PH-sensitive cationic lipid, lipid nanoparticles, pharmaceutical composition containing lipid nanoparticles, and use of lipid nanoparticles

Abstract

The present invention relates to a pH-sensitive cationic lipid, a lipid nanoparticle, a pharmaceutical composition containing the lipid nanoparticle, and a use of the lipid nanoparticle. A lipid nanoparticle according to the present invention is useful as a gene delivery carrier that can be selectively delivered to the liver or spleen, the lipid nanoparticle containing a pH-sensitive cationic lipid represented by the following formula (I): (In formula (I), a represents an integer of 3-5; b represents 0 or 1; r1 and R2 each independently represent a group represented by general formula (A); in formula (A), R11 and R12 each independently represent a linear or branched C2-15 alkyl group; c represents 0 or 1: v represents an integer from 4 to 12; x represents a group represented by general formula (B) or a 5-to 7-membered non-aromatic heterocyclic group; in formula (B), d represents an integer of 0-3; r3 and R4 each independently represent a C1-4 alkyl group or a C2-4 alkenyl group, and R3 and R4 may be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring.

Description

[0001] This application is a divisional application of the Chinese Patent Application No. 202180067522.1, filed on October 1, 2021, which claims priority from Chinese Patent Application No. 202010099593.9, filed on October 2, 2020, with the title of “Lipid Nanoparticle”. TECHNICAL FIELD

[0002] The present application relates to a lipid nanoparticle useful as a gene delivery carrier that is selectively delivered to the liver or spleen. BACKGROUND

[0003] As a carrier for encapsulating a fat-soluble drug, siRNA (short interfering RNA), mRNA, and the like, and delivering it to a target cell, a lipid nanoparticle (LNP) is used. For example, as a lipid nanoparticle for efficiently delivering a nucleic acid such as siRNA into a target cell, a lipid nanoparticle containing a pH-sensitive cationic lipid that is electrically neutral at physiological pH and is converted into a cationic property at a weakly acidic pH environment such as an endosome is reported as a constituent lipid (Patent Literature 1 and Non-Patent Literature 1).

[0004] As a pH-sensitive cationic lipid, for example, Jayaraman et al. developed DLin-MC3-DMA, which achieved an ED50 of 0.005 mg siRNA / kg in Factor VII (F7) knockdown in mouse liver 50 (Non-Patent Literature 2). The present inventors have also developed their own pH-sensitive cationic lipids YSK05 and YSK13-C3, which achieved ED50 of 0.06, 0.015 mg siRNA / kg, respectively, in F7 knockdown 50 (Non-Patent Literatures 3-5). In addition, Maier et al. developed L319 obtained by imparting biodegradability to MC3-DMA, and reported that it achieved an ED50 of 0.01 mg siRNA / kg 50 and high safety (Non-Patent Literatures 6-8). However, it has also been clarified that the endosome escape efficiency of the lipid nanoparticle containing these pH-sensitive cationic lipids is still only a few percent or so (Non-Patent Literature 9), and it is desired to develop a technology that can further improve bioavailability.

[0005] In addition, Dong et al. found their own lipid-like substance cKK-E12 through high-throughput screening, which achieved an ED50 of 0.002 mg siRNA / kg in F7 knockdown 50 (Non-Patent Literature 10). This technology is the most excellent in terms of activity on the literature level, but there is no insight into safety such as toxicity at high dose, biodegradability of lipids.

[0006] Prior Art Documents Patent Documents Patent Document 1: International Publication No. 2018 / 230710 Patent Document 2: International Publication No. 2018 / 190423 Non-Patent Documents Non-Patent Document 1: Sato et al., Journal of Controlled Release, 2019, vol.295, p.140-152. Non-Patent Document 2: Jayaraman et al., Angewandte Chemie International Edition, 2012, vol.51, p.8529-8533. Non-Patent Document 3: Watanabe et al., Scientific Reports, 2014, 4:4750, DOI: 10.1038 / srep04750. Non-Patent Document 4: Yamamoto et al., Journal of Hepatology, 2016, vol.64, p.547-555. Non-Patent Document 5: Sato et al., Molecular Therapy, 2016, vol.24, p.788-795. Non-Patent Document 6: Maier et al., Molecular Therapy, 2013, vol.21(8), p.1570-1578. Non-Patent Document 7: Wittrup et al., Nature Biotechnology, 2015, vol.33(8), p.870-876. Non-Patent Document 8: Xu et al., Molecular Pharmaceutics, 2014, vol.11, p.1424-1434. Non-Patent Document 9: Gilleron et al., Nature Biotechnology, 2013, vol.31(7), p.638-646. Non-Patent Literature 10: Dong, Proceedings of the National Academy of Sciences of the United States of America, 2014, vol. 111(11), p. 3955-3960. Non-Patent Literature 11: Leung et al., Journal of Physical Chemistry C SUMMARY

[0007] PROBLEMS TO BE SOLVED BY THE INVENTION An object of the present application is to provide a lipid nanoparticle which can be used as a gene delivery carrier selectively delivered to the liver or spleen, and to provide a lipid nanoparticle which is excellent in stability.

[0008] MEANS FOR SOLVING THE PROBLEMS The present inventors have found that a lipid nanoparticle containing a pH-sensitive cationic lipid having a branched hydrocarbon chain as a constituent lipid is high in selectivity for the liver or spleen, and is useful as a gene delivery carrier which is specifically highly expressed in the liver or spleen, thereby completing the present application.

[0009] That is, the present application provides a lipid nanoparticle represented by the following formula (I).

[0010] [1-1] A lipid nanoparticle containing a pH-sensitive cationic lipid represented by the following formula (I): [Chemical Formula 1] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represent a group represented by the following general formula (A); [Chemical Formula 2] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 5-15 alkyl group; c represents 0 or 1; and v represents an integer of 4 to 12). X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (wherein the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 3] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represents C 1-4 alkyl or C 2-4 alkenyl (in the C 1-4 alkyl or C 2-4 alkenyl, one or two hydrogen atoms can be substituted with phenyl), R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring can be substituted with C 1-4 alkyl or C 2-4 alkenyl).

[0011] [1-2] The lipid nanoparticle according to the above [1-1], further comprising a sterol and a polyalkylene glycol-modified lipid.

[0012] [1-3] The lipid nanoparticle according to the above [1-1] or [1-2], comprising a nucleic acid.

[0013] [1-4] The lipid nanoparticle according to the above [1-3], wherein the nucleic acid is siRNA.

[0014] [1-5] The lipid nanoparticle according to the above [1-3], wherein the nucleic acid is mRNA or plasmid DNA.

[0015] [1-6] The lipid nanoparticle according to any one of the above [1-3] to [1-5], wherein the nucleic acid is a gene to be expressed in a liver cell.

[0016] [1-7] A pharmaceutical composition comprising the lipid nanoparticle according to any one of the above [1-1] to [1-6] as an effective ingredient.

[0017] [1-8] The pharmaceutical composition according to the above [1-7], which is used for gene therapy.

[0018] [1-9] A method for expressing a foreign gene, wherein a lipid nanoparticle in which a target foreign gene to be expressed in a liver cell is encapsulated as the lipid nanoparticle according to any one of the above [1-1] to [1-6] is administered to a test animal (wherein a human is excluded), and the foreign gene is expressed in the liver of the test animal.

[0019] [2-1] A lipid nanoparticle comprising a pH-sensitive cationic lipid represented by the following formula (I), a stereoisomer thereof or a mixture of stereoisomers thereof, and a nucleic acid, wherein the nucleic acid is mRNA or plasmid DNA: [Chemical Formula 4] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represent a group represented by the following general formula (A); [Chemical Formula 5] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group; c represents 0 or 1; and v represents an integer of 4 to 12). X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (in which the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 6] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group (in which one or two hydrogen atoms of the C 1-4 alkyl group or the C 2-4 alkenyl group can be substituted with a phenyl group), and R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring (in which one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group).

[0020] The lipid nanoparticle according to [2-1], wherein the pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 7-1] [Chemical Formula 7-2] [Chemical Formula 7-3] [Chemical Formula 7-4] [Chemical Formula 7-5] [Chemical Formula 7-6] [Chemical Formula 7-7] [Chemical Formula 7-8] [3-1] A pharmaceutical composition for spleen delivery, comprising a pH-sensitive cationic lipid represented by the following formula (I), a stereoisomer thereof, or a mixture of stereoisomers thereof: [Chemical Formula 8] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represent a group represented by the following general formula (A); [Chemical Formula 9] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group; c represents 0 or 1; and v represents an integer of 4 to 12). X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (wherein the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 10] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group (one or two hydrogen atoms of the C 1-4 alkyl group or the C 2-4 alkenyl group can be substituted with a phenyl group), and R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group).

[0021] [3-2] The pharmaceutical composition according to [3-1], wherein the pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 11-1] [Chemical Formula 11-2] [Chemical Formula 11-3] [Chemical Formula 11-4] [Chemical Formula 11-5] [Chemical Formula 11-6] [Chemical Formula 11-7] [Chemical Formula 11-8] [4-1] A pH-sensitive cationic lipid represented by the following formula (I), a stereoisomer thereof or a mixture of stereoisomers thereof: [Chemical Formula 12] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represent a group represented by the following general formula (A); [Chemical Formula 13] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group; c represents 0 or 1; and v represents an integer of 4 to 12); X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (wherein the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO)b- through a carbon atom, and one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 14] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group (the C 1-4alkyl or C 2-4 In the alkenyl group, one or two hydrogen atoms can be substituted with a phenyl group, R 3 and R 4 may be mutually bonded to form a 5- to 7-membered non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring can be substituted with C 1-4 alkyl or C 2-4 alkenyl); wherein the pH-sensitive cationic lipid of the following formula is excluded: [Chemical Formula 15] .

[0022] [4-2] The pH-sensitive cationic lipid according to [4-1], a stereoisomer thereof or a mixture of stereoisomers thereof, wherein the pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 16-1] [Chemical Formula 16-2] [Chemical Formula 16-3] [Chemical Formula 16-4] [Chemical Formula 16-5] [Chemical Formula 16-6] [Chemical Formula 16-7] [Chemical Formula 16-8] [Chemical Formula 16-9] [Chemical Formula 16-10] [Chemical Formula 16-11] [5-1] 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) a pH-sensitive cationic lipid represented by the following formula (I), a stereoisomer thereof, or a mixture of stereoisomers thereof: [Chemical Formula 17] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represent a group represented by the following general formula (A); [Chemical Formula 18] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group; c represents 0 or 1; and v represents an integer of 4 to 12). X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (wherein the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO)b- via a carbon atom, and one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 19] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group (one or two hydrogen atoms of the C 1-4 alkyl group or the C 2-4 alkenyl group can be substituted with a phenyl group), and R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group).

[0023] [5-2] The lipid nanoparticle formulation according to [5-1], wherein the pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 20-1] [Chemical Formula 20-2] [Chemical Formula 20-3] [Chemical Formula 20-4] [Chemical Formula 20-5] [Chemical Formula 20-6] [Chemical Formula 20-7] [Chemical Formula 20-8] [Chemical Formula 20-9] [Chemical Formula 20-10] [Chemical Formula 20-11] [5-3] The lipid nanoparticle formulation according to [5-1] or [5-2], wherein the nucleic acid is mRNA.

[0024] [5-4] The lipid nanoparticle formulation according to any one of [5-1] to [5-3], wherein the lipid nanoparticle is suspended in an aqueous solution.

[0025] [5-5] The lipid nanoparticle formulation according to [5-4], wherein the concentration of the disaccharide is 1% to 20% by weight.

[0026] [5-6] The lipid nanoparticle formulation according to any one of [5-4] or [5-5], which is at pH 6.8 to pH 8.0 at 25°C.

[0027] [5-7] The lipid nanoparticle formulation according to any one of [5-1] to [5-3], which is freeze-dried.

[0028] [5-8] A re-suspension formulation, which is obtained by adding water or an aqueous solution to the lipid nanoparticle formulation of [5-7].

[0029] [6-1] A method for producing a pH-sensitive cationic lipid represented by the following formula (I), [Chemical Formula 21] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represents a group represented by the following general formula (A); [Chemical Formula 22] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group; c represents 0 or 1; v represents an integer of 4 to 12); X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (in which the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO) b- through a carbon atom, one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 23] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group (in the C 1-4 alkyl group or C 2-4 alkenyl group, one or two hydrogen atoms can be substituted with a phenyl group), R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group); wherein the chemical formula of the pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 24] The production method includes at least a step of reacting an alkyl carboxylic acid and a haloalkane in the presence of an organolithium, dimethylpropylene urea (DMPU), and tetrahydrofuran (THF) to obtain a branched fatty acid.

[0030] [6-2] The production method according to [6-1], wherein the volume ratio of the tetrahydrofuran (THF) to the dimethylpropylene urea (DMPU) in the step is 10: 1 to 1: 1 (v / v).

[0031] [6-3] The production method according to [6-1] or [6-2], wherein the organolithium is lithium diisopropylamide (LDA).

[0032] [6-4] The production method according to any one of [6-1] to [6-3], wherein the haloalkane is an iodoalkane.

[0033] [6-5] The production method according to any one of [6-1] to [6-4], further comprising a step of refining the branched fatty acid by reverse phase chromatography.

[0034] [6-6] A production method of a pH-sensitive cationic lipid represented by the following formula (I), a stereoisomer thereof, or a mixture of stereoisomers thereof, [Chemical Formula 25] (In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represent a group represented by the following general formula (A); [Chemical Formula 26] (In formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group; c represents 0 or 1; and v represents an integer of 4 to 12). X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group (wherein the 5- to 7-membered non-aromatic heterocyclic group can be bonded to (O-CO)b- via a carbon atom, and one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group): [Chemical Formula 27] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group (one or two hydrogen atoms of the C 1-4 alkyl group or the C 2-4 alkenyl group can be substituted with a phenyl group), and R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring can be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group); wherein the chemical formula of the pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 28-1] [Chemical Formula 28-2] [Chemical Formula 28-3] [Chemical Formula 28-4] [Chemical Formula 28-5] [Chemical Formula 28-6] [Chemical Formula 28-7] [Chemical Formula 28-8] [Chemical Formula 28-9] [Chemical Formula 28-10] [Chemical Formula 28-11] [Chemical Formula 28-12] The production method includes at least a step of obtaining a branched fatty acid by subjecting a reaction liquid obtained by reacting a malonic acid ester with a haloalkane in the presence of a base to a hydrolysis treatment and a heating treatment.

[0035] [6-7] The production method according to [6-6], wherein the malonic acid ester is dimethyl malonate.

[0036] [6-8] The production method according to [6-6] or [6-7], wherein the haloalkane is an iodoalkane.

[0037] [6-9] The production method according to any one of [6-6] to [6-8], wherein the base is selected from the group consisting of sodium hydride, calcium hydride, sodium ethoxide, and lithium bis(trimethylsilyl)amide.

[0038] [6-10] The production method according to any one of [6-6] to [6-9], wherein the hydrolysis treatment uses any one of sodium hydroxide, calcium hydroxide, and lithium hydroxide.

[0039] [6-11] The production method according to any one of [6-6] to [6-10], wherein the heating treatment is performed at 120°C to 170°C simultaneously with and / or after the hydrolysis treatment.

[0040] [6-12] The production method according to any one of [6-6] to [6-11], further comprising a step of refining the branched fatty acid by reverse phase chromatography.

[0041] Effects of the Invention The lipid nanoparticle of the present application can allow the encapsulated gene to be highly expressed in the liver or spleen. Therefore, the lipid nanoparticle is useful as a liver-specific gene delivery carrier or a spleen-specific gene delivery carrier used in gene therapy. In addition, the stability of the lipid nanoparticle of the present application is excellent. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a graph showing the results of measuring the pKa of each F7 siRNA-carrying lipid nanoparticle in Example 1. Figure 1 (A) is the results of the lipid nanoparticle prepared using CL4F6, CL4G6, or CL4H6, Figure 1 (B) is the results of the lipid nanoparticle prepared using CL15F6, CL15G6, or CL15H6.

[0043] Figure 2 is a graph showing the results of calculating the relative plasma F7 enzyme activity (%) of mice administered with each F7 siRNA-carrying lipid nanoparticle in Example 1. Figure 2 (A) is the results of mice administered with the lipid nanoparticle prepared using CL4F6, CL4G6, or CL4H6, Figure 2 (B) is the results of mice administered with the lipid nanoparticle prepared using CL15F6, CL15G6, or CL15H6.

[0044] Figure 3 is a graph showing the results of measuring the Nluc activity (RLU / mg protein) in the liver and spleen of mice administered with each Nluc mRNA-carrying lipid nanoparticle in Example 2.

[0045] Figure 4 is a graph showing the results of measuring the Fluc activity of HeLa-GFP cells into which each pFluc-carrying lipid nanoparticle was introduced in Example 3.

[0046] Figure 5 is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver and spleen of mice administered with each pFluc-carrying lipid nanoparticle in Example 3. DETAILED DESCRIPTION

[0047] Hereinafter, the embodiments of the present application will be specifically described. In the present application, "X1~X2 (X1 and X2 are real numbers satisfying X1X2)" means "X1 or more and X2 or less".

[0048] The lipid nanoparticle of the present application is a lipid nanoparticle containing a pH-sensitive cationic lipid represented by the following general formula (I) (hereinafter sometimes referred to as "pH-sensitive cationic lipid of the present application"). As a constituent lipid of the lipid nanoparticle, by having the pH-sensitive cationic lipid represented by the general formula (I), the lipid nanoparticle of the present application is high in selectivity to the liver or the spleen.

[0049] [Chemical Formula 29] In the general formula (I), a represents an integer of 3 to 5, and is preferably 4.

[0050] b represents 0 or 1. When b is 0, the -O-CO- group is not present, and means a single bond.

[0051] In the general formula (I), R 1 and R 2 each independently represent a group represented by the following general formula (A). In the general formula (A), R 11 and R 12 each independently represent a linear or branched C 2-15 alkyl group (alkyl group having 2 to 15 carbon atoms); c represents 0 or 1; and v represents an integer of 4 to 12.

[0052] [Chemical Formula 30] As the linear or branched C 2-15 alkyl group, the following can be mentioned: n-ethyl group; n-propyl group, 1-methylethyl group; n-butyl group, 1-methylpropyl group, 2-methylpropyl group, 1,1-dimethylethyl group; n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1-ethylpropyl group, 1,1-dimethylpropyl group, 2,2-dimethylpropyl group; n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutyl group, 3,3-dimethylbutyl group, 1,2-dimethylbutyl group, 1-methyl-2,2-dimethylbutyl group; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 9-methylnonyl, 1- ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 8,8-dimethyloctyl, 1- methyl-7,7-dimethyloctyl, 2-methyl-7,7-dimethyloctyl, 3-methyl-7,7- dimethyloctyl, 4-methyl-7,7-dimethyloctyl, 5-methyl-7,7-dimethyloctyl, 6-methyl- 7,7-dimethyloctyl, 7-methyl-7,7-dimethyloctyl, 8-methyl-7,7-dimethyloctyl; n-decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 9-methylnonyl, 1- ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 8,8-dimethyloctyl, 1- methyl-7,7-dimethyloctyl, 2-methyl-7,7-dimethyloctyl, 3-methyl-7,7- dimethyloctyl, 4-methyl-7,7-dimethyloctyl, 5-methyl-7,7-dimethyloctyl, 6-methyl- 7,7-dimethyloctyl, 7-methyl-7,7-dimethyloctyl, 8-methyl-7,7-dimethyloctyl; Tetradecyl, 1-methyltridecyl, 2-methyltridecyl, 3-methyltridecyl, 4-methyltridecyl, 5-methyltridecyl, 6-methyltridecyl, 7-methyltridecyl, 8-methyltridecyl, 9-methyltridecyl, 10-methyltridecyl, 11-methyltridecyl, 12-methyltridecyl, 1-ethyldodecyl, 1,1-dimethyldodecyl, 2,2-dimethyldodecyl, 3,3-dimethyldodecyl, 4,4-dimethyldodecyl, 5,5-dimethyldodecyl, 6,6-dimethyldodecyl, 7,7-dimethyldodecyl, 8,8-dimethyl Dodecyl, 9,9-dimethyldodecyl, 10,10-dimethyldodecyl, 11,11-dimethyldodecyl, 1-methyl-10,10-dimethylundecyl, 2-methyl-10,10-dimethylundecyl, 3-methyl-10,10-dimethylundecyl, 4-methyl-10,10-dimethylundecyl, 5-methyl-10,10-dimethylundecyl, 6-methyl-10,10-dimethylundecyl, 7-methyl-10,10-dimethylundecyl, 8-methyl-10,10-dimethylundecyl, 9-methyl-10,10-dimethylundecyl; Pentadecyl, 1-Methyltetradecyl, 2-Methyltetradecyl, 3-Methyltetradecyl, 4-Methyltetradecyl, 5-Methyltetradecyl, 6-Methyltetradecyl, 7-Methyltetradecyl, 8-Methyltetradecyl, 9-Methyltetradecyl, 10-Methyltetradecyl, 11-Methyltetradecyl, 12-Methyltetradecyl, 13-Methyltetradecyl, 1-Ethyltridecyl, 1,1-Dimethyltridecyl, 2,2-Dimethyltridecyl, 3,3-Dimethyltridecyl, 4,4-Dimethyltridecyl, 5,5-Dimethyltridecyl, 6,6-Dimethyltridecyl, 7,7-Dimethyltridecyl, 8,8-Dimethyltridecyl, 9,9-Dimethyl Tridecyl, 10,10-dimethyltridecyl, 11,11-dimethyltridecyl, 12,12-dimethyltridecyl, 1-methyl-11,11-dimethyldodecyl, 2-methyl-11,11-dimethyldodecyl, 3-methyl-11,11-dimethyldodecyl, 4-methyl-11,11-dimethyldodecyl, 5-methyl-11,11-dimethyldodecyl, 6-methyl-11,11-dimethyldodecyl, 7-methyl-11,11-dimethyldodecyl, 8-methyl-11,11-dimethyldodecyl, 9-methyl-11,11-dimethyldodecyl, 10-methyl-11,11-dimethyldodecyl, etc.

[0053] In general formula (A), R 11 and R 12each independently preferably a linear or branched C 2-12 alkyl (alkyl group having 2 to 12 carbon atoms), more preferably a linear or branched C 5-12 alkyl (alkyl group having 5 to 12 carbon atoms), further preferably a linear or branched C 5-10 alkyl (alkyl group having 5 to 10 carbon atoms), most preferably a linear or branched C 6-9 alkyl (alkyl group having 6 to 9 carbon atoms). In addition, in the pH-sensitive cationic lipid of the present application, R 1 and R 2 may be the same as each other or different from each other as long as they are a group represented by General Formula (A).

[0054] In General Formula (I), X represents a group represented by General Formula (B) below or a 5- to 7-membered non-aromatic heterocyclic group. The 5- to 7-membered non-aromatic heterocyclic group represented by X is bonded to (O-CO) b via a carbon atom.

[0055] [Chemical Formula 31] In General Formula (B), d represents an integer of 0 to 3. When d is 0, the - (CH2) - group is not present, and a single bond is meant.

[0056] In General Formula (B), R 3 and R 4 each independently represents a C 1-4 alkyl (alkyl group having 1 to 4 carbon atoms) or C 2-4 alkenyl (alkenyl group having 1 to 4 carbon atoms). R 3 and R 4 represented by R 1-4 alkyl or C 2-4 alkenyl. R 3 and R 4 may be the same as each other or different from each other as long as they are a C 1-4 alkyl or C 2-4 alkenyl. R

[0057] As the C 1-4 alkyl, there can be mentioned methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl. As the C 2-4 alkenyl, there can be mentioned vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl.

[0058] In General Formula (B), R 3 and R 4which can be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring. As R 3 which can be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring. As R 4 which can be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring. As R 3 which can be bonded to each other to form a 5- to 7-membered non-aromatic heterocyclic ring. As R 4 In the 5- to 7-membered non-aromatic heterocyclic ring which can be bonded to each other, one or two hydrogen atoms in the ring can be substituted with C 1-4 alkyl or C 2-4 alkenyl. When two hydrogen atoms in the ring are substituted with C 1-4 alkyl or C 2-4 alkenyl, the substituents can be the same or different from each other.

[0059] In General Formula (I), when X is a 5- to 7-membered non-aromatic heterocyclic group, as the heteroatom contained in the heterocyclic group, a nitrogen atom, an oxygen atom, or a sulfur atom, etc. can be given. The heteroatom constituting the heterocyclic ring in the heterocyclic group can be one, or two or more of the same or different heteroatoms. The heterocyclic ring in the heterocyclic group can be a saturated heterocyclic ring, or can contain one or two or more double bonds, but the heterocyclic ring will not be an aromatic ring.

[0060] As the pH-sensitive cationic lipid of the present application, in General Formula (I), a compound in which R 1 and R 2 each independently is R 11 and R 12 each independently is a linear or branched C 2-12 alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 1, and X is a 5- to 7-membered non-aromatic heterocyclic group (which can be bonded to (O-CO)b- via a carbon atom in the heterocyclic group), preferably a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (which can be bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom can be substituted with C 1-4 alkyl or C 2-4 alkenyl), or a compound in which R 1 and R 2 each independently is R 11 and R 12 each independently is a linear or branched C 2-12 alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 0, and X is a group in General Formula (B) in which d is 0, R 3 and R 4 each independently is a C 1-4 alkyl group or C 2-4 alkenyl group (R3 and R 4 C 1-4 alkyl or C 2-4 alkenyl (one or two hydrogen atoms in the C 1 and R 2 each independently R 11 and R 12 each independently a straight-chain or branched C 5-12 alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 1, and X is a 5- to 7-membered non-aromatic heterocyclic group (which can be bonded to (O-CO)b- via a carbon atom in the heterocyclic group), preferably 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl, or 1-piperazinyl (which can be bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom can be substituted with a C 1-4 alkyl or C 2-4 alkenyl group) ; or in the general formula (I), R 1 and R 2 each independently R 11 and R 12 each independently a straight-chain or branched C 5-12 alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 0, and X is R 3 and R 4 each independently a C 1-4 alkyl or C 2-4 alkenyl group (one or two hydrogen atoms in the C 3 and R 4 C 1-4 alkyl or C 2-4 alkenyl group). Among them, as the pH-sensitive cationic lipid of the present application, in the general formula (I), preferably R 1 and R 2 each independently R 11 and R 12 each independently a straight-chain or branched C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5, b is 1, and X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl, or 1-piperazinyl (which can be bonded to (O-CO)b- via a carbon atom in the ring, and one hydrogen atom can be substituted with a C 1-4 alkyl or C 2-4 alkenyl group) ; or in the general formula (I), R 1 and R 2 each independently R11 and R 12 each independently a straight-chain or branched C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5, b is 0, X is a group of the general formula (B) in which d is 0, R 3 and R 4 each independently a C 1-4 alkyl group.

[0061] As the pH-sensitive cationic lipid of the present application, more preferably a compound of the general formula (I) in which R 1 and R 2 each independently a group of the general formula (A) in which R 11 and R 12 each independently a straight-chain C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5, b is 1, X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (one hydrogen atom can be replaced with a C 1-4 alkyl group or a C 2-4 alkenyl group) ; R 1 and R 2 each independently a group of the general formula (A) in which R 11 and R 12 each independently a branched C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5, b is 1, X is a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (one hydrogen atom can be replaced with a C 1-4 alkyl group or a C 2-4 alkenyl group) ; R 1 and R 2 each independently a group of the general formula (A) in which R 11 and R 12 each independently a straight-chain C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5, b is 0, X is a group of the general formula (B) in which d is 0, R 3 and R 4 each independently a C 1-4 alkyl group; R 1 and R 2 each independently a group of the general formula (A) in which R 11 and R 12 each independently a branched C 6-9Alkyl groups, where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is the group in general formula (B) where d is 0 and R is 0. 3 and R 4 Each independently is C 1-4 Alkyl compounds.

[0062] As the pH-sensitive cationic lipid of the present invention, it is particularly preferred that R is in general formula (I). 1 and R 2 They are the same group and are R in general formula (A) 11 and R 12 Each is an independent linear C 6-9 Alkyl group, where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl, or 1-piperazinyl (which can be bonded to (O-CO)b- through a carbon atom in the ring, and one hydrogen atom can be bonded to C). 1-4 Alkyl or C 2-4 Compounds with alkenyl substitution; in general formula (I), R 1 and R 2 They are the same group and are R in general formula (A) 11 and R 12 Each is independently a branched C 6-9 Alkyl groups, where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, and X is 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl, or 1-piperazinyl (which can be bonded to (O-CO)b- through a carbon atom in the ring, and one hydrogen atom can be bonded to C). 1-4 Alkyl or C 2-4 Compounds with alkenyl substitution; in general formula (I), R 1 and R 2 They are the same group and are R in general formula (A) 11 and R 12 Each is an independent linear C 6-9 Alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, X is d in general formula (B) and R is 0. 3 and R 4 Each independently is C 1-4 Alkyl compounds; in general formula (I), R 1 and R 2 They are the same group and are R in general formula (A) 11 and R 12 Each is independently a branched C 6-9 Alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, X is d in general formula (B) and R is 0. 3and R 4 each independently is C 1-4 the compound of the alkyl group.

[0063] As the pH-sensitive cationic lipid of the present application, for example, a pH-sensitive cationic lipid having the following structure, a stereoisomer thereof or a mixture of stereoisomers thereof is exemplified: [Chemical Formula 32-1] [Chemical Formula 32-2] [Chemical Formula 32-3] [Chemical Formula 32-4] [Chemical Formula 32-5] [Chemical Formula 32-6] [Chemical Formula 32-7] [Chemical Formula 32-8] [Chemical Formula 32-9] [Chemical Formula 32-10] [Chemical Formula 32-11] [Chemical Formula 32-12] [Chemical Formula 32-13] [Chemical Formula 32-14] The present application relates to a pH-sensitive cationic lipid of the present application.

[0064] The pKa of the pH-sensitive cationic lipid represented by General Formula (I) is not particularly limited, and for example, can be selected in the range of about 4.0 to 9.0, preferably about 4.5 to 8.5, and preferably the kind of each substituent is selected in such a manner that the pKa in the range is imparted.

[0065] The pH-sensitive cationic lipids represented by General Formula (I) can be easily produced, for example, by the methods specifically described in the Examples of the present specification. By referring to the production methods, those skilled in the art can easily produce any lipids included in the scope of General Formula (I) by appropriately selecting raw material compounds, reagents, and reaction conditions, and the like.

[0066] The group represented by General Formula (A) is a group having a branched structure in which two hydrocarbon chains (R 11 and R 12 ) are connected to a -CO-O- group. That is, in the pH-sensitive cationic lipids of the present application, two branched hydrocarbon chains (R 1 and R 2 ) are present, and these hydrocarbon chains become hydrophobic scaffolds that are embedded in the lipid membrane of a lipid nanoparticle. The lipid nanoparticle of the present application has a characteristic of having high selectivity for the liver or the spleen by using the pH-sensitive cationic lipids of the present application having hydrophobic scaffolds composed of branched structures as a constituent component of the lipids.

[0067] The pH-sensitive cationic lipids of the present application that constitute the lipid nanoparticle of the present application can be only one kind, or two or more kinds. When the pH-sensitive cationic lipids of the present application that constitute the lipid nanoparticle of the present application are two or more kinds, the amount of the pH-sensitive cationic lipids of the present application refers to the total amount of the lipid molecules corresponding to the pH-sensitive cationic lipids of the present application among the lipid molecules that constitute the lipid nanoparticle.

[0068] The more the pH-sensitive cationic lipids of the present application are present in the lipid molecules that constitute the lipid nanoparticle, the higher the uptake efficiency of the lipid nanoparticle into target cells. Therefore, in the lipid nanoparticle of the present application, the proportion of the amount of the pH-sensitive cationic lipids of the present application with respect to the total amount of the lipids that constitute the lipid nanoparticle ([(amount (mol) of the pH-sensitive cationic lipids of the present application] / (amount (mol) of the total lipids that constitute the lipid nanoparticle)) x 100%) is preferably 20 mol% or more. On the other hand, if the proportion of the pH-sensitive cationic lipids in the lipid molecules that constitute the lipid nanoparticle is too high, it is sometimes difficult to sufficiently reduce the particle size. From the viewpoint of obtaining a lipid nanoparticle in which the uptake efficiency of the lipid nanoparticle into target cells is sufficiently high and the particle size is sufficiently small, the proportion of the amount of the pH-sensitive cationic lipids of the present application with respect to the total amount of the lipids that constitute the lipid nanoparticle of the present application is more preferably 30 mol% or more, further preferably 30 to 70 mol%, and even further preferably 40 to 60 mol%.

[0069] As the lipid other than the pH-sensitive cationic lipid of the present application in the constituent lipid of the lipid nanoparticle of the present application, a lipid used when a liposome is generally formed can be used. As such a lipid, for example, a phospholipid, a sterol or a sterol derivative, a glycolipid, or a saturated or unsaturated fatty acid, etc. can be exemplified. They can be used alone or two or more kinds thereof can be used in combination.

[0070] As the phospholipid, a glycerophospholipid such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, cardiolipin, plasmalogen, ceramide phosphoryl glycerophosphate, phosphatidic acid, etc.; a sphingomyelin such as sphingomyelin, ceramide phosphoryl glycerol, ceramide phosphoryl ethanolamine, etc. can be exemplified. In addition, a phospholipid derived from a natural product such as egg yolk lecithin, soybean lecithin, etc. can be used. The fatty acid residue in the glycerophospholipid and the sphingomyelin is not particularly limited, and for example, a saturated or unsaturated fatty acid residue having 12 to 24 carbon atoms, preferably a saturated or unsaturated fatty acid residue having 14 to 20 carbon atoms can be exemplified. Specifically, an acyl group derived from a fatty acid 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. can be exemplified. In the case where these glycerophospholipids or sphingolipids have two or more fatty acid residues, all of the fatty acid residues can be the same group, or can be the same group as each other.

[0071] As the sterol or the sterol derivative, for example, an animal-derived sterol such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, etc.; a plant-derived sterol (phytosterol) such as stigmasterol, sitosterol, β-sitosterol, campesterol, brassicasterol, etc.; a microorganism-derived sterol such as zymosterol, ergosterol, etc. can be exemplified. As the glycolipid, for example, a glyceroglycolipid such as sulfoxyribosyl glyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, glycosyldiglyceride, etc.; a sphingoglycolipid such as galactosylceramide, lactosylceramide, ganglioside, etc. can be exemplified. As the saturated or unsaturated fatty acid, for example, a saturated or unsaturated fatty acid having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, myristic acid, etc. can be exemplified.

[0072] As the constituent lipid of the lipid nanoparticle of the present application, in addition to the pH-sensitive cationic lipid of the present application, it is preferable that a neutral lipid is further contained, more preferable that a phospholipid or a sterol is further contained, further preferable that a sterol is further contained, and more further preferable that cholesterol is further contained.

[0073] The lipid nanoparticle of the present application preferably contains a polyalkylene glycol-modified lipid as a lipid component. The polyalkylene glycol is a hydrophilic polymer, and by using a polyalkylene glycol-modified lipid as a lipid to construct a lipid membrane to construct a lipid nanoparticle, the surface of the lipid nanoparticle can be modified with a polyalkylene glycol. By modifying the surface with a polyalkylene glycol, the stability of the lipid nanoparticle, such as blood retention, can sometimes be improved.

[0074] As the polyalkylene glycol, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, or the like can be used. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and further preferably about 1,000 to 5,000.

[0075] For example, in the modification of the lipid with polyethylene glycol, a stearylized polyethylene glycol (for example, stearic acid PEG45 (STR-PEG45), or the like) can be used. In addition, N- [carbonyl-methoxypolyethylene glycol-2000] -1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, n- [carbonyl-methoxypolyethylene glycol-5000] -1, 2-dipalmitoyl-sn-glycerol-3- phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol-750] -1, 2-distearyl-sn- glycerol-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol-2000] -1, 2- distearyl-sn-glycerol-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol- 5000] -1, 2-distearyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristyl-rac-glycerol-3- methoxypolyethylene glycol-2000 (PEG-DMG), and the like polyethylene glycol derivatives can also be used, but the polyalkylene glycol-modified lipid is not limited to these.

[0076] The proportion of the polyalkylene glycol-modified lipid to the total lipid amount constituting the lipid nanoparticle of the present application is not particularly limited as long as it does not impair the liver selectivity or the spleen selectivity of the pH-sensitive cationic lipid of the present application, and specifically, the liver-specific gene expression activity or the spleen-specific gene expression activity when the lipid nanoparticle of the present application is used as a gene carrier is not particularly limited. For example, the proportion of the polyalkylene glycol-modified lipid to the total lipid amount constituting the lipid nanoparticle is preferably 0.5 to 3 mol%.

[0077] The lipid nanoparticle of the present application can be appropriately surface-modified or the like as needed.

[0078] The lipid nanoparticle of the present application can improve blood retention by modifying the surface with a hydrophilic polymer or the like. By using a lipid modified with such a modification group as a constituent lipid of a lipid nanoparticle, surface modification can sometimes also be performed.

[0079] In the production of the lipid nanoparticles of the present application, as a lipid derivative for improving blood retention, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivative, glutamic acid derivative, polyglycerophospholipid derivative, and the like can also be used. In addition, as a hydrophilic polymer for improving blood retention, in addition to polyalkylene glycol, dextran, pullulan, Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, glycogen, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, and the like can also be used in surface modification.

[0080] In addition, in order to promote the intranuclear transfer of the lipid nanoparticles of the present application, for example, the lipid nanoparticles can also be surface-modified with an oligosaccharide compound of 3 or more sugars. The kind of the oligosaccharide compound of 3 or more sugars is not particularly limited, and for example, an oligosaccharide compound having 3 to 10 or so sugar units bonded thereto can be used, and an oligosaccharide compound having 3 to 6 or so sugar units bonded thereto is preferably used. Among them, an oligosaccharide compound belonging to a 3-mer or 6-mer of glucose is preferably used, and an oligosaccharide compound belonging to a 3-mer or 4-mer of glucose is further preferably used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose, and the like can be preferably used, and maltotriose, maltotetraose, maltopentaose, or maltohexaose having glucose bonded thereto via an a 1-4 bond is further preferably used. Maltotriose or maltotetraose is particularly preferably used, and maltotriose is most preferably used. The amount of surface modification of the lipid nanoparticles with the oligosaccharide compound is not particularly limited, and for example, 1 to 30 mol% or so, preferably 2 to 20 mol% or so, and more preferably 5 to 10 mol% or so, relative to the total lipid amount.

[0081] The method of surface-modifying the lipid nanoparticles with the oligosaccharide compound is not particularly limited, and for example, a liposome in which a monosaccharide such as galactose or mannose is known to be surface-modified (International Publication No. 2007 / 102481), and thus the surface modification method described in this publication can be employed. The entire disclosure of the above publication is incorporated by reference in the disclosure of the present application.

[0082] In addition, the lipid nanoparticles of the present application can be given any one or two or more of, for example, a temperature change sensitivity function, a membrane permeation function, a gene expression function, and a pH sensitivity function. By appropriately adding these functions, the retention of the lipid nanoparticles in the blood can be improved, the lipid nanoparticles can be efficiently released from the pinocytosis vesicle after endocytosis in the target cells, and the enclosed nucleic acid can be more efficiently expressed in the liver cells or in the liver cells.

[0083] The lipid nanoparticles of the present application can contain one or two or more substances selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charge substances, and membrane polypeptides. As charge substances that impart a positive charge, for example, saturated or unsaturated aliphatic amines such as stearylamine, oleylamine, and the like can be given; as charge substances that impart a negative charge, for example, dicetyl phosphate, cholesterol succinate monoester, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and the like can be given. As membrane polypeptides, for example, membrane surface polypeptides, or membrane intrinsic polypeptides, and the like can be given. The amount of these substances to be combined is not particularly limited, and can be appropriately selected according to the purpose.

[0084] The size of the lipid nanoparticles of the present application is preferably 400 nm or less in average particle diameter, more preferably 300 nm or less in average particle diameter, and further preferably 200 nm or less in average particle diameter, and still further preferably 150 nm or less in average particle diameter, from the aspect of easily obtaining a high delivery efficiency to liver cells or spleen cells in the body. The average particle diameter of the lipid nanoparticles refers to the number average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed by a conventional method using a commercially available DLS device or the like.

[0085] The polydispersity index (PDI) of the lipid nanoparticles of the present application is about 0.01 to about 0.7, preferably about 0.01 to about 0.6, and further preferably about 0.03 to about 0.3. The zeta potential at pH 7.4 can be in the range of -50 mV to 5 mV, and preferably in the range of -45 mV to 5 mV.

[0086] The form of the lipid nanoparticles of the present application is not particularly limited, and for example, as a form dispersed in an aqueous solvent, unilamellar liposomes, multilamellar liposomes, spherical micelles, or amorphous lamellar structures, and the like can be given. As the lipid nanoparticles of the present application, unilamellar liposomes, multilamellar liposomes are preferred.

[0087] The lipid nanoparticles of the present application preferably encapsulate a target component that needs to be delivered into a target cell inside the particle covered with a lipid membrane. The component encapsulated inside the particle of the lipid nanoparticles of the present application is not particularly limited as long as it is of a size that can be encapsulated. Any substance such as a nucleic acid, a saccharide, a peptide, a low molecular compound, a metal compound, and the like can be enclosed in the lipid nanoparticles of the present application.

[0088] As the component enclosed in the lipid nanoparticle of the present application, a nucleic acid is preferable. As the nucleic acid, DNA, RNA, or an analog or derivative thereof (e.g., peptide nucleic acid (PNA) or phosphorothioate DNA, etc.) can be used. The nucleic acid enclosed in the lipid nanoparticle of the present application can be a single-stranded nucleic acid or a double-stranded nucleic acid, and can be linear or circular.

[0089] In one embodiment of the present application, the lipid nanoparticle of the present application comprises the pH-sensitive cationic lipid of the present application, a stereoisomer thereof or a mixture of stereoisomers, and a nucleic acid.

[0090] The nucleic acid enclosed in the lipid nanoparticle of the present application preferably comprises a foreign gene for expression in a target cell, and more preferably is a nucleic acid that functions to express a foreign gene in a cell by being taken into the cell. The foreign gene can be a gene originally contained in the genomic DNA of the target cell (preferably a liver cell and a spleen cell), or can be a gene not contained in the genomic DNA. As such a nucleic acid, a gene expression vector comprising a nucleic acid consisting of a base sequence encoding a target gene to be expressed can be mentioned. The gene expression vector can exist as an extrachromosomal gene in the introduced cell, or can be incorporated into the genomic DNA by homologous recombination.

[0091] As the gene expression vector enclosed in the lipid nanoparticle of the present application, there is no particular limitation, and a vector generally used in gene therapy and the like can be used. As the gene expression vector enclosed in the lipid nanoparticle of the present application, a nucleic acid vector such as a plasmid vector is preferable. The plasmid vector can be enclosed in the lipid nanoparticle of the present application in a circular form, or can be enclosed in a state of being previously cut into a linear form. The gene expression vector can be designed by a conventional method using generally used molecular biological tools based on the base sequence information of the target gene to be expressed, and can be produced by various publicly known methods.

[0092] The nucleic acid enclosed in the lipid nanoparticle of the present application is also preferably a functional nucleic acid that controls the expression of a target gene present in a target cell. As the functional nucleic acid, an antisense oligonucleotide, antisense DNA, antisense RNA, siRNA, microRNA, mRNA, etc. can be mentioned. In addition, it can be plasmid DNA (pDNA) that is a siRNA expression vector for expressing siRNA in a cell. As the siRNA expression vector, a commercially available siRNA expression vector can be used, or it can be appropriately modified. As the nucleic acid enclosed in the lipid nanoparticle of the present application, particularly from the viewpoint of good selectivity for the liver or the spleen, mRNA or pDNA is preferable.

[0093] In one embodiment of the present application, the lipid nanoparticle of the present application comprises the pH-sensitive cationic lipid of the present application, a stereoisomer thereof, or a mixture of stereoisomers, and a nucleic acid, wherein the nucleic acid is mRNA or plasmid DNA.

[0094] The method for producing the lipid nanoparticle of the present application is not particularly limited, and any method available to those skilled in the art can be employed. For example, the lipid nanoparticle can be produced by dissolving all the lipid components in an organic solvent such as chloroform, performing vacuum drying using an evaporator, spray drying using a spray drier to form a lipid film, adding the above mixture obtained by drying a water-based solvent containing components to be encapsulated in the lipid nanoparticle, such as a nucleic acid, and further emulsifying using an emulsifying machine such as a homogenizer, an ultrasonic emulsifying machine, or a high-pressure jet emulsifying machine. Alternatively, the lipid nanoparticle can be produced by a known method such as a reverse phase evaporation method. In the case where the size of the lipid nanoparticle is to be controlled, the lipid nanoparticle can be extruded (extrusion filtration) under high pressure using a membrane filter having a uniform pore size.

[0095] The composition of the water-based solvent (dispersion medium) is not particularly limited, and examples include a phosphate buffer, a citric acid buffer, a buffer such as a phosphate buffered saline, a physiological saline, a medium for cell culture, and the like. These water-based solvents (dispersion media) can stably disperse the lipid nanoparticle, but a sugar (aqueous solution) such as glucose, galactose, mannose, fructose, inositol, ribose, xylose, a disaccharide such as lactose, sucrose, cellobiose, trehalose, maltose, a trisaccharide such as raffinose, melezitose, a polysaccharide such as a cyclodextrin, erythritol, xylitol, sorbitol, mannitol, maltitol, a polyol (aqueous solution) such as glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butanediol, and the like can be further added. In order to stably and long-term store the lipid nanoparticle dispersed in the water-based solvent, it is preferable to exclude as much as possible an electrolyte in the water-based solvent from the viewpoint of physical stability such as inhibition of aggregation. In addition, it is preferable to set the pH of the water-based solvent to be neutral around the vicinity of weakly acidic (pH 3.0 to 8.0 or so) and / or to remove dissolved oxygen by nitrogen bubbling or the like from the viewpoint of chemical stability of the lipid.

[0096] The lipid nanoparticles of the present application can also be produced by an alcohol dilution method using a flow path. This method is a method in which a solution in which a lipid component is dissolved in an alcohol solvent and a solution in which a water-soluble component contained in the lipid nanoparticles is dissolved in an aqueous solvent are introduced from respective flow paths, caused to flow together, and thereby the lipid nanoparticles are produced. By using a three-dimensional micro-mixer-embedded micro-flow path capable of achieving instantaneous mixing of two liquids, lipid nanoparticles having a diameter of about 30 nm can be produced with good reproducibility (non-patent document 11). As the flow path used in the production, from the viewpoint of being able to form a lipid particle formation system in which the particle size control property is high, a flow path structure in which a baffle having a certain width with respect to the width of the flow path is arranged in a simple two-dimensional structure so as to be different in distance from both side surfaces is preferably used, as described in patent document 2. As the aqueous solvent used in the alcohol dilution method, the above-described solvents can be used.

[0097] In the case where the obtained aqueous dispersion of the lipid nanoparticles is subjected to freeze-drying or spray-drying, when a sugar (aqueous solution) such as monosaccharides of glucose, galactose, mannose, fructose, inositol, ribose, xylose, disaccharides of lactose, sucrose, cellobiose, trehalose, maltose, trisaccharides of raffinose, melezitose, polysaccharides of cyclodextrin, erythritol, xylitol, sorbitol, mannitol, maltitol, and the like is used, the stability can sometimes be improved. In the case where the above-described aqueous dispersion is frozen, when a polyhydric alcohol (aqueous solution) such as the above-described sugars, glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butanediol, and the like is used, the stability can sometimes be improved.

[0098] In one embodiment of the present application, the lipid nanoparticles of the present application are freeze-dried.

[0099] The present application relates to a lipid nanoparticle formulation containing a pH-sensitive cationic lipid of the present application, a stereoisomer thereof, or a mixture of stereoisomers thereof. The present application relates to a lipid nanoparticle formulation containing (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) a pH-sensitive cationic lipid of the present application, a stereoisomer thereof, or a mixture of stereoisomers thereof.

[0100] As the sterol or the sterol derivative, for example, cholesterol, sitosterol, and the like can be given, and cholesterol is preferred.

[0101] As the polyalkylene glycol-modified lipid, for example, a polyethylene glycol-modified lipid, a polypropylene glycol-modified lipid, and the like can be given, and a polyethylene glycol-modified lipid is preferred.

[0102] As the nucleic acid, for example, siRNA, pDNA, mRNA, etc. can be given, and mRNA is preferred.

[0103] As the buffer, for example, HEPES buffer, phosphate buffer, Tris buffer, etc. can be given.

[0104] As the disaccharide, for example, lactose, sucrose, cellobiose, trehalose, maltose, etc. can be given, and sucrose is 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.

[0105] The molar ratio of the sterol or sterol derivative to the pH-sensitive cationic lipid, the stereoisomer thereof, or the mixture of stereoisomers is, for example, 68.5:20 to 28.5:60.

[0106] In the present application, the lipid nanoparticle formulation can be prepared by suspending the lipid nanoparticle in an aqueous solution.

[0107] The pH of the lipid nanoparticle formulation of the present application is, for example, 5.5 to 8.5, preferably 6.8 to 8.0 at 25°C.

[0108] The present application relates, in one aspect, to a resuspension formulation which is obtained by resuspending the lipid nanoparticle formulation by adding water or an aqueous solution.

[0109] The lipid nanoparticle of the present application is excellent in stability. The lipid nanoparticle of the present application is stable for 1 week or more under storage at -80°C, and / or for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more under storage at 5°C, and / or for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more under storage at 25°C, and / or for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more under storage at 40°C.

[0110] As for the quality of the lipid nanoparticle, for example, the lipid nanoparticle can be left to stand at a prescribed temperature, the average particle diameter, PDI, and nucleic acid encapsulation rate after storage for a prescribed period of time can be compared with the values immediately after preparation, and the lipid nanoparticle which satisfies all of the three conditions of maintaining the average particle diameter within ±20 nm from the preparation day of the lipid nanoparticle, PDI of 0.2 or less and maintaining high uniformity, and nucleic acid encapsulation rate of 80% or more is defined as a lipid nanoparticle which is good in quality retention.

[0111] For example, the evaluation can be performed according to the following criteria: Good: the particle diameter is within ±20 nm from the particle diameter immediately after preparation, and PDI is 0.2 or less, and the encapsulation rate is 80% or more; Not good enough: particle size is more than ± 20 nm from the particle size immediately after preparation, or the encapsulation efficiency is less than 80%.

[0112] For example, a lipid nanoparticle that retains quality for 1 week or more when left standing at 5°C, or a lipid nanoparticle that retains quality for 1 week or more when left standing at 40°C, can be evaluated as a lipid nanoparticle excellent in stability.

[0113] When the lipid nanoparticle of the present application, in which a gene expression vector is encapsulated, is administered to an animal subject, the gene expression vector encapsulated in the lipid nanoparticle is selectively expressed in the liver or the spleen as compared to other organs. Likewise, when the lipid nanoparticle of the present application, in which an siRNA expression vector is encapsulated, is administered to an animal subject, the siRNA expression vector encapsulated in the lipid nanoparticle is selectively expressed in the liver or the spleen as compared to other organs, and the expression of a gene targeted by the expression vector is inhibited. For example, when the lipid nanoparticle of the present application, in which a target foreign gene that is expressed in a liver cell or a spleen cell is encapsulated, is administered to a test animal, the foreign gene can be expressed in the liver or the spleen of the test animal.

[0114] By the gene expression activity highly selective to the liver or the spleen, the lipid nanoparticle of the present application functions as a gene expression vector targeting the liver or the spleen. In the lipid nanoparticle of the present application, after a target foreign gene that needs to be expressed in a liver cell or a spleen cell is encapsulated, the foreign gene is expressed in the liver or the spleen of a test animal by administration to the test animal. Therefore, the lipid nanoparticle of the present application is useful as an effective ingredient of a pharmaceutical composition used in gene therapy, and particularly, as an effective ingredient of a pharmaceutical composition used in gene therapy targeting the liver or the spleen.

[0115] The present application relates to a pharmaceutical composition for liver delivery, which contains the pH-sensitive cationic lipid of the present application, a stereoisomer thereof, or a mixture of stereoisomers.

[0116] The present application relates to a pharmaceutical composition for spleen delivery, which contains the pH-sensitive cationic lipid of the present application, a stereoisomer thereof, or a mixture of stereoisomers.

[0117] The animal to which the lipid nanoparticle of the present application is administered is not particularly limited, and can be a human or an animal other than a human. As the non-human animal, there can be mentioned a mammal such as a cow, a pig, a horse, a sheep, a goat, a monkey, a dog, a cat, a rabbit, a mouse, a rat, a hamster, a guinea pig, and the like, a bird such as a chicken, a quail, a duck, and the like.

[0118] The pH-sensitive cationic lipid, the stereoisomer thereof, or the mixture of stereoisomers of the present application is synthesized, for example, by condensing 7-(4-(dipropylamino)butyl)tridecan-1,7,13-triol or 5,11-dihydroxy 5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate as a basic skeleton with a branched fatty acid.

[0119] The present application relates to a method for producing the pH-sensitive cationic lipid, the stereoisomer thereof, or the mixture of stereoisomers of the present application. In one embodiment of the present application, the method for producing the pH-sensitive cationic lipid of the present application comprises at least a step (Step A) of obtaining a branched fatty acid by reacting an alkyl carboxylic acid with a halogenated alkane in the presence of an organolithium, dimethylpropylene urea (DMPU), and tetrahydrofuran (THF).

[0120] In the method, the alkyl carboxylic acid is, for example, caprylic acid, capric acid, tridecanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, palmitoleic acid.

[0121] In the method, the halogenated alkane is, for example, 1-iodohexane, 1-iodobutane, 2-iodohexane, 1-bromohexane, methyl iodide, ethyl iodide, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1-iodohexane, 1-iodoheptane, 1-iodooctane, 1-iodononane, 1-iodododecane, 1-iodoundecane, 1-iodododecane, 1-iodotridecane, 1-iodotetradecane, 1-iodopentadecane, 1-iodohexadecane.

[0122] In the method, the organolithium is, for example, lithium diisopropylamide (LDA), tert-butyllithium, n-butyllithium.

[0123] In one embodiment of the present application, the method for producing the pH-sensitive cationic lipid of the present application comprises at least a step (Step B) of obtaining a branched fatty acid by subjecting a reaction solution obtained by reacting a malonic acid ester with a halogenated alkane in the presence of a base to hydrolysis treatment and heating treatment.

[0124] In the method, the malonic acid ester is, for example, dimethyl malonate, diethyl malonate, diisopropyl malonate, and preferably dimethyl malonate.

[0125] In the method, the halogenated alkane is, for example, an iodoalkane, and the iodoalkane is, for example, 1-iodohexane, 1-iodopropane, 2-iodohexane.

[0126] In the method, the base is, for example, sodium hydride, calcium hydride, sodium ethoxide, and lithium bis(trimethylsilyl)amide, and preferably sodium hydride.

[0127] In the method, the hydrolysis treatment is performed using any one of sodium hydroxide, calcium hydroxide, and lithium hydroxide.

[0128] In the method, the heat treatment is performed simultaneously with and / or after the hydrolysis treatment, preferably at 120°C to 170°C, more preferably at 150°C to 170°C.

[0129] The method further includes a step of purifying the branched fatty acid by reverse phase chromatography.

[0130] In the branched fatty acid used for synthesizing the following compound, the branched fatty acid is more favorably obtained by the process A than by the process B in terms of yield.

[0131] [Chemical Formula 33] In the branched fatty acid used for synthesizing the following compound, the branched fatty acid is more favorably obtained by the process B than by the process A in terms of yield.

[0132] [Chemical Formula 34-1] [Chemical Formula 34-2] [Chemical Formula 34-3] [Chemical Formula 34-4] [Chemical Formula 34-5] [Chemical Formula 34-6] [Chemical Formula 34-7] [Chemical Formula 34-8] [Chemical Formula 34-9] [Chemical Formula 34-10] [Chemical Formula 34-11] [Chemical Formula 34-12] In one embodiment of the present application, the branched fatty acid used for synthesizing the pH-sensitive cationic lipid of the present application can be obtained, for example, by the method described in Japanese Patent No. 2756756.

[0133] Examples Next, the present application is described in more detail by showing examples, but the present application is not limited to the following examples.

[0134] I. Synthesis of CL4F6, CL4G6, CL15F6 and CL15G6 [Synthesis Example 1] Synthesis of CL4F6 Synthesized 7-(4-(dipropylamino)butyl)tridecan-1,7,13-triol (1.0 mmol) by the method described in Patent Literature 1, dissolved in 5 mL of dichloromethane, then added decanoic acid 2-hexyl ester (2.20 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.20 mmol) and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol), and allowed to react at room temperature for one night. After distilling off the solvent using a rotary evaporator, suspended with ethyl acetate, and removed the insolubles by filtration. The filtrate was partitioned washed with 0.5 N aqueous sodium oxide solution and saturated brine. Added anhydrous sodium sulfate to the organic layer to dehydrate. After filtering, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was refined by subjecting it to silica gel chromatography [elution solvent; dichloromethane:methanol (continuous gradient)] to obtain 7-(4-(dipropylamino)butyl)-7-hydroxytridecan-1,13-diyl bis(2-hexyl decanoate) (CL4F6).

[0135] [Synthesis Example 2] Synthesis of CL4G6 The same operation as in Synthesis Example 1 was performed except that 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate was used instead of decanoic acid 2-hexyl ester to obtain 7-(4-(dipropylamino)butyl)-7-hydroxytridecan-1,13-diyl bis(2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate) (CL4G6).

[0136] [Synthesis Example 3] Synthesis of CL15F6 Synthesized 5,11-dihydroxy 5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate (1.00 mmol) synthesized by the method described in Patent Literature 1 was dissolved in 10 mL of dichloromethane. Next, decanoic acid 2-hexyl ester (2.20 mmol), DMAP (0.20 mmol), and EDCI (3.0 mmol) were added, and it was allowed to react at room temperature overnight. After distilling off the solvent using a rotary evaporator, it was suspended with ethyl acetate, and insolubles were removed by filtration. The filtrate was partitioned washed with a 0.5 N aqueous sodium oxide solution and saturated brine. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtering it, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography [elution solvent; dichloromethane:methanol (continuous gradient)] to obtain 7-hydroxy-7-(4-((1-methylpiperidin-4-yl)carbonyl)oxy)butyl)tridecane-1,13-diyl bis(2-hexyl decanoate) (CL15F6).

[0137] [Synthesis Example 4] Synthesis of CL15G6 The same operation as in Synthesis Example 3 was performed except that 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate was used instead of decanoic acid 2-hexyl ester to obtain 7-hydroxy-7-(4-((1-methylpiperidin-4-yl)carbonyl)oxy)butyl)tridecane-1,13-diyl bis(2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate) (CL15G6).

[0138] II. Preparation and evaluation of lipid nanoparticles using CL4F6, CL4G6, CL15F6, and CL15G6 <Preparation of lipid nanoparticles> In the following experiments, unless otherwise specified, lipid nanoparticles were prepared by the alcohol dilution method using a flow path. As the flow path, a microfluidic device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.) was used.

[0139] Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and an acetic acid buffer (25 mM, pH 4.0) adjusted to an siRNA concentration of 71.1 μg / mL were sent to the microchannel at 0.375 mL / min and 1.125 mL / min, respectively, and the lipid nanoparticle solution discharged from the channel was recovered. The lipid nanoparticle solution was added to a dialysis membrane (MWCO: 12,000 to 14,000) so that the external water phase was 20 mM MES buffer (pH 6.0), and dialyzed at 4°C for 2 hours or more. Then, the external water phase was replaced with PBS (-) (pH 7.4), and further dialyzed at 4°C for 2 hours or more, and the lipid nanoparticle solution was recovered from the dialysis membrane.

[0140] <Composition of the lipid nanoparticle> CL4F6, CL4G6, CL15F6, and CL15G6 used the compounds synthesized in Synthesis Examples 1 to 4.

[0141] 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6) and 7-hydroxy-7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diyl dioleate (CL15H6) used the compounds synthesized by the method described in Patent Literature 1.

[0142] [Chemical Formula 35-1] [Chemical Formula 35-2] In addition, as neutral lipids, cholesterol (chol) and polyethylene glycol 2000-modified dimyristyl glycerol (PEG-DMG) were used.

[0143] <Measurement of average particle diameter, PDI (polydispersity index), and zeta potential of the lipid nanoparticle> The average particle diameter (number average) and PDI of the lipid nanoparticle in PBS (-) and the zeta potential in 10 mM HEPES buffer (pH 7.4) were measured using an analysis device "Zetasizer Nano ZS ZEN3600" (manufactured by Malvern) using a dynamic light scattering method.

[0144] <Measurement of pKa of the lipid nanoparticle> The pKa of the lipid nanoparticles was determined using p-toluidino-2-naphthalenesulfonic acid (TNS). First, TNS (final concentration: 0.75 μM) and the lipid nanoparticles (final concentration: 30 mM) were mixed in a buffer adjusted to each pH. The fluorescence intensity of the prepared mixture was measured with a microplate reader. The highest value and the lowest value in the measured values were taken as 100% and 0% of the charge rate, respectively, and the pH indicating 50% of the charge rate was calculated as the pKa.

[0145] <Encapsulation rate of nucleic acid into lipid nanoparticles> The encapsulation rate of siRNA, mRNA, and pDNA into the lipid nanoparticles was determined using Ribogreen (manufactured by life technologies).

[0146] [Example 1] F7 siRNA-loaded lipid nanoparticles were produced by the alcohol dilution method using a pH-sensitive cationic lipid, cholesterol, and PEG-DMG in a molar ratio of 50:50:1. As the pH-sensitive cationic lipid, CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 was used. Hereinafter, the lipid nanoparticles produced using a pH-sensitive cationic lipid X will be referred to as X-LNP. For example, the lipid nanoparticles produced using the pH-sensitive cationic lipids CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 will be referred to as CL4F-LNP, CL4G6-LNP, CL4H6-LNP, CL15F-LNP, CL15G6-LNP, or CL15H6-LNP, respectively. In addition, the base sequence of the siRNA against F7 is shown in Table 1.

[0147] In the table, the capital letters represent natural type RNA (T is only natural type DNA), and the small letters represent 2'-fluoro modifier, indicates a phosphorothioate bond.

[0148] The average particle diameter of each of the produced lipid nanoparticles was 80 to 120 nm, and the siRNA encapsulation rate was 90% or more. The results obtained by measuring the pKa of each of the lipid nanoparticles are shown in Figure 1 (A) and Figure 1 (B). As shown in Figure 1 , the lipid nanoparticles produced using CL4F6, CL4G6, CL15F6, or CL15G6 having a branched structure as the scaffold structure showed a lower pKa than the lipid nanoparticles produced using CL4H6 or CL15H6 having a linear structure as the scaffold structure.

[0149] Next, the prepared each F7 siRNA-loaded liposome was administered to ICR mice (4 weeks old, female) to investigate F7 knockdown activity in vivo. Specifically, each F7 siRNA-loaded liposome was intravenously administered to ICR mice at 0.003 to 0.1 mg siRNA / kg, and F7 enzyme activity in the plasma 24 hours later was measured. The F7 enzyme activity in the plasma of untreated mice was taken as 100%, and the relative F7 enzyme activity (%) in the plasma of mice administered with each F7 siRNA-loaded liposome was calculated. The results are shown in Figure 2 (A) and Figure 2 (B). Figure 2 (B) is the result of intravenous administration of each F7 siRNA-loaded liposome at 0.1 mg siRNA / kg. As shown in Figure 2 , the liposome produced using CL4F6, CL4G6, CL15F6, or CL15G6 having a branched structure as the scaffold structure showed equivalent F7 knockdown activity in vivo as compared with the liposome produced using CL4H6 or CL15H6 having a linear structure as the scaffold structure. From these results, it was found that CL4F6, CL4G6, CL15F6, and CL15G6 are useful as the liposome produced using the lipids as siRNA delivery carriers.

[0150] [Example 2] Instead of siRNA, mRNA-loaded liposomes were prepared to investigate gene expression activity in vivo. The mRNA used was mRNA (Nluc mRNA) prepared by an in vitro transcription reaction on pDNA encoding NanoLuc (registered trademark) luciferase (Nluc) (manufactured by Promega Corporation).

[0151] First, a liposome loaded with Nluc mRNA (Nluc mRNA-loaded liposome) was produced using a pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG in a molar ratio of 60:10:40:1 by the alcohol dilution method. As the pH-sensitive cationic lipid, CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 was used.

[0152] The average particle diameter, PDI, zeta potential, and mRNA encapsulation efficiency of the prepared lipid nanoparticles were investigated. The measurement results are shown in Table 2. In Table 2, "CL" means a cationic lipid. The average particle diameter calculated by the dynamic light scattering method was 70 to 130 nm (Table 2). Of the prepared lipid nanoparticles, only CL15H6-LNP formed particles having a large PDI and low uniformity (Table 2). As for the mRNA encapsulation efficiency, CL4H6-LNP was less than 80%, but LNP containing other cationic lipids all showed 90% or more.

[0153] Next, the prepared each Nluc mRNA-carrying lipid nanoparticle was administered to ICR mice (4 weeks old, female), and the in vivo gene expression activity was investigated. Specifically, each Nluc mRNA-carrying lipid nanoparticle was intravenously administered to ICR mice at 0.04 mg mRNA / kg, and the Nluc activity in the liver and spleen 24 hours later was measured. The Nluc activity was measured using a luminometer (RLU), and was corrected with the amount of protein quantified by the BCA method.

[0154] The measurement results of the Nluc activity (RLU / mg protein) in the liver and spleen of the mice administered with each Nluc mRNA-carrying lipid nanoparticle are shown in Figure 3 . Figure 3 (A) is the measurement results of the Nluc activity in the liver, Figure 3 (B) is the measurement results of the Nluc activity in the spleen. Furthermore, the liver selectivity of gene expression was calculated by dividing the gene expression activity in the liver by the gene expression activity in the spleen. Figure 3 (C) is a graph showing the calculation results of the [Nluc activity in the liver] / [Nluc activity in the spleen] of the mice administered with each Nluc mRNA-carrying lipid nanoparticle. As Figure 3 (A) shows, in the liver, in the mice administered with CL4F6-LNP, CL15F6-LNP, and CL15G6-LNP, Nluc activity equivalent to that in the mice administered with CL4H6-LNP was shown. In addition, as Figure 3(C) shows that mice administered with CL4F6-LNP and CL4G6-LNP showed higher liver selectivity than mice administered with CL4H6-LNP. Similarly, mice administered with CL15F6-LNP and CL15G6-LNP showed higher liver selectivity than mice administered with CL15H6-LNP. From these results, it was found that lipid nanoparticles containing a pH-sensitive cationic lipid having a branched chain structure as a scaffold structure, when encapsulating mRNA, showed higher liver selectivity than lipid nanoparticles containing a pH-sensitive cationic lipid having a linear chain structure as a scaffold structure, and were useful as a delivery carrier for specifically delivering to the liver.

[0155] [Example 3] Instead of siRNA, lipid nanoparticles carrying pDNA were prepared, and in vivo gene expression activity was investigated. pDNA used a plasmid (pFluc) expressing firefly luciferase (Fluc) under a CMV promoter.

[0156] <In vitro gene expression activity> First, lipid nanoparticles carrying pFluc (pFluc-carrying lipid nanoparticles) were prepared by the alcohol dilution method using pH-sensitive cationic lipids, DSPC, cholesterol, and PEG-DMG in a molar ratio of 50:10:40:1.5. As the pH-sensitive cationic lipid, CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 was used. In addition, the N / P ratio in the microchannel was set to 9.

[0157] The average particle diameter, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were investigated. The results of the measurement are shown in Table 3. In Table 3, "CL" means a cationic lipid. The average particle diameter calculated by the dynamic light scattering method was 90 to 150 nm (Table 3). Regarding the pDNA encapsulation rate, CL4H6-LNP and CL15H6-LNP were 70% and 82%, respectively. On the other hand, the pDNA encapsulation rate of the lipid nanoparticles containing other cationic lipids showed a good value of 90% or more.

[0158] The pFluc-carrying lipid nanoparticles were introduced into cultured cells, and the gene expression activity in vitro was investigated. Specifically, the pFluc-carrying lipid nanoparticles were transfected into HeLa-GFP cells cultured in a 96-well plate at 0.0625 pg pDNA / well, and the Fluc activity after 24 hours was measured. As a positive control, pFluc was introduced into HeLa-GFP cells using the introduction reagent "Lipofectamine 3000" (manufactured by Thermo Fisher Scientific). The Fluc activity was measured using a luminometer (RLU), and was corrected with the amount of protein quantified by the BCA method.

[0159] The results of measuring the Fluc activity of HeLa-GFP cells into which each pFluc-carrying lipid nanoparticle was introduced are shown in Figure 4 . In the figure, "Lipo3K" means a positive control in which gene introduction was performed using Lipofectamine 3000. As shown in Figure 4 , the cells into which CL15F6-LNP was introduced showed higher activity than the cells into which CL4H6-LNP, CL15H6-LNP, and the positive control were introduced.

[0160] "In Vitro Gene Expression Activity" The pFluc-carrying lipid nanoparticles were prepared in the same manner as described above except that the N / P ratio in the microchannel was made to be 6.

[0161] The average particle diameter, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were investigated. The results of the measurement are shown in Table 4. In Table 4, "CL" means a cationic lipid. The average particle diameter calculated by the dynamic light scattering method was 70 to 125 nm (Table 4). Regarding the pDNA encapsulation rate, CL4F6-LNP, CL4G6-LNP, CL15F6-LNP, and CL15G6-LNP all showed good values of 90% or more.

[0162] Next, each of the prepared pFluc-carrying lipid nanoparticles was administered to ICR mice (4 weeks old, female), and the gene expression activity in vivo was investigated. Specifically, each of the pFluc-carrying lipid nanoparticles was intravenously administered to ICR mice at 0.5 mg mRNA / kg, and the Fluc activity in the liver and spleen after 6 hours was measured. The Fluc activity was measured using a luminometer (RLU), and was corrected with the amount of protein quantified by the BCA method.

[0163] The results of measuring the Fluc activity (RLU / mg protein) in the liver and spleen of mice to which each of the pFluc-carrying lipid nanoparticles was administered are shown inFigure 5 . Figure 5 (A) is a result of measurement of Fluc activity in the liver, Figure 5 (B) is a result of measurement of Fluc activity in the spleen. In addition, the liver selectivity of gene expression was calculated by dividing the gene expression activity in the liver by the gene expression activity in the spleen. Figure 5 (C) is a graph showing the calculation results of [Fluc activity in the liver] / [Fluc activity in the spleen] of mice administered with each pFluc-carrying lipid nanoparticle. As shown in Figure 5 (A), the Fluc activity in the liver was more excellent in mice administered with CL4F6-LNP and CL15F6-LNP than in mice administered with CL4H6-LNP and CL15H6-LNP. In addition, as shown in Figure 5 (C), mice administered with CL4F6-LNP and CL4G6-LNP showed higher liver selectivity than mice administered with CL4H6-LNP. Similarly, mice administered with CL15F6-LNP and CL15G6-LNP showed higher liver selectivity than mice administered with CL15H6-LNP. From these results, it was found that, in the case where pDNA was encapsulated, the lipid nanoparticle containing a pH-sensitive cationic lipid having a branched chain type scaffold structure showed higher liver selectivity than the lipid nanoparticle containing a pH-sensitive cationic lipid having a linear type scaffold structure, and was useful as a delivery carrier for specifically delivering to the liver.

[0164] III. Synthesis of CL4F6 derivatives and CL15F6 derivatives < Synthesis of CL4F6 and CL4F6 derivatives > The synthesis of CL4F6 and CL4F6 derivatives, CL4F 6-2, CL4F 6-4, CL4F 7-3, CL4F 7-4, CL4F 7-5, CL4F 8-4, CL4F 8-5, CL4F 8-6, CL4F 9-3, CL4F 9-4, CL4F 9-5, CL4F 9-6, CL4F 9-7, CL4F10-2, CL4F 10-4, CL4F 10-5, CL4F 10-7, CL4F 10-8, CL4F 11-5, CL4F 11-6, CL4F 11-7, CL4F 11-9, CL4F 12-4, CL4F 12-6, CL4F 12-10, CL4F 13-3, CL4F 14-2, CL4F 16-0, and CL4F16-1 was carried out as follows.

[0165] Synthesized 7-(4-(dimethylamino)butyl)tridecane-1,7,13-triol (1.0 mmol) synthesized by the method described in Patent Literature 1 was dissolved in 5 mL of dichloromethane, followed by addition of a branched fatty 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 allowed to react at room temperature for one night. After distilling off the solvent using a rotary evaporator, the resultant was suspended with ethyl acetate, and then subjected to liquid-liquid separation using 0.5 N aqueous sodium hydroxide and saturated brine. Anhydrous sodium sulfate was added to the organic layer to remove water. After filtration, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by ODS silica gel chromatography [elution solvent; acetonitrile / isopropanol (50:50): water (0.1% TFA) (continuous gradient)] and silica gel chromatography [elution solvent; dichloromethane:methanol (continuous gradient)] to obtain CL4F6 or a CL4F6 derivative.

[0166] Synthesis of CL15F6 and CL15F6 derivatives CL15F6 and CL15F 6-2, CL15F 6-4, CL15F 7-3, CL15F 7-5, CL15F 8-6, CL15F 9-3, CL15F 9-5, CL15F 9-7, CL15F 10-4, CL15F 10-5, CL15F 10-8, CL15F 11-5, CL15F 11-6, CL15F 11-7, CL15F 11-9, CL15F 12-4, CL15F 12-10, CL15F 13-3, CL15F 14-2, CL15F 16-0, and CL15F 16-1 as CL15F6 derivatives were synthesized as follows.

[0167] ​Synthesized 5, 11-dihydroxy-5- (6-hydroxyhexyl) undecyl 1-methylpiperidine-4-carboxylate (1.00 mmol) synthesized by the method described in Patent Literature 1 was dissolved in 10 mL of dichloromethane. Next, decanoic acid 2-hexyl ester (2.40 mmol), DMAP (0.10 mmol), and EDCI (3.0 mmol) were added, and it was allowed to react at room temperature overnight. After distilling off the solvent using a rotary evaporator, it was suspended with ethyl acetate, and then partitioned and washed with a 0.5 N aqueous sodium hydroxide solution and saturated brine. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtering it, the solvent was distilled off using a rotary evaporator to obtain a crude product. Purification was performed by subjecting the crude product to ODS silica gel chromatography [elution solvent; acetonitrile / isopropanol (50:50): water (0.1% TFA) (continuous gradient)] and silica gel chromatography [elution solvent; dichloromethane:methanol (continuous gradient)] to obtain CL15F6 or a CL15F6 derivative.

[0168] The above branched fatty acid was synthesized from a straight-chain fatty acid or dimethyl malonate as follows.

[0169] <Synthesis of branched fatty acid from straight-chain fatty acid> A straight-chain fatty acid (10.28 mmol) was dissolved in 36 mL of THF, and then lithium diisopropylamide (24 mmol) was added dropwise at -20°C or lower, and it was stirred at 0°C for 30 minutes. Next, DMPU (18 mL) was added, and it was stirred at 0°C for 60 minutes. Next, iodocane (23.2 mmol) was added, and it was allowed to react at 10°C overnight. After quenching with 2 N hydrochloric acid, it was diluted with dimethyl ether, and partitioned and washed with saturated brine. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtering it, the solvent was distilled off using a rotary evaporator to obtain a crude product. Purification was performed by subjecting the crude product to ODS silica gel chromatography [elution solvent; acetonitrile / isopropanol (50:50): water (10 mM ammonium acetate) (continuous gradient)] to obtain a branched fatty acid.

[0170] <Synthesis of branched fatty acid from dimethyl malonate> NaH (7.56 mmol) was dissolved in 18 mL of THF and stirred at 0°C for 10 minutes. Next, dimethyl malonate (7.56 mmol) was added and stirred at 0°C for 10 minutes. Next, the arbitrary iodoalkane (7.56 mmol) was added and allowed to react at room temperature overnight. NaH (11.34 mmol) was added and stirred at 0°C for 10 minutes. Next, the arbitrary iodoalkane (11.34 mmol) was added and allowed to react at room temperature overnight. After quenching with acetic acid, diluted with ethyl acetate, and partitioned with saturated brine. Dehydrated with anhydrous sodium sulfate in the organic layer. After filtration, the solvent was distilled off using a rotary evaporator. The component from which the solvent was removed was dissolved in 16 mL of ethanol, 5 mL of 8N aqueous sodium hydroxide solution was added, and allowed to react at 60°C overnight. After neutralization with 6N hydrochloric acid, diluted with ethyl acetate, and partitioned with saturated brine. Dehydrated with anhydrous sodium sulfate in the organic layer. After filtration, the solvent was distilled off using a rotary evaporator, heated at 160°C for 2 hours, and the crude product was obtained. The crude product was refined by ODS silica gel chromatography [elution solvent; acetonitrile / isopropyl alcohol (50:50): water (10 mM ammonium acetate) (continuous gradient)] to obtain a branched fatty acid.

[0171] As for the branched fatty acid used in the synthesis of CL4F 7-4, CL4F 8-5, CL4F 9-6, and CL4F 10-7, the method of using a straight-chain fatty acid in the raw material can yield the branched fatty acid more favorably than the method of using dimethyl malonate in the raw material.

[0172] As for the branched fatty acid used in the synthesis of CL4F 6-2, CL4F 6-4, CL4F 7-3, CL4F 7-5, CL4F 8-4, CL4F 8-6, CL4F 9-3, CL4F 9-4, CL4F 9-5, CL4F 9-7, CL4F 10-2, CL4F 10-4, CL4F 10-5, CL4F 6, CL4F 10-8, CL4F 11-5, CL4F 11-6, CL4F 11-7, CL4F 11-9, CL4F 12-4, CL4F 12-6, CL4F 12-10, CL4F 13-3, CL4F 14-2, CL4F 16-1, CL15F 6-2, CL15F 6-4, CL15F 7-3, CL15F 7-5, CL15F 8-6, CL15F 9-3, CL15F 9-5, CL15F 9-7, CL15F 10-4, CL15F 10-5, CL15F 6, CL15F 10-8, CL15F 11-5, CL15F 11-6, CL15F 11-7, CL15F 11-9, CL15F 12-4, CL15F 12-10, CL15F 13-3, CL15F 14-2, and CL15F 16-1, the method using dimethyl malonate in the raw material can obtain the branched fatty acid in a more favorable yield compared to the method using a straight-chain fatty acid in the raw material.

[0173] IV. Preparation and evaluation of lipid nanoparticles using CL4F6 derivatives and CL15F6 derivatives 1. Preparation and evaluation of mRNA-carrying lipid nanoparticles Preparation of mRNA-carrying lipid nanoparticles (mRNA-LNP) The lipid nanoparticles were prepared by an alcohol dilution method using a flow path. As the flow path, a mixer-embedded microfluidic device "NanoAssemblr" (manufactured by Precision NanoSystems) was used.

[0174] Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citric acid buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were sent to the microflow path at 3 mL / min and 9 mL / min, respectively, and a lipid nanoparticle solution discharged from the flow path was recovered. After the lipid nanoparticle solution was diluted 10-fold with a 20 mM HEPES buffer (9% sucrose, pH 7.45), it was concentrated with an ultrafiltration unit, and a lipid nanoparticle solution was recovered.

[0175] <Composition of the lipid nanoparticle> Fluc mRNA-carrying liposomal nanoparticles (Fluc mRNA-carrying liposomal nanoparticles) were produced by the alcohol dilution method using pH-sensitive cationic lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Oily Industries), cholesterol (Nacalai Tesque), and DMG-PEG2K (Oily Industries) in a molar ratio of 50:10:38.5:1.5. Fluc mRNA (5 moU) was used CleanCap (registered trademark) FLuc mRNA (5 moU) from TriLink Biotechnologies, Inc.

[0176] Measurement of average particle diameter and PDI of liposomal nanoparticles The average particle diameter (z-average) and PDI of the liposomal nanoparticles in PBS (-) were measured using an analysis device "Zetasizer Nano ZSP" (manufactured by Malvern) using a dynamic light scattering method.

[0177] Measurement of pKa of liposomal nanoparticles The pKa of the liposomal nanoparticles was measured using p-toluidino-2-naphthalenesulfonic acid (TNS). First, TNS (final concentration: 0.75 µM) and liposomal nanoparticles (final concentration: 60 µM) were mixed in a buffer adjusted to each pH. The fluorescence intensity of the prepared mixture was measured with a microplate reader. Regarding the excitation wavelength, the measured value at pH 3.5 in the measured value was taken as 100% charge rate, the measured value at pH 9.5 was taken as 0% charge rate, and the pH indicating 50% charge rate was calculated as pKa.

[0178] Nucleic acid encapsulation rate of liposomal nanoparticles The siRNA and mRNA encapsulation rates of the liposomal nanoparticles were measured with Ribogreen reagent. A solution obtained by diluting the liposomal nanoparticles with TE buffer so that the concentration of the liposomal nanoparticles would be 8 µg / mL in terms of nucleic acid concentration was prepared as a solution for measuring the nucleic acid concentration on the surface of the nanoparticles. In addition, a total nucleic acid concentration measurement solution was prepared by adding X-triton 100 so that the concentration of the liposomal nanoparticles would be 1.2 µg / mL in terms of nucleic acid concentration and X-triton 100 would be 1% (w / w). 100 µL of Ribogreen (registered trademark) reagent (Quant-iT TM RiboGreen TMThe RNA Reagent (manufactured by Thermofisher Scientific) was mixed with each solution at 100 μL, and the fluorescence intensity at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm was measured using a microplate reader. The nucleic acid concentration of a nucleic acid solution containing 1% X-triton 100 (nucleic acid concentration: 0 to 2.5 μg / mL) was calculated using a standard curve prepared in the same manner as described above. The nucleic acid encapsulation efficiency of each lipid nanoparticle was calculated by the following formula.

[0179] Encapsulation efficiency % = (total nucleic acid concentration of the solution for measuring the nucleic acid concentration (μg / mL) - nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of the nanoparticles (μg / mL)) ÷ total nucleic acid concentration of the solution for measuring the nucleic acid concentration (μg / mL) x 100 <Results> The average particle diameter, PDI, and mRNA encapsulation efficiency of the prepared lipid nanoparticles were investigated. The results of the CL4F6 derivative nanoparticle measurements are shown in Tables 5 and 6, and the results of the CL15F6 derivative nanoparticle measurements are shown in Table 7. As a control, D-Lin-MC3-DMA (MC3) having the following structure (manufactured by MedChemExpress) was used.

[0180] [Chemical Formula 36] The average particle diameters of the CL4F6 derivative nanoparticles of Table 5 calculated by dynamic light scattering were all 60 to 220 nm, and particles with small PDI and high uniformity were formed except for CL4F 16-0-LNP. Regarding the mRNA encapsulation efficiency, CL4F 6-2-LNP and CL4F 16-1-LNP were less than 80%, but LNP containing other cationic lipids all showed more than 80%.

[0181] Regarding the average particle diameters of the CL15F6 derivative nanoparticles of Table 7, CL15F 6-4-LNP and CL15F 7-3-LNP were more than 300 nm, but LNP containing other cationic lipids were all 90 to 200 nm. Particles with small PDI and high uniformity were formed except for CL15F 6-4-LNP, CL15F 7-3-LNP, CL15F 16-0-LNP, and CL15H6-LNP. The mRNA encapsulation efficiency was less than 80% in CL15F 6-4-LNP and CL15F 7-3-LNP, but LNP containing other cationic lipids all showed more than 80%.

[0182] Next, the prepared each Fluc mRNA-loaded lipid nanoparticle was administered to Balb / c mice (Japan Charles River, 7 weeks old, female), and the in vivo gene expression activity was investigated. Specifically, each Fluc mRNA-loaded lipid nanoparticle was intravenously administered to Balb / c mice at 0.1 mg mRNA / kg, and the Fluc activity in the liver and spleen 6 hours later was measured. The Fluc activity was measured by administering 1.5 mg of VivoGlo Luciferin, In Vivo Grade (Promega, P1041) dissolved in PBS to 15 mg / mL from the tail vein of each mouse, and then by an in vivo imaging system (Perkin Elmer, IVIS200). The unit of the Fluc activity is the luminescence intensity per unit area (Avg Radiance [p / s / cm 2 / sr] at a maximum luminescence wavelength of about 560 nm).

[0183] The results of measuring the Fluc activity (Avg Radiance [p / s / cm 2 / sr] in the liver and spleen of mice administered with each Fluc mRNA-loaded lipid nanoparticle are shown in Tables 5 to 7.

[0184] The Fluc activity in the liver of the CL4F6 derivative nanoparticles of Tables 5 and 6 showed higher Fluc activity in mice administered with Fluc mRNA-loaded lipid nanoparticles containing CL4F8-6, CL4F 9-7, or CL4F 11-6 than in mice administered with Fluc mRNA lipid nanoparticles containing CL4F6. On the other hand, the Fluc activity of the CL4F6 derivative nanoparticles in the spleen showed higher Fluc activity in mice administered with Fluc mRNA lipid nanoparticles containing CL4F 7-5, CL4F 8-4, CL4F 9-3, CL4F 10-2, CL4F 8-6, CL4F 10-4, CL4F 10-5, CL4F 12-4, CL4F 13-3, CL4F 14-2, CL4F 7-4, CL4F 8-5, CL4F 9-4, or CL4F 9-5 than in mice administered with Fluc mRNA lipid nanoparticles containing CL4F6.

[0185] The Fluc activity of the CL15F6 derivative nanoparticles in the liver of Table 7 indicates that the Fluc activity was higher in mice administered with Fluc mRNA lipid nanoparticles containing CL15F9-7, CL15F 11-5, CL15F 11-6, CL15F 10-8, CL15F 11-7, CL15F 11-9, CL15F 12-10, or CL15F 14-2 than in mice administered with Fluc mRNA lipid nanoparticles containing CL15F6. The Fluc activity of CL15F6 in the spleen of Table 7 indicates that the Fluc activity was higher in mice administered with Fluc mRNA lipid nanoparticles containing CL15F 6-4, CL15F 7-3, CL15F 7-5, CL15F 9-3, CL15F 9-5, CL15F 10-5, CL15F 13-3, CL15F 11-6, CL15F 10-8, CL15F 11-7, CL15F 11-9, or CL15F 14-2 than in mice administered with Fluc mRNA lipid nanoparticles containing CL15F6.

[0186] 2. Preparation and evaluation of siRNA-loaded lipid nanoparticles Preparation of siRNA-loaded lipid nanoparticles Instead of mRNA, siRNA-loaded lipid nanoparticles were prepared, and F7 knockdown activity in vivo was investigated. The siRNA-loaded lipid nanoparticles were prepared in the same manner as described above, except that the N / P ratio in the microflow channel was set to 6. The base sequence of the siRNA against F7 is shown in Table 8. In the table, the capital letters represent natural type RNA (only T is natural type DNA), and the small letters represent 2’-fluoro modification, represents a phosphorothioate bond.

[0187] First, lipid nanoparticles loaded with siRNA against F7 (F7 siRNA-loaded lipid nanoparticles) were prepared by the alcohol dilution method using pH-sensitive cationic lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG in a molar ratio of 50:10:38.5:1.5.

[0188] Results The average particle diameter, PDI, siRNA encapsulation rate, and pKa of the prepared lipid nanoparticles were investigated. The results of the CL4F6 derivative nanoparticle measurements are shown in Tables 9 and 10, and the results of the CL15F6 derivative nanoparticle measurements are shown in Table 11. The average particle diameter of the CL4F6 derivative nanoparticles was 60 to 280 nm, and the siRNA encapsulation rate was less than 90% for CL4F 6-2-LNP, but was 90% or more for the LNP containing other cationic lipids. Except for CL4F 16-2-LNP, particles having a small PDI and high uniformity were formed.

[0189] The average particle diameter of the CL15F6 derivative nanoparticles was more than 300 nm for CL15F 6-2-LNP, but was 85 to 290 nm for the LNP containing other cationic lipids. The siRNA encapsulation rate was 90% or more for all of the LNP. Except for CL15F 6-2-LNP, CL15F 6-4-LNP, CL15F 7-3-LNP, and CL15F 16-0-LNP, particles having a small PDI and high uniformity were formed.

[0190] Next, the F7 knockdown activity in vivo was investigated by administering each of the prepared F7 siRNA-carrying lipid nanoparticles to Balb / c mice (Charles River, Japan, 5 weeks old, female). Specifically, the F7 siRNA-carrying lipid nanoparticles containing the lipids described in Table 9 were intravenously administered to BALB / c mice at 0.025 mg siRNA / kg, the F7 siRNA-carrying lipid nanoparticles containing the lipids described in Tables 10 and 11 were intravenously administered to BALB / c mice at 0.025 mg siRNA / kg, and the F7 enzyme activity in the plasma 24 hours later was measured using BIOPHEN FVII (Biophen, A221304). The F7 enzyme activity in the plasma of the untreated group of mice was taken as 100%, and the relative F7 enzyme activity in the plasma of the mice administered with each of the F7 siRNA-carrying lipid nanoparticles was calculated (%). In addition, the relative F7 enzyme activity in the plasma of the mice administered with the F7 siRNA-carrying lipid nanoparticles containing MC3 was taken as 1, and the ratio was calculated. The results of the CL4F6 derivative nanoparticle measurements are shown in Tables 9 and 10, and the results of the CL15F6 derivative nanoparticle measurements are shown in Table 11.

[0191] The F7 siRNA-carrying lipid nanoparticles containing CL4F 10-4, CL4F 8-5, CL4F 9-5, or CL4F 12-6 of Tables 9 and 10 showed higher knockdown activity than the F7 siRNA-carrying lipid nanoparticles containing MC3.

[0192] The F7 siRNA-loaded lipid nanoparticles of Table 11 containing CL15F 9-7, CL15F 11-5, CL15F 12-4, CL15F 11-6, CL15F 10-8, CL15F 11-7, CL15F 11-9, or CL15F 12-10 showed higher knockdown activity than the F7 siRNA-loaded lipid nanoparticles containing MC3.

[0193] 3. Preparation and evaluation of pDNA-loaded lipid nanoparticles Preparation of pDNA-loaded lipid nanoparticles Instead of mRNA, lipid nanoparticles loaded with pDNA encoding eGFP were prepared, and the luminescence of eGFP in vitro was investigated. The pDNA encoding eGFP was prepared by inserting the gene encoding eGFP into pCMV-LacI from LacSwitch II Mammalian Expression System (Agilent Technologies).

[0194] The pDNA-loaded lipid nanoparticles were prepared in the same manner as the mRNA-loaded lipid nanoparticles.

[0195] Lipid constituting the lipid nanoparticles The pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, NOF Corporation), cholesterol (Nacalai Tesque), and DMG-PEG2K (NOF Corporation) were used in a molar ratio of 50:10:38.5:1.5.

[0196] Measurement of average particle diameter and PDI of the lipid nanoparticles The measurement of the average particle diameter and PDI of the lipid nanoparticles was performed in the same manner as the measurement of the average particle diameter of mRNA-LNP.

[0197] Nucleic acid encapsulation efficiency of the lipid nanoparticles The pDNA encapsulation efficiency of the lipid nanoparticles was measured using Picogreen (registered trademark) reagent (Quant-i T TMassay kit, broad range, manufactured by Thermofisher Scientific) was used. In the pDNA entrapment rate assay, the nucleic acid concentration of the nanoparticle surface nucleic acid concentration assay solution was made to be 1.6 pg / mL, the nucleic acid concentration of the total nucleic acid concentration assay solution was made to be 24 ng / mL, and the nucleic acid concentration used in the standard curve was made to be 0 to 0.5 pg / mL, and as the assay reagent, Picogreen (registered trademark) reagent (Quant-iT dsDNA Assay Kit, broad range, manufactured by Thermofisher Scientific) was used. TM The dsDNA Assay Kit, broad range, manufactured by Thermofisher Scientific) was used. In the pDNA entrapment rate assay, the nucleic acid concentration of the nanoparticle surface nucleic acid concentration assay solution was made to be 1.6 pg / mL, the nucleic acid concentration of the total nucleic acid concentration assay solution was made to be 24 ng / mL, and the nucleic acid concentration used in the standard curve was made to be 0 to 0.5 pg / mL, and as the assay reagent, Picogreen (registered trademark) reagent (Quant-iT dsDNA Assay Kit, broad range, manufactured by Thermofisher Scientific) was used.

[0198] Entrapment rate (%) = (nucleic acid concentration of total nucleic acid concentration assay solution (pg / mL) - nucleic acid concentration of nanoparticle surface nucleic acid concentration assay solution (pg / mL)) ÷ (nucleic acid concentration of total nucleic acid concentration assay solution (pg / mL)) x 100 <Results> The results are shown in the table below. The prepared lipid nanoparticles were left to stand at 40°C, and after storage for each period, whether the quality was well maintained was evaluated according to the following criteria: Good (O): The particle diameter was within ± 20 nm of the particle diameter immediately after preparation, the PDI was 0.2 or less, and the entrapment rate was 80% or more; Not good enough (X): The particle diameter was more than ± 20 nm of the particle diameter immediately after preparation, or the entrapment rate was less than 80%.

[0199] For the lipid nanoparticles whose quality was well maintained when left to stand at 40°C for one week or more, it was evaluated that the stability was excellent.

[0200] 4. Storage stability evaluation of lipid nanoparticles <Preparation of lipid nanoparticles> The lipid nanoparticles were prepared by the alcohol dilution method using a flow path. As the flow path, a mixer built-in microfluidic device "NanoAssemblr" (manufactured by Precision NanoSystems) was used.

[0201] Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citric acid buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were sent at 3 mL / min and 9 mL / min, respectively, into the microchannel, and a lipid nanoparticle solution discharged from the channel was recovered. The lipid nanoparticle solution was diluted 10-fold with a 20 mM HEPES buffer (9% sucrose, pH 7.45) and concentrated with an ultrafiltration unit, and a lipid nanoparticle solution was recovered.

[0202] <Composition of the lipid nanoparticle> A lipid nanoparticle (Fluc mRNA-loaded lipid nanoparticle) loaded with Fluc mRNA was produced by an alcohol dilution method using a pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG in a molar ratio of 50:10:38.5:1.5. Fluc mRNA was CleanCap (registered trademark) FLuc mRNA (5 moU) from TriLink Biotechnologies.

[0203] <Measurement of average particle diameter and PDI of the lipid nanoparticle> The average particle diameter (z-average) and PDI of the lipid nanoparticle in PBS (-) were measured using an analysis device "Zetasizer Nano ZSP" (manufactured by Malvern) using a dynamic light scattering method.

[0204] <Encapsulation rate of nucleic acid of the lipid nanoparticle> The encapsulation rate of mRNA of the lipid nanoparticle was measured using Ribogreen (manufactured by life technologies).

[0205] <Evaluation of storage stability> The lipid nanoparticle was left to stand at each of -80°C, 5°C, 25°C, and 40°C, and after storage for each period, the average particle diameter, PDI, and nucleic acid encapsulation rate were measured. The lipid nanoparticle satisfying all of the three conditions of maintaining the average particle diameter within ±20 nm from the day of LNP preparation, PDI being 0.2 or less and maintaining high uniformity, and the nucleic acid encapsulation rate being maintained at 80% or more was judged to be of good quality.

[0206] <Results> The Fluc mRNA each loaded in the lipid nanoparticles was left to stand at -80°C, 5°C, 25°C, and 40°C, and after each storage period, the average particle diameter, PDI, and nucleic acid encapsulation efficiency were measured, and the results are shown in Tables 13 and 14 as the period during which the quality of the lipid nanoparticles was maintained to be good. For the lipid nanoparticles whose quality was maintained to be good at 5°C for one week or more, the stability was evaluated to be excellent.

[0207] 5. Production and evaluation of lipid nanoparticles having various compositions Preparation of lipid nanoparticles The lipid nanoparticles were prepared by an alcohol dilution method using a flow path. As the flow path, a microfluidic device "NanoAssemblr" (manufactured by Precision NanoSystems) was used.

[0208] Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citric acid buffer (50 mM, pH 3.5) adjusted to a nucleic acid concentration of 46.1 μg / mL were each fed at 3 mL / min and 9 mL / min into the microflow path, and a lipid nanoparticle solution discharged from the flow path was recovered. The lipid nanoparticle solution was diluted 10-fold with a 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated with an ultrafiltration unit, and a lipid nanoparticle solution was recovered.

[0209] Composition of lipid nanoparticles Lipids were prepared according to the compositions described in Tables 15 to 18. As the pH-sensitive cationic lipids, CL4F 10-5, CL4F 9-7, CL4F 8-4, CL4F 6, CL15 10-5, and CL15F 6 were used. As other lipids, cholesterol (Nacalai Tesque Co.), β-sitosterol (22, 23-Dihydrostigmasterol, beta-Sitosterol, 5-Stigmasten-3β-ol, α-Dihydrofucosterol, 24α-Ethylcholesterol, Sigma-All), DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine, COATSOME MC-8080, Oily Chemical Industries), DMG-PEG2K (1,2-Dimyrixyl-rac-glycero-3-methylpolyoxyethylen, SUNBRIGHT GM-020, Oily Chemical Industries, Inc.), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine, COATSOME MC-8181, Oily Chemical Industries), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, COATSOME ME-8181, Oily Chemical Industries), DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, COATSOME MC-6060, Oily Chemical Industries) were used. As the mRNA, Fluc-expressing mRNA (CleanCap Fluc mRNA (5moU), TriLink Biotechnologies Inc.), mCherry-expressing mRNA (CleanCap mCherry mRNA (5moU), TriLink Biotechnologies Inc.) were used.

[0210] <Stability Evaluation> The prepared lipid nanoparticles were left to stand at 40°C, and after each period of storage, the average particle diameter, PDI, and nucleic acid encapsulation rate were measured. Whether the quality was maintained well was evaluated according to the following criteria: Good (O): The particle diameter was within ± 20 nm of the particle diameter immediately after preparation, the PDI was 0.2 or less, and the encapsulation rate was 80% or more; Not good enough (X): The particle diameter was more than ± 20 nm of the particle diameter immediately after preparation, or the encapsulation rate was less than 80%.

[0211] For the lipid nanoparticles whose quality was maintained well for 1 week or more at 40°C, it can be evaluated that the stability is excellent.

[0212] <Measurement of luciferase expression activity in vitro> As the medium for cell culture, a medium obtained by adding 10% fetal bovine serum (Fetal Bovine Serum Characterized, Corning) and 10% antibiotics (penicillin-streptomycin (10,000 U / mL), Thermo Fisher) to E-MEM medium (containing L-glucose, phenol red, sodium pyruvate, non-essential amino acids, 1,500 mg / L sodium bicarbonate) (product number 055-08975, Nacalai Tesque) was used. Human embryonic kidney cells 293 (HEK293 cells) were added at 2.0 x 10 4 cells / well to a 96-well white plate (SIGMA), and after incubation at 37°C under a 5% CO2 atmosphere for one night, 100 ng / well of the lipid nanoparticles were added in terms of mRNA content. After incubation for 24 hours under the same conditions, 100 μg / well of a luciferin solution (Beetle luciferin, Promega) at 300 μg / mL was added, and the luminescence intensity was measured using a multifunctional enzyme marker (EnSight multimode plate reader, Perkin Elmer). At this time, the luciferin solution was added to wells to which only the medium had been added and to wells in which cells had been added without the lipid nanoparticles, and the measurement was performed in the same manner, and the value obtained by subtracting the luminescence intensity of the medium wells as background. Note that the luminescence intensity of the wells in which cells had been added without the lipid nanoparticles was below the background.

[0213] <Measurement of mCherry expression activity in vitro> As a culture medium for cell culture, a medium obtained by adding 10% fetal bovine serum (Fetal Bovine Serum Characterized, Corning) and 1% antibiotics (penicillin-streptomycin (10,000 U / mL), Thermo Fisher) to E-MEM medium (containing L-glucose, phenol red, sodium pyruvate, non-essential amino acids, 1,500 mg / L sodium bicarbonate) (product number 055-08975, and Wako Pure Chemical Industries, Ltd.) was used. Human embryonic kidney cells 293 (HEK293 cells) were added at 2.0 x 10 4 cells / well to a 96-well black plate (SIGMA) and, after incubation at 37°C under a 5% CO2 atmosphere overnight, 1000 ng / well of the lipid nanoparticles was added in terms of mRNA content. After incubation for 24 hours under the same conditions, the fluorescence intensity (excitation wavelength 587 nm / emission wavelength 610 nm) was measured with a multifunctional enzyme marker (Ensight multimode plate reader, Perkin Elmer). At this time, wells to which only the culture medium was added and wells of cells to which no lipid nanoparticles were added were also prepared, and the measurement was performed in the same manner, using the value obtained by subtracting the luminescence intensity of the culture medium wells as background. Note that the fluorescence intensity of the wells of cells to which no lipid nanoparticles were added was below the background.

[0214] Table 15 <Results> The results are shown in Tables 19 to 23 below.

[0215]

Claims

1. A pH-sensitive cationic lipid, its stereoisomers, or a mixture of stereoisomers, represented by formula (I): [Chemical Formula 1] In equation (I), a represents an integer from 3 to 5; b represents 0 or 1; R 1 and R 2 Each of the groups represented by the general formula (A) below can be represented independently; [Chemical Formula 2] In formula (A), R 11 and R 12 Each can independently represent a linear or branched C. 2-15 Alkyl; c represents 0 or 1; v represents an integer from 4 to 12; X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group, wherein, The 5-7 membered non-aromatic heterocyclic group can be bonded to a carbon atom via (O-CO)b-, and one or two hydrogen atoms of the ring can be bonded to a carbon atom via a carbon atom. 1-4 Alkyl or C 2-4 Alkenyl substitution: [Chemical Formula 3] In equation (B), d represents an integer from 0 to 3; R 3 and R 4 Each represents C independently. 1-4 Alkyl or C 2-4 Alkenyl, the C 1-4 Alkyl or C 2-4 In alkenyl groups, one or two hydrogen atoms can be replaced by phenyl groups, R 3 and R 4 They can bond with each other to form 5-7 membered non-aromatic heterocycles, wherein one or two hydrogen atoms of the ring can be bonded by C. 1-4 Alkyl or C 2-4 Alkenyl substitution, The pH-sensitive cationic lipids represented by formula (I) are excluded from the pH-sensitive cationic lipids of the following formulas: [Chemical Formula 15] 。 2. The pH-sensitive cationic lipid, its stereoisomers, or mixtures thereof according to claim 1, wherein, The pH-sensitive cationic lipid is represented by the following formula: [Chemical Formula 16-1] [Chemical Formula 16-2] [Chemical Formula 16-3] [Chemical Formula 16-4] [Chemical Formula 16-5] [Chemical Formula 16-6] [Chemical Formula 16-7] [Chemical Formula 16-8] [Chemical Formula 16-9] [Chemical Formula 16-10] [Chemical Formula 16-11] 。 3. A lipid nanoparticle comprising a pH-sensitive cationic lipid represented by formula (I), a stereoisomer or mixture thereof, and nucleic acid. [Chemical Formula 1] In equation (I), a represents an integer from 3 to 5; b represents 0 or 1; R 1 and R 2 Each of the groups represented by the general formula (A) below can be represented independently; [Chemical Formula 2] In formula (A), R 11 and R 12 Each can independently represent a linear or branched C. 2-15 Alkyl; c represents 0 or 1; v represents an integer from 4 to 12; X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group, wherein, The 5-7 membered non-aromatic heterocyclic group can be bonded to a carbon atom via (O-CO)b-, and one or two hydrogen atoms of the ring can be bonded to a carbon atom via a carbon atom. 1-4 Alkyl or C 2-4 Alkenyl substitution: [Chemical Formula 3] In equation (B), d represents an integer from 0 to 3; R 3 and R 4 Each represents C independently. 1-4 Alkyl or C 2-4 Alkenyl, the C 1-4 Alkyl or C 2-4 In alkenyl groups, one or two hydrogen atoms can be replaced by phenyl groups, R 3 and R 4 They can bond with each other to form 5-7 membered non-aromatic heterocycles, wherein one or two hydrogen atoms of the ring can be bonded by C. 1-4 Alkyl or C 2-4 Alkenyl substitution, The nucleic acid is mRNA or plasmid DNA.

4. The lipid nanoparticles according to claim 3, wherein, The pH-sensitive cationic lipids represented by formula (I) are excluded from the pH-sensitive cationic lipids of the following formulas: [Chemical Formula 0] 。 5. The lipid nanoparticles according to claim 3, wherein, The pH-sensitive cationic lipid represented by formula (I) is a pH-sensitive cationic lipid represented by the following formula: [Chemical Formula 7-1] [Chemical Formula 7-2] [Chemical Formula 7-3] [Chemical Formula 7-4] [Chemical Formula 7-5] [Chemical Formula 7-6] [Chemical Formula 7-7] [Chemical Formulas 7-8] 。 6. The lipid nanoparticles according to any one of claims 3 to 5, further comprising lipids modified with sterols and polyalkylene glycols.

7. The lipid nanoparticles according to any one of claims 3 to 6, wherein, The nucleic acid is a gene expressed in liver cells.

8. A pharmaceutical composition comprising, as described in any one of claims 3 to 7, lipid nanoparticles as the active ingredient.

9. The pharmaceutical composition according to claim 8, for use in gene therapy.

10. Use of the lipid nanoparticles according to any one of claims 3 to 7 in the manufacture of a medicament for expressing foreign genes in liver cells.

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