluted 25 times with PBS (-) (Nacalai Tesque, #14249-24) to prepare a measurement solution. The mean particle size (z) and polydispersity index (PDI) of the nanoparticles in the measurement solution were measured using an analyzer "Zetasizer Nano ZSP" (Malvern). The results are shown in Tables 2 and 3.

[0094]

[0095]

[0096] 5. Transfection of mRNA-encapsulated lipid nanoparticles into in vitro cultured cells. 24 hours before transfection, HepG2 cells or CHO-K1 cells were seeded at 5,000 cells / well in a 96-well plate (Corning, #3904). The medium used was DMEM, low glucose, pyruvate (Gibco, #11885-084) or DMEM / F-12 (Gibco, #11320-033) adjusted to a final concentration of 10% FBS and 1% penicillin / streptomycin. FLuc mRNA (TriLink BioTechnologies, #L-7202)-encapsulated lipid nanoparticles were transfected to a final concentration of 10 ng mRNA / well, and the cells were incubated at 37°C and 5% CO. ２ The cells were incubated at 37°C, 5% CO. 24 hours after transfection, the luminescence intensity per cell was quantified using the ONE-Glo + Tox Luciferase Reporter and Cell Viability Assay (Promega, #E7120) to evaluate the efficiency of mRNA delivery by lipid nanoparticles. Specifically, a fluorescent peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC) was added according to the kit's protocol, and the cells were incubated at 37°C, 5% CO. ２ After 2 hours of incubation in the 100% CI 0.01 to 0.01 environment, the fluorescence intensity was measured and a viable cell count assay was performed. Next, the substrate for the ONE-Glo Luciferase Assay was added, and 3 minutes later, the luminescence intensity was measured. The luminescence intensity was corrected for the number of viable cells to calculate the relative luminescence intensity.

[0097] The results are shown in Figure 1. The higher the relative luminescence intensity, the higher the in vitro Fluc activity, indicating that the Fluc-encoding mRNA was efficiently introduced into living cells and that the encapsulated gene was more efficiently expressed in vitro. All six types of lipid nanoparticles containing pH-sensitive cationic lipids with asymmetric hydrophobic scaffolds exhibited higher in vitro FLuc activity than CL4F12-10, which contains symmetric hydrophobic scaffolds. These results indicate that lipid nanoparticles containing CL4F12-10 / Ste, CL4F12-10 / Ole, CL4F12-10 / Lin, CL4F12-10 / Myr, CL4F12-10 / Myristole, and CL4F12-10 / Dec as constituent lipids are useful as mRNA delivery carriers.

[0098] 6. mRNA Delivery to the Liver and Spleen by mRNA-Encapsulated Lipid Nanoparticles. Lipid nanoparticles encapsulating FLuc mRNA (TriLink Biotechnologies, #L-7202) were intravenously administered to 7-week-old female Balb / c mice at 0.1 mg mRNA / kg, and 6 hours later, Fluc activity was measured in the liver or spleen. VivoGlo Luciferin, In Vivo Grade (Promega, #P1041), dissolved in PBS at 15 mg / mL, was intravenously administered to each mouse at a dose of 1.5 mg. Luminescence intensity in the liver or spleen was measured using an in vivo imaging system (Perkin Elmer, IVIS200). The unit of Fluc activity is the luminescence intensity per unit area (Avg Radiance [p / s / cm² / sr]) at a maximum emission wavelength of approximately 560 nm. The results for the liver are shown in Figure 2(A), and the results for the spleen are shown in Figure 2(B). The vertical axis shows the luminescence intensity per unit area in the liver or spleen. The higher the luminescence intensity, the higher the in vivo Fluc activity, indicating that the mRNA encoding Fluc was efficiently introduced into the liver and spleen, and that the encapsulated gene was more expressed in the liver and spleen. All six types of lipid nanoparticles containing pH-sensitive cationic lipids with asymmetric hydrophobic scaffolds as their constituent lipids showed higher Fluc activity in the liver and spleen compared to CL4F12-10, which has a symmetric hydrophobic scaffold. From these results, it can be said that lipid nanoparticles containing CL4F12-10 / Ste, CL4F12-10 / Ole, CL4F12-10 / Lin, CL4F12-10 / Myr, CL4F12-10 / Myristole, and CL4F12-10 / Dec as constituent lipids are useful as mRNA delivery carriers.

[0099] 7. In vivo gene knockdown in hepatic parenchymal cells by siRNA-encapsulated lipid nanoparticles. 0.5 mg siRNA / kg of siF7-specific siRNA (siF7)-encapsulated lipid nanoparticles were intravenously administered to C57BL / 6J mice (5-6 weeks old, female). 24 hours later, F7 knockdown activity in the liver was measured by quantitative real-time PCR. Seventy-two hours prior to lipid nanoparticle administration, C57BL / 6J mice received an intraperitoneal injection of 10 mL / kg of carbon tetrachloride (Fujifilm Wako Pure Chemical Industries, Ltd., #035-01273) diluted 1:10 with olive oil (Fujifilm Wako Pure Chemical Industries, Ltd., #150-00276). Organs were homogenized using QIAzol Lysis Reagent (QIAGEN, #79306) and a homogenizer (Multi Bead Shocker, Yasui Machinery). RNA was extracted using the RNeasy Plus Universal Mini Kit (QIAGEN, #73404). After reverse transcription using the PrimeScript RT reagent Kit (TAKARA, #RR037), F7 mRNA was quantified as the target gene and Atp5f1 mRNA as an internal control using TB Green Fast qPCR Mix (TAKARA, #RR430) and the QuantStudio5 Real-Time PCR System. The primer sequences used are listed in Table 4. The amount of F7 mRNA in the livers of untreated mice was set at 100%, and the percentage of F7 mRNA in the livers of mice administered siRNA-encapsulated lipid nanoparticles was calculated.

[0100] The results are shown in Figure 3. The vertical axis shows the amount of F7 mRNA relative to the amount of Atp5f1 mRNA as an internal standard (F7 / Atp5f1). The lower the F7 / Atp5f1 value, the higher the F7 knockdown activity. All six types of lipid nanoparticles containing pH-sensitive cationic lipids with an asymmetric hydrophobic scaffold as their constituent lipids exhibited higher F7 knockdown activity compared to CL4F12-10, whose hydrophobic scaffold has a symmetric structure. From these results, it can be said that lipid nanoparticles containing CL4F12-10 / Ste, CL4F12-10 / Ole, CL4F12-10 / Lin, CL4F12-10 / Myr, CL4F12-10 / Myristole, and CL4F12-10 / Dec as constituent lipids are useful as siRNA delivery carriers.

[0101] 8. Evaluation of Liver Damage Markers and Chemokine Production Following High-Dose LNP Administration. Lipid nanoparticles encapsulating siRNA against human PLK1 (siPLK1) were prepared in the same manner as lipid nanoparticles encapsulating siRNA against F7 (siF7). The sequence of siPLK1 is shown in Table 5. siPLK1-encapsulated lipid nanoparticles were intravenously administered to ICR mice (5-8 weeks old, female) at 5 mg siRNA / kg, and plasma ALT, AST, and MCP-1 (monocyte chemotactic protein-1) were measured 24 hours later. ALT and AST were measured using a Fujifilm VET Systems system, and MCP-1 was measured using the Mouse CCL2 / JE / MCP-1 Quantikine ELISA Kit 2nd Gen (R&D Systems, #MJE00B).

[0102] The results are shown in Figure 4. Compared to CL4F12-10, which has a symmetric hydrophobic scaffold, lipid nanoparticles composed of CL4F12-10 / Ole, which has an asymmetric hydrophobic scaffold, showed lower values ​​for all of the ALT, AST, and MCP-1 items. From these results, it can be said that compared to CL4F12-10, which has a symmetric hydrophobic scaffold, lipid nanoparticles composed of pH-sensitive cationic lipids with an asymmetric hydrophobic scaffold, even at high doses, showed lower values ​​for ALT and AST, which are said to reflect inflammation in the liver, and also lower values ​​for MCP-1, which is said to be produced when the inflammatory state is enhanced, making it less likely to cause an inflammatory reaction as an unintended side effect.

Claims

1. A compound of the following formula (I) (R １ ), (R ２ )C(OH),(CH ２ )a-(O-CO)b-X (I) where, R １ is the following formula (A) X ０ -COO-(CH ２ )p-(A) is a group represented by R ２ is represented by the following formula (B): ０ -COO-(CH ２ )q-(B) is a group represented by the formula: ０ and Y ０ is different, X ０ is represented by the following formula (C): -(CH ２ )r-CH(R １ａ ), (R ２ａ ), (C), r is an integer of 0 to 6, R １ａ and R ２ａ are each independently C １－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, a is an integer of 3 to 5, b is 0 or 1, and X is selected from any one of the following formulae (X-a) to (X-d): In the formula, d is an integer of 0 to 3; ３ａ and R ４ａ are each independently hydrogen or C １－５ is an alkyl group, where R ３ａ and R ４ａ do not bond to each other to form a ring, and Z １ , CH, CR ５ａ or N; Z ２ ~Z ７ are, when present, each independently NH, NR ５ａ , C.R. ６ａ R ６ｂ or O; R ５ａ is C １－５ is an alkyl group, R ６ａ and R ６ｂ are each independently hydrogen or C １－５ is an alkyl group, provided that in formula (X-b), Z １ ~Z ５ At least one of the groups contains a nitrogen atom, and in (X-c), Z １ ~Z ６ At least one of (X-d) contains a nitrogen atom, and Z １ ~Z ７ at least one of the groups contains a nitrogen atom, and does not form an N-N bond, an N-O bond, or an O-O bond in formulae (X-b) to (X-d), p is an integer of 4 to 12, and q is an integer of 4 to 12, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

2. Y ０ The compound according to claim 1 , or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein is a linear saturated or unsaturated hydrocarbon group.

3. R １ａ and R ２ａ At least one of ９－１６ The compound according to claim 1, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein the compound is an alkyl group.

4. R １ａ and R ２ａ However, both are C ９－１６ The compound according to claim 1, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein the compound is an alkyl group.

5. The compound according to claim 1, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein r is 0.

6. X is formula (X-a), d is 0, and R ３ａ and R ４ａ are each independently hydrogen or C １－５ or X is a group represented by formula (X-c), d is 0, and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , and Z ６ is CH ２ and Z ４ NR ５ａ and R ５ａ is C １－５ The compound according to claim 5 , wherein R is an alkyl group, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

7. X is formula (X-a), and R ３ａ and R ４ａ are each independently hydrogen or C １－５ or X is a group represented by formula (X-c) and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , Z ６ is CH ２ and Z ４ NR ５ａ and R ５ａ is C １－５ The compound according to claim 1 , wherein R is an alkyl group, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

8. X is formula (X-a), and R ３ａ and R ４ａ are each independently hydrogen or C １－５ or X is a group represented by formula (X-c) and Z is an alkyl group. １ is CH, and Z ２ , Z ３ , Z ５ , Z ６ is CH ２ and Z ４ NR ５ａ and R ５ａ is C １－５ is an alkyl group, ０ The compound according to claim 1 , wherein is a linear saturated or unsaturated hydrocarbon group, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

9. Y ０ But linear C ９－２１ 2. The compound according to claim 1, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, which is a saturated or unsaturated hydrocarbon group.

10. Y ０ But linear C １４－１８ 2. The compound according to claim 1, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, which is a saturated or unsaturated hydrocarbon group.

11. A compound represented by the following formula (II) or (III): In the formula, ０ is -(CH ２ )r-CH(R １ａ ), (R ２ａ ) is a group represented by the formula: r is an integer of 0 to 6; １ａ and R ２ａ are each independently C １－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, ０ and Y ０ or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein 12. A compound represented by the following formula (II) or (III): In the formula, ０ is -(CH ２ )r-CH(R １ａ ), (R ２ａ ) is a group represented by the formula: r is an integer of 0 to 6; １ａ and R ２ａ are each independently C ９－１６ is an alkyl group, ０ is a linear or branched, saturated or unsaturated hydrocarbon group, ０ and Y ０ or a stereoisomer thereof, or a salt thereof, or a mixture thereof, wherein 13. Which of the following compounds The compound according to claim 1, or a stereoisomer thereof, or a salt thereof, or a mixture thereof, selected from:

14. Lipid nanoparticles comprising the compound according to any one of claims 1 to 13, or a stereoisomer thereof, or a salt thereof, or a mixture thereof.

15. The lipid nanoparticle of claim 14, which contains a nucleic acid.

16. The lipid nanoparticle of claim 15, wherein the nucleic acid is siRNA.

17. The lipid nanoparticle of claim 15, wherein the nucleic acid is mRNA.

18. The lipid nanoparticle of claim 17, wherein the nucleic acid is a gene to be expressed in liver or spleen cells.

19. A pharmaceutical composition containing the lipid nanoparticles of claim 14 as an active ingredient.

20. A method for expressing a foreign gene, comprising administering the lipid nanoparticles described in claim 14, which encapsulate a foreign gene to be expressed in liver or spleen cells, to a subject animal (excluding humans), and expressing the foreign gene in the liver or spleen of the subject animal.

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