Lipid nanoparticles

Abstract

The present invention addresses the problem of providing lipid nanoparticles which function as gene transfer carriers capable of selective transfer to the liver or spleen. Lipid nanoparticles which contain a pH-sensitive cationic lipid represented by 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) (R11 and R12 each independently represent a straight-chain or branched-chain C2-15 alkyl group; c represents 0 or 1; v represents an integer of 4-12); and X represents a group represented by general formula (B) (d represents an integer of 0-3; and R3 and R4 each independently represent a C1-4 alkyl group or C2-4 alkenyl group, while R3 and R4 may form a 5- to 7-membered non-aromatic heterocycle) or represents a 5- to 7-membered non-aromatic heterocyclic group]. (I): (R1)(R2)C(OH)-(CH2)a-(O-CO)b-X. (A): (R11)(R12)-CH-(CO-O)c-(CH2)v-. (B): -(CH2)d-N(R3)(R4).

Description

[Technical Field]

[0001] This invention relates to lipid nanoparticles useful as gene delivery carriers that can be selectively delivered to the liver or spleen. [Background technology]

[0002] Lipid nanoparticles (LNPs) are used as carriers to encapsulate lipid-soluble drugs and nucleic acids such as siRNA (short interfering RNA) and mRNA, and to deliver them to target cells. For example, lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids such as siRNA into target cells have been reported, and these contain pH-sensitive cationic lipids as constituent lipids that are electrically neutral at physiological pH and change to cationic in weakly acidic pH environments such as endosomes (Patent Document 1 and Non-Patent Document 1).

[0003] As an example of pH-sensitive cationic lipids, Jayaraman et al. developed DLin-MC3-DMA and used it in factor VII (F7) knockdown in mouse liver to achieve ED 50 A dose of 0.005 mg siRNA / kg was achieved (Non-Patent Literature 2). The inventors have also developed their own pH-sensitive cationic lipids YSK05 and YSK13-C3, and have achieved ED in F7 knockdown. 50 They achieved 0.06 and 0.015 mg siRNA / kg respectively (Non-Patent Documents 3-5). In addition, Maier et al. developed L319, which is MC3-DMA with biodegradability, and ED 50 The study has reported achieving both a high level of safety and a concentration of 0.01 mg siRNA / kg (Non-Patent Literature 6-8). However, it has been revealed that the endosome escape efficiency of lipid nanoparticles containing these pH-sensitive cationic lipids is still only a few percent (Non-Patent Literature 9), and the development of technologies that can further improve bioavailability is desired.

[0004] Furthermore, Dong et al. discovered a unique lipid-like substance, cKK-E12, through high-throughput screening, and achieved ED in F7 knockdown. 50 A dose of 0.002 mg siRNA / kg was achieved (Non-Patent Literature 10). While this technology is the most effective in terms of activity according to the literature, there is no information on safety aspects such as toxicity at high doses or lipid biodegradability. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2018 / 230710 [Patent Document 2] International Publication No. 2018 / 190423 [Non-patent literature]

[0006] [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 Document 10] Dong, Proceedings of the National Academy of Sciences of the United States of America, 2014, vol.111(11), p.3955-3960. [Non-Patent Document 11] Leung et al.,Journal of Physical Chemistry C Nanomater Interfaces,2012,vol.116(34),p.18440-18450. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to provide lipid nanoparticles that can serve as gene delivery carriers selectively delivered to the liver or spleen, and to provide lipid nanoparticles with excellent stability. [Means for solving the problem]

[0008] The inventors of the present invention have found that lipid nanoparticles containing a pH-sensitive cationic lipid having a branched hydrocarbon chain as a constituent lipid are highly selective for the liver or spleen and useful as a gene delivery carrier that is specifically highly expressed in the liver or spleen, and have completed the present invention.

[0009] That is, the present invention provides the following lipid nanoparticles. [1-1] The following formula (I): [Chemical formula] [In formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R 1 and R 2 each independently represents the following general formula (A): [Chemical formula] (In formula (A), R 11 and R 12 each independently represents a linear or branched C 5-15 alkyl group; c represents 0 or 1; v represents an integer of 4 to 12) represents a group represented by; X represents the following general formula (B): [Chemical formula] (In formula (B), d represents an integer of 0 to 3; R 3 and R 4 each independently represents a C 1-4 alkyl group or a C 2-4 alkenyl group (the C 1-4 alkyl group or C 2-4 alkenyl group may have one or two hydrogen atoms substituted with a phenyl group), but R 3 and R 4 are bonded to each other to form a 5- to 7-member non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring are C 1-4 alkyl group or C 2-4A group represented by (which may be substituted with an alkenyl group) or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 (May be substituted with an alkenyl group) Lipid nanoparticles containing pH-sensitive cationic lipids represented by [formula].

[0010] [1-2] The lipid nanoparticles of [1-1] further comprising sterols and polyalkylene glycol-modified lipids. [1-3] Lipid nanoparticles of [1-1] or [1-2] containing nucleic acids. [1-4] The lipid nanoparticles of [1-3] wherein the nucleic acid is siRNA. [1-5] The lipid nanoparticles of [1-3] wherein the nucleic acid is mRNA or plasmid DNA.

[0011] [1-6] Lipid nanoparticles of any of [1-3] to [1-5], wherein the nucleic acid is a gene expressed in liver cells. [1-7] A pharmaceutical composition comprising any of the lipid nanoparticles described in [1-1] to [1-6] above as an active ingredient. [1-8] The pharmaceutical composition of [1-7] used in gene therapy. [1-9] A method for expressing an exogenous gene, comprising administering a lipid nanoparticle containing an exogenous gene intended to be expressed in liver cells, which is any of the lipid nanoparticles described in [1-1] to [1-6] above, to a test animal (excluding humans), thereby expressing the exogenous gene in the liver of the test animal.

[0012] [2-1] The following equation (I): [ka] [In formula (I), a represents an integer between 3 and 5; b represents 0 or 1; R 1 and R 2 Each of these independently corresponds to the following general formula (A): [ka] (In formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-15 (Indicates an alkyl group; c indicates 0 or 1; v indicates an integer between 4 and 12) The base is represented by the following general formula (B): [ka] (In equation (B), d represents an integer from 0 to 3; R 3 and R 4 Each is independently C 1-4 Alkyl alkyl group or C 2-4 Alkenyl group (the C 1-4 Alkyl alkyl group or C 2-4 The alkenyl group may have one or two hydrogen atoms substituted for a phenyl group, but R 3 and R 4 These are bonded to each other to form a 5-7 member non-aromatic heteroring (one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 A group represented by (which may be substituted with an alkenyl group) or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 (May be substituted with an alkenyl group) pH-sensitive cationic lipids represented by these, their stereoisomers or mixtures of stereoisomers, and nucleic acids Lipid nanoparticles containing, The lipid nanoparticle wherein the nucleic acid is mRNA or plasmid DNA.

[0013] [2-2] The pH-sensitive cationic lipid is represented by the following formula, and is a lipid nanoparticle of [2-1]: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0014] [3-1] The following equation (I): [ka] [In formula (I), a represents an integer between 3 and 5; b represents 0 or 1; R 1 and R 2 Each of these independently corresponds to the following general formula (A): [ka] (In formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-15 (Indicates an alkyl group; c indicates 0 or 1; v indicates an integer between 4 and 12) The base is represented by the following general formula (B): [ka] (In equation (B), d represents an integer from 0 to 3; R 3 and R 4 Each is independently C 1-4 Alkyl alkyl group or C 2-4 Alkenyl group (the C 1-4 Alkyl alkyl group or C 2-4The alkenyl group may have one or two hydrogen atoms substituted for a phenyl group, but R 3 and R 4 These are bonded to each other to form a 5-7 member non-aromatic heteroring (one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 A group represented by (which may be substituted with an alkenyl group) or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 (May be substituted with an alkenyl group) pH-sensitive cationic lipids, their stereoisomers, or mixtures of stereoisomers represented by these lipids. A pharmaceutical composition for spleen delivery containing the following ingredients.

[0015] [3-2] The pharmaceutical composition of [3-1] wherein the pH-sensitive cationic lipid is represented by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0016] [4-1] The following equation (I): [ka] [In formula (I), a represents an integer between 3 and 5; b represents 0 or 1; R 1 and R 2 Each of these independently corresponds to the following general formula (A): [ka] (In formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-15 (Indicates an alkyl group; c indicates 0 or 1; v indicates an integer between 4 and 12) The base is represented by the following general formula (B): [ka] (In equation (B), d represents an integer from 0 to 3; R 3 and R 4 Each is independently C 1-4 Alkyl alkyl group or C 2-4 Alkenyl group (the C 1-4 Alkyl alkyl group or C 2-4 The alkenyl group may have one or two hydrogen atoms substituted for a phenyl group, but R 3 and R 4 These are bonded to each other to form a 5-7 member non-aromatic heteroring (one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 A group represented by (which may be substituted with an alkenyl group) or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 (May be substituted with an alkenyl group) pH-sensitive cationic lipids represented by (excluding pH-sensitive cationic lipids of the following formula): [ka] ), its stereoisomer, or mixture of stereoisomers.

[0017] [4-2] The pH-sensitive cationic lipid is represented by the following formula: the pH-sensitive cationic lipid of [4-1], its stereoisomer, or a mixture of stereoisomers: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0018] [5-1] (i) sterols or sterol derivatives, (ii) Polyalkylene glycol-modified lipids, (iii) nucleic acid; (iv) buffering agent, (v) disaccharides and (vi) The following equation (I): [ka] [In formula (I), a represents an integer between 3 and 5; b represents 0 or 1; R 1 and R 2Each of these independently corresponds to the following general formula (A): [ka] (In formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-15 (Indicates an alkyl group; c indicates 0 or 1; v indicates an integer between 4 and 12) The base is represented by the following general formula (B): [ka] (In equation (B), d represents an integer from 0 to 3; R 3 and R 4 Each is independently C 1-4 Alkyl alkyl group or C 2-4 Alkenyl group (the C 1-4 Alkyl alkyl group or C 2-4 The alkenyl group may have one or two hydrogen atoms substituted for a phenyl group, but R 3 and R 4 These are bonded to each other to form a 5-7 member non-aromatic heteroring (one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 A group represented by (which may be substituted with an alkenyl group) or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring are C 1-4 Alkyl alkyl group or C 2-4 (May be substituted with an alkenyl group) pH-sensitive cationic lipids represented by, their stereoisomers or mixtures of stereoisomers, A lipid nanoparticle formulation containing these nanoparticles.

[0019] [5-2] The pH-sensitive cationic lipid is represented by the following formula [5-1] in the lipid nanoparticle formulation: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0020] [5-3] A lipid nanoparticle formulation of [5-1] or [5-2], wherein the nucleic acid is mRNA. [5-4] A lipid nanoparticle formulation according to any of [5-1] to [5-3], wherein lipid nanoparticles are suspended in an aqueous solution. [5-5] A lipid nanoparticle formulation of [5-4] wherein the concentration of the disaccharide is 1% to 20% by weight.

[0021] [5-6] A lipid nanoparticle formulation of either [5-4] or [5-5] having a pH of 6.8 to 8.0 at 25°C. [5-7] A lyophilized lipid nanoparticle formulation of any of [5-1] to [5-3]. [5-8] A resuspended preparation obtained by adding water or an aqueous solution to the lipid nanoparticle preparation of [5-7].

[0022] [6-1] The following equation (I): [ka] [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] (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) and X represents a group represented by the following general formula (B): [Chemical formula] (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 1-4 alkyl group or the C 2-4 alkenyl group may have one or two hydrogen atoms substituted by a phenyl group), provided that R 3 and R 4 may be bonded to each other to form a 5- to 7-member non-aromatic heterocyclic ring (one or two hydrogen atoms of the ring may be substituted by a C 1-4 alkyl group or a C 2-4 alkenyl group)) or a 5- to 7-member non-aromatic heterocyclic group (however, the group is bonded to (O-CO)b- by a carbon atom, and one or two hydrogen atoms of the ring may be substituted by a C 1-4 alkyl group or a C 2-4 alkenyl group))] A method for producing a pH-sensitive cationic lipid represented by, a stereoisomer thereof, or a mixture of stereoisomers , comprising: The chemical formula of the pH-sensitive cationic lipid is represented by the following formula: [Chemical formula] ​​​​A method for producing branched fatty acids, comprising at least the step of reacting an alkyl carboxylic acid with an alkyl halide in the presence of organolithium, dimethylpropylene urea (DMPU), and tetrahydrofuran (THF).

[0023] [6-2] The volume ratio of tetrahydrofuran (THF) to dimethylpropylene urea (DMPU) in the above step is 10:1 to 1:1 (v / v) The method for producing [6-1]. [6-3] A method for producing [6-1] or [6-2], wherein the organolithium is lithium diisopropylamide (LDA). [6-4] A method for producing any of the methods described in [6-1] to [6-3], wherein the alkyl halide is an iodoalkyl group. [6-5] A method for producing branched fatty acids, any of the methods described in [6-1] to [6-4], further comprising the step of purifying branched fatty acids by reverse-phase chromatography.

[0024] [6-6] The following equation (I): [ka] [In equation (I), a represents an integer between 3 and 5; b represents 0 or 1; and R1 and R2 are independently given by the following general equation (A): [ka] (In formula (A), R11 and R12 each independently represent a linear or branched C2-15 alkyl group; c represents 0 or 1; and v represents an integer from 4 to 12.) The base is represented by the following general formula (B): [ka] (In formula (B), d represents an integer from 0 to 3; R3 and R4 each independently represent a C1-4 alkyl group or a C2-4 alkenyl group (the C1-4 alkyl group or C2-4 alkenyl group may have one or two hydrogen atoms substituted with a phenyl group), but R3 and R4 may be bonded to each other to form a 5-7 membered non-aromatic heterocycle (the ring may have one or two hydrogen atoms substituted with a C1-4 alkyl group or a C2-4 alkenyl group).) [The group represented by or a 5-7 membered non-aromatic heterocyclic group (where the group is bonded to (O-CO)b- by a carbon atom, and one or two hydrogen atoms of the ring may be substituted with a C1-4 alkyl group or a C2-4 alkenyl group)] pH-sensitive cationic lipids, their stereoisomers, or mixtures of stereoisomers represented by these lipids. A method for manufacturing, The chemical formula for the pH-sensitive cationic lipid is as follows: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is represented as, A method for producing branched fatty acids, comprising at least the step of reacting a malonic acid ester with an alkyl halide in the presence of a base, and then subjecting the resulting reaction solution to hydrolysis and heat treatment.

[0025] [6-7] A method for producing [6-6], wherein the malonic acid ester is dimethyl malonate. [6-8] A method for producing [6-6] or [6-7], wherein the alkyl halide is iodoalkyl. [6-9] A method of production according to any of [6-6] to [6-8], wherein the base is selected from the group consisting of sodium hydride, calcium hydride, sodium ethoxide, and bis(trismethylsilyl)amide lithium. [6-10] A method of production according to any of [6-6] to [6-9], wherein the hydrolysis treatment is performed using sodium hydroxide, calcium hydroxide, or lithium hydroxide. [6-11] A manufacturing method according to any of [6-6] to [6-10], wherein the heat treatment is performed at 120°C to 170°C simultaneously with and / or after the hydrolysis treatment. [6-12] A method of production according to any of [6-6] to [6-11], further comprising a step of purifying branched fatty acids by reverse-phase chromatography. [Effects of the Invention]

[0026] The lipid nanoparticles according to the present invention can enable high expression of encapsulated genes in the liver or spleen. For this reason, these lipid nanoparticles are useful as liver-specific or spleen-specific gene delivery carriers used in gene therapy. Furthermore, the lipid nanoparticles according to the present invention exhibit excellent stability. [Brief explanation of the drawing]

[0027] [Figure 1]This is a diagram showing the results of measuring the pKa of each lipid nanoparticle loaded with F7 siRNA in Example 1. Figure 1(A) shows the results of lipid nanoparticles prepared using CL4F6, CL4G6, or CL4H6, and Figure 1(B) shows the results of lipid nanoparticles prepared using CL15F6, CL15G6, or CL15H6. [Figure 2] This is a diagram showing the results of calculating the relative plasma F7 enzyme activity (%) in mice administered with each lipid nanoparticle loaded with F7 siRNA in Example 1. Figure 2(A) shows the results of mice administered with lipid nanoparticles prepared using CL4F6, CL4G6, or CL4H6, and Figure 2(B) shows the results of mice administered with lipid nanoparticles prepared using CL15F6, CL15G6, or CL15H6. [Figure 3] This is a diagram showing the measurement results of Nluc activity (RLU / mg protein) in the liver and spleen of mice administered with each lipid nanoparticle loaded with Nluc mRNA in Example 2. [Figure 4] This is a diagram showing the measurement results of Fluc activity in HeLa-GFP cells transfected with each lipid nanoparticle loaded with pFluc in Example 3. [Figure 5] This is a diagram showing the measurement results of Fluc activity (RLU / mg protein) in the liver and spleen of mice administered with each lipid nanoparticle loaded with pFluc in Example 3.

Mode for Carrying Out the Invention

[0028] Hereinafter, embodiments of the present invention will be specifically described. In the specification of the present application, "X1 to X2 (X1 and X2 are real numbers satisfying X1 < X2)" means "X1 or more and X2 or less".

[0029] The lipid nanoparticles according to the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid represented by the following general formula (I) (hereinafter sometimes referred to as "the pH-sensitive cationic lipid of the present invention"). By comprising the pH-sensitive cationic lipid represented by general formula (I) as a constituent lipid of the lipid nanoparticles, the lipid nanoparticles according to the present invention exhibit high selectivity for the liver or spleen.

[0030] [ka]

[0031] In general formula (I), a represents an integer between 3 and 5, but is preferably 4. b represents either 0 or 1. If b is 0, it means that there is no -O-CO- group and that it is a single bond.

[0032] In general formula (I), R 1 and R 2 Each of these independently represents a group represented by the following general formula (A). In general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-15 It represents an alkyl group (an alkyl group with 2 to 15 carbon atoms); c represents 0 or 1; and v represents an integer from 4 to 12.

[0033] [ka]

[0034] Linear or branched C 2-15 As alkyl groups, 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-heptyl group, 1-methylhexyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl group, 1-ethylpentyl group, 1,1-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 4,4-dimethylpentyl group, 1-methyl-3,3-dimethylbutyl group, 2-methyl-3,3-dimethylbutyl group; n-octyl group, 1-methylheptyl group, 2-methylheptyl group, 3-methylheptyl group, 4-methylheptyl group, 5-methylheptyl group, 6-methylheptyl group, 1-ethylhexyl group, 1,1-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 5,5-dimethylhexyl group, 1-methyl-4,4-dimethylpentyl group, 2-methyl-4,4-dimethylpentyl group, 3-methyl-4,4-dimethylpentyl group; n-nonyl group, 1-methyloctyl group, 2-methyloctyl group, 3-methyloctyl group, 4-methyloctyl group, 5-methyloctyl group, 6-methyloctyl group, 7-methyloctyl group, 1-ethylheptyl group, 1,1-dimethylheptyl group, 2,2-dimethylheptyl group, 3,3-dimethylheptyl group, 4,4-dimethylheptyl group, 5,5-dimethylheptyl group, 6,6-dimethylheptyl group, 1-methyl-5,5-dimethylhexyl group, 2-methyl-5,5-dimethylhexyl group, 3-methyl-5,5-dimethylhexyl group, 4-methyl-5,5-dimethylhexyl group; n-decyl group, 1-methylnonyl group, 2-methylnonyl group, 3-methylnonyl group, 4-methylnonyl group, 5-methylnonyl group, 6-methylnonyl group, 7-methylnonyl group, 8-methylnonyl group, 1-ethyloctyl group, 1,1-dimethyloctyl group, 2,2-dimethyloctyl group, 3,3-dimethyloctyl group, 4,4-dimethyloctyl group, 5,5-dimethyloctyl group, 6,6-dimethyloctyl group, 7,7-dimethyloctyl group, 1-methyl-6,6-dimethylheptyl group, 2-methyl-6,6-dimethylheptyl group, 3-methyl-6,6-dimethylheptyl group, 4-methyl-6,6-dimethylheptyl group, 5-methyl-6,6-dimethylheptyl group; n-undecyl group, 1-methyldecyl group, 2-methyldecyl group, 3-methyldecyl group, 4-methyldecyl group, 5-methyldecyl group, 6-methyldecyl group, 7-methyldecyl group, 8-methyldecyl group, 9-methyldecyl group, 1-ethylnonyl group, 1,1-dimethylnonyl group, 2,2-dimethylnonyl group, 3,3-dimethylnonyl group, 4,4-dimethylnonyl group, 5,5-dimethylnonyl group, 6,6-dimethylnonyl group, 7,7-dimethylnonyl group, 8,8-dimethylnonyl group, 1-methyl-7,7-dimethyloctyl group, 2-methyl-7,7-dimethyloctyl group, 3-methyl-7,7-dimethyloctyl group, 4-methyl-7,7-dimethyloctyl group, 5-methyl-7,7-dimethyloctyl group, 6-methyl-7,7-dimethyloctyl group; n-dodecyl group, 1-methylundecyl group, 2-methylundecyl group, 3-methylundecyl group, 4-methylundecyl group, 5-methylundecyl group, 6-methylundecyl group, 7-methylundecyl group, 8-methylundecyl group, 9-methylundecyl group, 10-methylundecyl group, 1-ethyldecyl group, 1,1-dimethyldecyl group, 2,2-dimethyldecyl group, 3,3-dimethyldecyl group, 4,4-dimethyldecyl group, 5,5 -dimethyldecyl group, 6,6-dimethyldecyl group, 7,7-dimethyldecyl group, 8,8-dimethyldecyl group, 9,9-dimethyldecyl group, 1-methyl-8,8-dimethylnonyl group, 2-methyl-8,8-dimethylnonyl group, 3-methyl-8,8-dimethylnonyl group, 4-methyl-8,8-dimethylnonyl group, 5-methyl-8,8-dimethylnonyl group, 6-methyl-8,8-dimethylnonyl group, 7-methyl-8,8-dimethylnonyl group; n-tridecyl group, 1-methyldodecyl group, 2-methyldodecyl group, 3-methyldodecyl group, 4-methyldodecyl group, 5-methyldodecyl group, 6-methyldodecyl group, 7-methyldodecyl group, 8-methyldodecyl group, 9-methyldodecyl group, 10-methyldodecyl group, 11-methyldodecyl group, 1-ethylundecyl group, 1,1-dimethylundecyl group, 2,2-dimethylundecyl group, 3,3-dimethylundecyl group, 4,4-dimethylundecyl group, 5,5-dimethylundecyl group, 6,6-di Methyl undecyl group, 7,7-dimethyl undecyl group, 8,8-dimethyl undecyl group, 9,9-dimethyl undecyl group, 10,10-dimethyl undecyl group, 1-methyl-9,9-dimethyldecyl group, 2-methyl-9,9-dimethyldecyl group, 3-methyl-9,9-dimethyldecyl group, 4-methyl-9,9-dimethyldecyl group, 5-methyl-9,9-dimethyldecyl group, 6-methyl-9,9-dimethyldecyl group, 7-methyl-9,9-dimethyldecyl group, 8-methyl-9,9-dimethyldecyl group; n-tetradecyl group, 1-methyltridecyl group, 2-methyltridecyl group, 3-methyltridecyl group, 4-methyltridecyl group, 5-methyltridecyl group, 6-methyltridecyl group, 7-methyltridecyl group, 8-methyltridecyl group, 9-methyltridecyl group, 10-methyltridecyl group, 11-methyltridecyl group, 12-methyltridecyl group, 1-ethyldodecyl group, 1,1-dimethyldodecyl group, 2,2-dimethyldodecyl group, 3,3-dimethyldodecyl group, 4,4-dimethyldodecyl group, 5,5-dimethyldodecyl group, 6,6-dimethyldodecyl group, 7,7-dimethyldodecyl group, 8,8-dimethyldodecyl group, 9,9-dimethyldodecyl group, 10,10-dimethyldodecyl group, 11,11-dimethyldodecyl group, 1-methyl-10,10-dimethylundecyl group, 2-methyl-10,10-dimethylundecyl group, 3-methyl-10,10-dimethylundecyl group, 4-methyl-10,10-dimethylundecyl group, 5-methyl-10,10-dimethylundecyl group, 6-methyl-10,10-dimethylundecyl group, 7-methyl-10,10-dimethylundecyl group, 8-methyl-10,10-dimethylundecyl group, 9-methyl-10,10-dimethylundecyl group; n-pentadecyl group, 1-methyltetradecyl group, 2-methyltetradecyl group, 3-methyltetradecyl group, 4-methyltetradecyl group, 5-methyltetradecyl group, 6-methyltetradecyl group, 7-methyltetradecyl group, 8-methyltetradecyl group, 9-methyltetradecyl group, 10-methyltetradecyl group, 11-methyltetradecyl group, 12-methyltetradecyl group, 13-methyltetradecyl group, 1-ethyltridecyl group, 1,1-dimethyltridecyl group, 2,2-dimethyltridecyl group, 3,3-dimethyltridecyl group, 4,4-dimethyltridecyl group, 5,5-dimethyltridecyl group, 6,6-dimethyltridecyl group, 7,7-dimethyltridecyl group, 8,8- Examples include dimethyltridecyl group, 9,9-dimethyltridecyl group, 10,10-dimethyltridecyl group, 11,11-dimethyltridecyl group, 12,12-dimethyltridecyl group, 1-methyl-11,11-dimethyldodecyl group, 2-methyl-11,11-dimethyldodecyl group, 3-methyl-11,11-dimethyldodecyl group, 4-methyl-11,11-dimethyldodecyl group, 5-methyl-11,11-dimethyldodecyl group, 6-methyl-11,11-dimethyldodecyl group, 7-methyl-11,11-dimethyldodecyl group, 8-methyl-11,11-dimethyldodecyl group, 9-methyl-11,11-dimethyldodecyl group, and 10-methyl-11,11-dimethyldodecyl group.

[0035] In general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-12 Preferably, it is an alkyl group (an alkyl group having 2 to 12 carbon atoms), and is linear or branched. 5-12 It is more preferably an alkyl group (an alkyl group having 5 to 12 carbon atoms), and is linear or branched C 5-10 It is more preferably an alkyl group (an alkyl group having 5 to 10 carbon atoms), and is linear or branched in shape. 6-9 It is most preferable that the alkyl group be an alkyl group having 6 to 9 carbon atoms. In addition, in the pH-sensitive cationic lipid of the present invention, R 1 and R 2The groups can be any group represented by general formula (A), and they may be the same group or different groups.

[0036] In general formula (I), X represents a group represented by the following general formula (B) 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 a carbon atom by (O-CO)b-.

[0037] [ka]

[0038] In general formula (B), d is an integer between 0 and 3. When d is 0, it means that there is no -(CH2)- group and that it is a single bond. In general formula (B), R 3 and R 4 Each is independently C 1-4 Alkyl group (alkyl group with 1 to 4 carbon atoms) or C 2-4 This indicates an alkenyl group (an alkenyl group with 1 to 4 carbon atoms). 3 and R 4 C, which is shown 1-4 Alkyl alkyl group or C 2-4 The alkenyl group may have one or two hydrogen atoms substituted with phenyl groups. 3 and R 4 C 1-4 Alkyl alkyl group or C 2-4 Any alkenyl group will suffice; they may be the same group or different groups.

[0039] C 1-4 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl groups. 2-4 Examples of alkenyl groups include vinyl group, 1-propenyl group, 2-propenyl group, 1-methylvinyl group, 2-methyl-1-propenyl group, 1-butenyl group, 2-butenyl group, and 3-butenyl group.

[0040] In general formula (B), R 3 and R 4These may be bonded to each other to form a 5-7 member non-aromatic heterocycle. 3 and R 4 Examples of 5- to 7-membered non-aromatic heterocycles formed by the bonding of these groups include 1-pyrrolidinyl groups, 1-piperidinyl groups, 1-morpholinyl groups, and 1-piperazinyl groups. 3 and R 4 A 5-7 membered non-aromatic heteroring is formed by the bonding of these atoms to one another, in which one or two hydrogen atoms in the ring are C 1-4 Alkyl alkyl group or C 2-4 It may be substituted with an alkenyl group. Two hydrogen atoms in the ring are C 1-4 Alkyl alkyl group or C 2-4 If the groups are substituted with alkenyl groups, they may be substituted with the same group or with different groups.

[0041] In general formula (I), when X is a 5- to 7-membered non-aromatic heterocyclic group, examples of heteroatoms included in the heterocyclic group include nitrogen atoms, oxygen atoms, or sulfur atoms. The heteroatoms constituting the heterocycle in the heterocyclic group may be one, or two or more identical or different heteroatoms. The heterocycle in the heterocyclic group may be a saturated heterocycle and may contain one or more double bonds, but the heterocycle will not be an aromatic ring.

[0042] The pH-sensitive cationic lipid of the present invention is R in general formula (I). 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-12 The group is an alkyl group in which 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 a 5-7 member non-aromatic heterocyclic group (where the carbon atoms in the heterocyclic group are bonded to (O-CO)b-), preferably a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (where the carbon atoms in the ring are bonded to (O-CO)b-, and one hydrogen atom is C 1-4Alkyl or C 2-4 Compounds that are substituted with an alkenyl group, or in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 2-12 It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, X is the general formula (B), d is 0, R 3 and R 4 Each of them independently C 1-4 Alkyl alkyl group or C 2-4 Alkenyl group (R 3 and R 4 C, which is shown 1-4 Alkyl alkyl group or C 2-4 A compound in which the alkenyl group may have one or two hydrogen atoms substituted with phenyl groups is preferred. Also, in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 5-12 The group is an alkyl group in which 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 a 5-7 member non-aromatic heterocyclic group (where the carbon atoms in the heterocyclic group are bonded to (O-CO)b-), preferably a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (where the carbon atoms in the ring are bonded to (O-CO)b-, and one hydrogen atom is C 1-4 Alkyl or C 2-4 Compounds that are substituted with an alkenyl group, or in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 5-12It is an alkyl group where c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 0, X is the general formula (B), d is 0, R 3 and R 4 Each of them independently C 1-4 Alkyl alkyl group or C 2-4 Alkenyl group (R 3 and R 4 C, which is shown 1-4 Alkyl alkyl group or C 2-4 Compounds in which the alkenyl group may have one or two hydrogen atoms substituted with phenyl groups are preferred. In particular, as the pH-sensitive cationic lipid of the present invention, R in general formula (I) 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 6-9 It is an alkyl group in which 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 a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C 1-4 Alkyl or C 2-4 Compounds that are substituted with an alkenyl group, or in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently a linear or branched C 6-9 It is an alkyl group 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 general formula (B) where d is 0, R 3 and R 4 Each of them independently C 1-4 Compounds that are alkyl groups are preferred.

[0043] The pH-sensitive cationic lipid of the present invention is more preferably R in general formula (I). 1 and R 2Each of them independently, in the general formula (A), R 11 and R 12 Each of them is independently a linear C 6-9 It is an alkyl group in which 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 a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C 1-4 Alkyl or C 2-4 Compounds that may be substituted with an alkenyl group; in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently branched in a chain-like structure. 6-9 It is an alkyl group in which 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 a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C 1-4 Alkyl or C 2-4 Compounds that may be substituted with an alkenyl group; in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of them is independently a linear C 6-9 It is an alkyl group 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 general formula (B) where d is 0, R 3 and R 4 Each of them independently C 1-4 Compounds that are alkyl groups; in general formula (I), R 1 and R 2 Each of them independently, in the general formula (A), R 11 and R 12 Each of these is independently branched in a chain-like structure. 6-9It is an alkyl group 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 general formula (B) where d is 0, R 3 and R 4 Each of them independently C 1-4 It is a compound that is an alkyl group.

[0044] The pH-sensitive cationic lipid of the present invention is R in general formula (I). 1 and R 2 The same group, and of the general formula (A), R 11 and R 12 Each of them is independently a linear C 6-9 It is an alkyl group in which 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 a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C 1-4 Alkyl or C 2-4 Compounds that may be substituted with an alkenyl group; in general formula (I), R 1 and R 2 The same group, and of the general formula (A), R 11 and R 12 Each of these is independently branched in a chain-like structure. 6-9 It is an alkyl group in which 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 a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, or a 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, with one hydrogen atom being C 1-4 Alkyl or C 2-4 Compounds that may be substituted with an alkenyl group; in general formula (I), R 1 and R 2 The same group, and of the general formula (A), R 11 and R 12 Each of them is independently a linear C 6-9It is an alkyl group 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 general formula (B) where d is 0, R 3 and R 4 Each of them independently C 1-4 Compounds that are alkyl groups; in general formula (I), R 1 and R 2 The same group, and of the general formula (A), R 11 and R 12 Each of these is independently branched in a chain-like structure. 6-9 It is an alkyl group 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 general formula (B) where d is 0, R 3 and R 4 Each of them independently C 1-4 Compounds that are alkyl groups are particularly preferred.

[0045] Examples of pH-sensitive cationic lipids of the present invention include pH-sensitive cationic lipids having the following structure, stereoisomers thereof, or mixtures of stereoisomers: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [Chemical] [Chemical] [Chemical] [Chemical] [Chemical] In one aspect, the present invention relates to the pH-sensitive cationic lipid of the present invention.

[0046] The pKa of the pH-sensitive cationic lipid represented by the general formula (I) is not particularly limited, but can be selected, for example, in the range of about 4.0 to 9.0, preferably about 4.5 to 8.5. It is preferable to select the types of each substituent so as to give a pKa within this range.

[0047] The pH-sensitive cationic lipid represented by the general formula (I) can be easily produced, for example, by the method specifically shown in the examples of this specification. By referring to this production method and appropriately selecting raw material compounds, reagents, reaction conditions, etc., those skilled in the art can easily produce any lipid included in the range of the general formula (I).

[0048] The group represented by the general formula (A) is a group having a branched structure in which two hydrocarbon chains (R 11 and R 12 ) are linked to the -CO-O- group. That is, in the pH-sensitive cationic lipid of the present invention, two branched-chain hydrocarbon chains (R 1 and R 2 ) are provided, and these hydrocarbon chains serve as hydrophobic scaffolds embedded in the lipid membrane of the lipid nanoparticles. The lipid nanoparticles according to the present invention have the characteristic of high selectivity for the liver or spleen by using the pH-sensitive cationic lipid of the present invention having a hydrophobic scaffold composed of a branched-chain structure as a constituent component of the lipid.

[0049] The pH-sensitive cationic lipid constituting the lipid nanoparticles according to the present invention may be one type or two or more types. When there are two or more pH-sensitive cationic lipids constituting the lipid nanoparticles according to the present invention, the amount of the pH-sensitive cationic lipids according to the present invention means the total amount of lipid molecules that correspond to the pH-sensitive cationic lipids according to the present invention among the lipid molecules constituting the lipid nanoparticles.

[0050] The higher the proportion of the pH-sensitive cationic lipid of the present invention among the lipid molecules constituting the lipid nanoparticles, the higher the efficiency of uptake of the lipid nanoparticles into target cells. For this reason, in the lipid nanoparticles according to the present invention, the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipid constituting the lipid nanoparticles ([amount of pH-sensitive cationic lipid of the present invention (mol)] / ([amount of total lipid constituting the lipid nanoparticles (mol)]) × 100%) is preferably 20 mol% or more. On the other hand, if the proportion of the pH-sensitive cationic lipid among the lipid molecules constituting the lipid nanoparticles is too high, it may be difficult to sufficiently reduce the particle size. In order to obtain lipid nanoparticles with sufficient uptake efficiency into target cells and sufficiently small particle size, the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipid constituting the lipid nanoparticles in the lipid nanoparticles according to the present invention is more preferably 30 mol% or more, even more preferably 30 to 70 mol%, and even more preferably 40 to 60 mol%.

[0051] Among the constituent lipids of the lipid nanoparticles according to the present invention, lipids other than the pH-sensitive cationic lipids of the present invention can be lipids that are generally used when forming liposomes. Examples of such lipids include phospholipids, sterols or sterol derivatives, glycolipids, or saturated or unsaturated fatty acids. These can be used individually or in combination of two or more.

[0052] Examples of phospholipids include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phospharidylcholine, cardiolipin, plasmalogen, ceramidephosphorylglycerol phosphate, and phosphatidic acid; and sphingophospholipids such as sphingomyelin, ceramidephosphorylglycerol, and ceramidephosphorylethanolamine. Natural phospholipids such as egg yolk lecithin and soy lecithin can also be used. The fatty acid residues in glycerophospholipids and sphingophospholipids are not particularly limited, but examples include saturated or unsaturated fatty acid residues with 12 to 24 carbon atoms, with saturated or unsaturated fatty acid residues with 14 to 20 carbon atoms being preferred. Specifically, examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid. When these glycerolipids or sphingolipids have two or more fatty acid residues, all fatty acid residues may be the same group or they may be different groups.

[0053] Examples of sterols or sterol derivatives include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, β-sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as thymosterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; and sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside. Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids with 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0054] The constituent lipids of the lipid nanoparticles according to the present invention preferably include neutral lipids in addition to the pH-sensitive cationic lipids of the present invention, more preferably include phospholipids or sterols, even more preferably include sterols, and still more preferably include cholesterol.

[0055] The lipid nanoparticles according to the present invention preferably contain polyalkylene glycol-modified lipids as lipid components. Polyalkylene glycol is a hydrophilic polymer, and by constructing lipid nanoparticles using polyalkylene glycol-modified lipids as lipid membrane constituent lipids, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may enhance the stability of the lipid nanoparticles, such as their blood retention.

[0056] Examples of polyalkylene glycols that can be used include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.

[0057] For example, stearylated polyethylene glycol (e.g., PEG-45 stearate (STR-PEG45)) can be used for modifying lipids with polyethylene glycol. Other examples include N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and N-[carbonyl-methoxypolyethylene glycol Polyethylene glycol derivatives such as [N-[Carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[Carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) can also be used, but polyalkylene glycolated lipids are not limited to these.

[0058] The ratio of polyalkylene glycol-modified lipids to the total lipid content of the lipid nanoparticles according to the present invention is not particularly limited as long as it does not impair the liver selectivity or spleen selectivity of the pH-sensitive cationic lipids of the present invention, specifically, the liver-specific gene expression activity or spleen-specific gene expression activity when the lipid nanoparticles according to the present invention are used as gene carriers. For example, the ratio of polyalkylene glycol-modified lipids to the total lipid content of the lipid nanoparticles is preferably 0.5 to 3 mol%.

[0059] The lipid nanoparticles according to the present invention can be subjected to appropriate surface modification as needed. The lipid nanoparticles according to the present invention can have their blood retention enhanced by modifying their surface with a hydrophilic polymer or the like. In some cases, surface modification can also be performed by using lipids modified with these modifying groups as constituent lipids of the lipid nanoparticles.

[0060] In producing lipid nanoparticles according to the present invention, lipid derivatives that enhance blood retention can be used, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, and polyglycerol phospholipid derivatives. In addition, hydrophilic polymers that enhance blood retention can be used for surface modification, such as polyalkylene glycol, dextran, pullulan, ficol, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, and carrageenan.

[0061] In addition, in order to promote the nuclear translocation of the lipid nanoparticles according to the present invention, for example, the lipid nanoparticles can be surface-modified with an oligosaccharide compound having three or more sugars. The type of the oligosaccharide compound having three or more sugars is not particularly limited. For example, an oligosaccharide compound having about 3 to 10 sugar units bonded thereto can be used, and preferably an oligosaccharide compound having about 3 to 6 sugar units bonded thereto can be used. Among them, preferably, an oligosaccharide compound that is a trimer to hexamer of glucose can be used, and more preferably an oligosaccharide compound that is a trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose, etc. can be preferably used. Among these, maltotriose, maltotetraose, maltopentaose, or maltohexaose in which glucose is α1-4-bonded is more preferable. Particularly preferable is maltotriose or maltotetraose, and most preferable is maltotriose. The amount of surface modification of the lipid nanoparticles with the oligosaccharide compound is not particularly limited. For example, it is about 1 to 30 mol%, preferably about 2 to 20 mol%, more preferably about 5 to 10 mol% based on the total lipid amount.

[0062] The method for surface-modifying the lipid nanoparticles with the oligosaccharide compound is not particularly limited. For example, since liposomes (International Publication No. 2007 / 102481) in which the lipid nanoparticles are surface-modified with a monosaccharide such as galactose or mannose are known, the surface modification method described in this publication can be adopted. The entire disclosure of the above publication is incorporated herein by reference and included as part of the disclosure of the present specification.

[0063] Furthermore, the lipid nanoparticles according to the present invention can be endowed with one or more functions, such as temperature-sensitive function, membrane permeability function, gene expression function, and pH-sensitive function. By appropriately adding these functions, the retention of lipid nanoparticles in the bloodstream can be improved, allowing the lipid nanoparticles to efficiently escape from endosomes after endocytosis in target cells, and enabling more efficient expression of encapsulated nucleic acids in or within liver cells.

[0064] The lipid nanoparticles according to the present invention may contain one or more substances selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides. Examples of charged substances that impart a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and examples of charged substances that impart a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Examples of membrane polypeptides include superficial polypeptides and endogenous polypeptides. The amounts of these substances are not particularly limited and can be appropriately selected depending on the purpose.

[0065] The size of the lipid nanoparticles according to the present invention is preferably such that the average particle diameter is 400 nm or less, more preferably 300 nm or less, even more preferably 200 nm or less, and even more preferably 150 nm or less, as this facilitates 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 using a commercially available DLS device or the like in a conventional manner.

[0066] The polydispersity index (PDI) of the lipid nanoparticles according to the present invention is approximately 0.01 to 0.7, preferably 0.01 to 0.6, and more preferably 0.03 to 0.3. The zeta potential at pH 7.4 can be in the range of -50mV to 5mV, preferably -45mV to 5mV.

[0067] The morphology of the lipid nanoparticles according to the present invention is not particularly limited, but examples of morphologies when dispersed in an aqueous solvent include single-layer liposomes, multilayer liposomes, spherical micelles, or amorphous layered structures. The lipid nanoparticles according to the present invention are preferably single-layer liposomes or multilayer liposomes.

[0068] The lipid nanoparticles according to the present invention preferably contain a component intended for delivery into target cells within a lipid membrane-covered particle. The component contained within the lipid nanoparticles according to the present invention is not particularly limited as long as it is of a size that can be encapsulated. Any substance such as nucleic acids, sugars, peptides, low molecular weight compounds, and metal compounds can be encapsulated in the lipid nanoparticles according to the present invention.

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

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

[0071] The gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is not particularly limited, and vectors commonly used in gene therapy and the like can be used. Preferably, the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is a nucleic acid vector such as a plasmid vector. The plasmid vector may remain circular, or it may be pre-cut into a linear shape and then encapsulated in the lipid nanoparticles according to the present invention. The gene expression vector can be designed by conventional methods using commonly used molecular biological tools based on the base sequence information of the gene to be expressed, and can be manufactured by various known methods.

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

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

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

[0075] Lipid nanoparticles according to the present invention can also be produced by an alcohol dilution method using a channel. This method involves introducing a solution in which lipid components are dissolved in an alcohol solvent and a solution in which water-soluble components to be incorporated into lipid nanoparticles are dissolved in an aqueous solvent from separate channels and then combining them to produce lipid nanoparticles. By using a microchannel with a built-in three-dimensional micromixer capable of instantaneous mixing of the two liquids, lipid nanoparticles with a diameter of approximately 30 nm can be produced with good reproducibility (Non-Patent Literature 11). As for the channel used for production, it is preferable to use a simple two-dimensional channel structure, such as the one described in Patent Literature 2, in which baffles (obstruction plates) of a constant width relative to the channel width are arranged alternately on both sides of a micro-sized channel through which the raw material solution flows. This allows for the formation of a nano-sized lipid particle formation system with high particle size controllability.

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

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

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

[0079] The lipid nanoparticles of the present invention have excellent stability. For example, the lipid nanoparticles of the present invention are stable for one week or more when stored at -80°C, and / or stable for one week, two weeks, three weeks, four weeks, five weeks or more when stored at 5°C, and / or stable for one week, two weeks, three weeks, four weeks, five weeks or more when stored at 25°C, and / or stable for three days, one week, two weeks, three weeks, four weeks, five weeks or more when stored at 40°C. Regarding the quality of lipid nanoparticles, for example, lipid nanoparticles can be defined as those that meet all three conditions: the average particle size, PDI, and nucleic acid encapsulation rate after being left to stand at a predetermined temperature for a predetermined period are compared to the values ​​immediately after preparation, and the average particle size is maintained within ±20 nm of the date of preparation, the PDI is 0.2 or less to maintain high uniformity, and the nucleic acid encapsulation rate is maintained at 80% or higher. For example, evaluation may be conducted according to the following criteria: Good: Particle size is within ±20 nm of the particle size immediately after preparation, PDI is 0.2 or less, and encapsulation rate is 80% or higher; Poor: Particle size is more than ±20 nm of the particle size immediately after preparation, or the encapsulation rate is less than 80%. For example, lipid nanoparticles that maintain good quality for more than a week when left standing at 5°C, or lipid nanoparticles that maintain good quality for more than a week when left standing at 40°C, may be evaluated as lipid nanoparticles with excellent stability.

[0080] When lipid nanoparticles containing a gene expression vector are administered to an animal, the gene expression vector encapsulated in the lipid nanoparticles is selectively expressed in the liver or spleen rather than in other organs. Similarly, when lipid nanoparticles containing an siRNA expression vector are administered to an animal, the siRNA expression vector encapsulated in the lipid nanoparticles is selectively expressed in the liver or spleen rather than in other organs, and the expression of the gene targeted by the expression vector is suppressed. For example, when lipid nanoparticles containing an exogenous gene intended to be expressed in liver cells or spleen cells are administered to a test animal, the exogenous gene can be expressed in the liver or spleen of the test animal.

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

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

[0083] The pH-sensitive cationic lipids of the present invention, their stereoisomers, or mixtures of stereoisomers are synthesized, for example, by condensing a branched fatty acid with a basic skeleton, such as 7-(4-(dipropylamino)butyl)tridecane-1,7,13-triol or 5,11-dihydroxy5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate. In one aspect, the present invention relates to a method for producing pH-sensitive cationic lipids, their stereoisomers, or mixtures of stereoisomers. In one embodiment of the present invention, the method for producing pH-sensitive cationic lipids includes at least a step (step A) of reacting an alkyl carboxylic acid with an alkyl halide in the presence of organolithium, dimethylpropylene urea (DMPU), and tetrahydrofuran (THF) to obtain a branched fatty acid. In this method, the alkylcarboxylic acid is, for example, octanoic acid, decanoic acid, tridecanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, or hexadecenoic acid. In this method, the alkyl halide is, for example, 1-iodohexane, 1-iodobutane, 2-iodohexane, 1-bromohexane, iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1-iodohexane, 1-iodoheptane, 1-iodooctane, 1-iodononane, 1-iodododecane, 1-iodonundecane, 1-iodododecane, 1-iodotridecane, 1-iodotetradecane, 1-iodopentadecane, or 1-iodohexadecane. In this method, the organolithium is, for example, lithium diisopropylamide lithium (LDA), t-butyllithium, or n-butyllithium.

[0084] In one embodiment of the present invention, the method for producing pH-sensitive cationic lipids of the present invention includes at least a step (step B) of hydrolyzing and heat-treating a reaction solution obtained by reacting a malonic acid ester with an alkyl halide in the presence of a base to obtain a branched fatty acid. In this method, the malonic acid ester is, for example, dimethyl malonate, diethyl malonate, or diisopropyl malonate, and is preferably dimethyl malonate. In this method, the alkyl halide is, for example, an iodoalkyl, and the iodoalkyl is, for example, 1-iodohexane, 1-iodopropane, or 2-iodohexane. In this method, the base is, for example, sodium hydride, calcium hydride, sodium ethoxide, and bis(trismethylsilyl)amide lithium, and is preferably sodium hydride. In this method, the hydrolysis treatment is carried out using, for example, sodium hydroxide, calcium hydroxide, and lithium hydroxide. In this method, the heat treatment is carried out simultaneously with and / or after the hydrolysis treatment, preferably at 120°C to 170°C, more preferably at 150°C to 170°C. The method further includes a step of purifying branched fatty acids by reverse-phase chromatography. The following compounds: [ka] In the synthesis of branched fatty acids, step A yields branched fatty acids more efficiently than step B.

[0085] The following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] In the synthesis of branched fatty acids, step B yields branched fatty acids more efficiently than step A.

[0086] In one embodiment of the present invention, the branched fatty acid used in the synthesis of the pH-sensitive cationic lipid of the present invention may be obtained, for example, by the method described in Japanese Patent No. 2756756. [Examples]

[0087] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0088] I. Synthesis of CL4F6, CL4G6, CL15F6, and CL15G6 [Synthesis Example 1] Synthesis of CL4F6 7-(4-(dipropylamino)butyl)tridecane-1,7,13-triol (1.0 mmol), synthesized by the method described in Patent Document 1, was dissolved in 5 mL of dichloromethane. Subsequently, 2-hexyldecanoic acid (2.20 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.20 mmol), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol) were added, and the mixture was reacted overnight at room temperature. After removing the solvent using a rotary evaporator, the mixture was suspended in ethyl acetate, and insoluble matter was removed by filtration. The filtrate was separated and washed with 0.5 N sodium oxide aqueous solution and saturated saline solution. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtration, the solvent was removed using a rotary evaporator to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to obtain 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-hexyldecanoate)(CL4F6).

[0089] [Synthesis Example 2] Synthesis of CL4G6 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diylbis(2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate)(CL4G6) was obtained in the same manner as in Synthesis Example 1, except that 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate) was used instead of 2-hexyldecanoic acid.

[0090] [Synthesis Example 3] Synthesis of CL15F6 5,11-dihydroxy5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate (1.00 mmol), synthesized by the method described in Patent Document 1, was dissolved in 10 mL of dichloromethane. Subsequently, 2-hexyldecanoic acid (2.20 mmol), DMAP (0.20 mmol), and EDCI (3.0 mmol) were added, and the mixture was reacted overnight at room temperature. After removing the solvent using a rotary evaporator, the mixture was suspended in ethyl acetate, and insoluble matter was removed by filtration. The filtrate was separated and washed with 0.5 N sodium oxide aqueous solution and saturated saline solution. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtration, the solvent was removed using a rotary evaporator to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: dichloromethane: methanol (continuous gradient)] to obtain 7-hydroxy-7-(4-((1-methylpiperidine)-4-carbonyl)oxy)butyl)tridecane-1,13-diylbis(2-hexyldecanoate)(CL15F6).

[0091] [Synthesis Example 4] Synthesis of CL15G6 7-hydroxy-7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diylbis(2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate (CL15G6) was obtained in the same manner as in Synthesis Example 3, except that 2-hexyldecanoic acid was replaced with 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoate (CL15G6).

[0092] II. Preparation and evaluation of lipid nanoparticles using CL4F6, CL4G6, CL15F6, and CL15G6 <Preparation of lipid nanoparticles> In subsequent experiments, unless otherwise specified, lipid nanoparticles were prepared by alcohol dilution using a channel. The channel used was the mixer-integrated microfluidic device "iLiNP" (Lilac Pharma Co., Ltd.). Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and an acetate buffer (25 mM, pH 4.0) adjusted to an siRNA concentration of 71.1 μg / mL were delivered into a microfluidic channel at 0.375 mL / min and 1.125 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was placed in a dialysis membrane (MWCO 12,000-14,000), and the outer aqueous phase was dialyzed with 20 mM MES buffer (pH 6.0) at 4°C for more than 2 hours. After that, the outer aqueous phase was replaced with PBS(-) (pH 7.4), and dialyzed again at 4°C for more than 2 hours, after which the lipid nanoparticle solution was collected from the dialysis membrane.

[0093] <Constituent lipids of lipid nanoparticles> CL4F6, CL4G6, CL15F6, and CL15G6 were synthesized using synthesis examples 1-4. 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) were synthesized using the method described in Patent Document 1. [ka] [ka]

[0094] Furthermore, cholesterol (chol) and polyethylene glycol 2000-modified dimyristoyl glycerol (PEG-DMG) were used as neutral lipids.

[0095] <Measurement of average particle size, PDI (polydispersion index), and zeta potential of lipid nanoparticles> The average particle size (number mean) and PDI of lipid nanoparticles in PBS(-) and the zeta potential in 10 mM HEPES buffer (pH 7.4) were measured using the dynamic light scattering analyzer "Zetasizer Nano ZS ZEN3600" (Malvern).

[0096] <Measurement of pKa of lipid nanoparticles> The pKa of lipid nanoparticles was measured using p-toluidino-2-naphthalenesulfonic acid (TNS). First, TNS (final concentration: 0.75 μM) and lipid nanoparticles (final concentration: 30 mM) were mixed in buffer solutions adjusted to various pH levels. The fluorescence intensity of the prepared mixtures was measured using a microplate reader. The highest and lowest measured values ​​were defined as 100% and 0% charge levels, respectively, and the pH corresponding to 50% charge level was calculated as the pKa. <Nucleic acid encapsulation rate of lipid nanoparticles> The encapsulation rates of siRNA, mRNA, and pDNA in lipid nanoparticles were measured using Ribogreen (Life Technologies).

[0097] [Example 1] Lipid nanoparticles (F7siRNA-loaded lipid nanoparticles) carrying siRNA against F7 were prepared using a pH-sensitive cationic lipid, cholesterol, and PEG-DMG in a molar ratio of 50:50:1 by alcohol dilution. CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 were used as the pH-sensitive cationic lipid. Hereinafter, lipid nanoparticles produced using pH-sensitive cationic lipid X will be referred to as X-LNP. For example, lipid nanoparticles produced using 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. The base sequences of the siRNA against F7 are shown in Table 1. In the table, uppercase letters indicate natural RNA (only T is natural DNA), and lowercase letters indicate 2'-fluoro modified forms. * These represent phosphorothioate bonds, respectively.

[0098] [Table 1]

[0099] Each prepared lipid nanoparticle had an average particle size of 80–120 nm and an siRNA encapsulation rate of 90% or more. The results of measuring the pKa of each lipid nanoparticle are shown in Figures 1(A) and 1(B). As shown in Figure 1, lipid nanoparticles produced using branched scaffold structures (CL4F6, CL4G6, CL15F6, or CL15G6) showed lower pKas compared to lipid nanoparticles produced using linear scaffold structures (CL4H6 or CL15H6).

[0100] Next, each prepared F7siRNA-loaded lipid nanoparticle was administered to ICR mice (4 weeks old, female) and its in vivo F7 knockdown activity was examined. Specifically, each F7siRNA-loaded lipid nanoparticle was intravenously administered to ICR mice at a dose of 0.003 to 0.1 mg siRNA / kg, and plasma F7 enzyme activity was measured 24 hours later. The relative plasma F7 enzyme activity (%) of mice administered each F7siRNA-loaded lipid nanoparticle was calculated, with the plasma F7 enzyme activity of untreated mice set as 100%. The results are shown in Figures 2(A) and 2(B). Figure 2(B) shows the results of intravenous administration of each F7siRNA-loaded lipid nanoparticle at a dose of 0.1 mg siRNA / kg. As shown in Figure 2, lipid nanoparticles produced using CL4F6, CL4G6, CL15F6, or CL15G6, which have a branched scaffold structure, showed comparable in vivo F7 knockdown activity to lipid nanoparticles produced using CL4H6 or CL15H6, which have a linear scaffold structure. These results demonstrate that lipid nanoparticles containing CL4F6, CL4G6, CL15F6, and CL15G6 as constituent lipids are useful as siRNA delivery carriers.

[0101] [Example 2] Lipid nanoparticles loaded with mRNA instead of siRNA were prepared, and their in vivo gene expression activity was investigated. The mRNA used was prepared by performing an in vitro transcription reaction on pDNA encoding NanoLuc® luciferase (Nluc) (Promega Corporation) (Nluc mRNA).

[0102] First, lipid nanoparticles loaded with NlucmRNA (NlucmRNA-loaded lipid nanoparticles) were prepared 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 alcohol dilution. CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 were used as the pH-sensitive cationic lipid.

[0103] [Table 2]

[0104] The average particle size, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were investigated. The measurement results are shown in Table 2. In Table 2, "CL" refers to cationic lipids. The average particle size calculated by dynamic light scattering was 70-130 nm for all samples (Table 2). Of the prepared lipid nanoparticles, only CL15H6-LNP had a high PDI and formed particles with low uniformity (Table 2). The mRNA encapsulation rate for CL4H6-LNP was below 80%, while all other LNPs containing cationic lipids showed encapsulation rates of 90% or higher.

[0105] Next, each prepared NlucmRNA-loaded lipid nanoparticle was administered to ICR mice (4 weeks old, female) and its in vivo gene expression activity was examined. Specifically, each NlucmRNA-loaded lipid nanoparticle was intravenously administered to ICR mice at a dose of 0.04 mg mRNA / kg, and Nluc activity in the liver and spleen was measured 24 hours later. Nluc activity was measured using a luminometer (RLU) and corrected for protein levels quantified by the BCA method.

[0106] The measurement results of Nluc activity (RLU / mg protein) in the liver and spleen of mice administered with each Nluc mRNA-loaded lipid nanoparticle are shown in Figure 3. Figure 3(A) shows the measurement results of Nluc activity in the liver, and Figure 3(B) shows the measurement results of 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 diagram showing the calculation results of [Nluc activity in the liver] / [Nluc activity in the spleen] of mice administered with each Nluc mRNA-loaded lipid nanoparticle. As shown in Figure 3(A), in the liver, mice administered with CL4F6-LNP, CL15F6-LNP, and CL15G6-LNP showed Nluc activity equivalent to that of mice administered with CL4H6-LNP. Also, as shown in Figure 3(C), mice administered with CL4F6-LNP and CL4G6-LNP showed higher liver selectivity compared to mice administered with CL4H6-LNP. Similarly, mice administered with CL15F6-LNP and CL15G6-LNP showed higher liver selectivity compared to mice administered with CL15H6-LNP. From these results, it was found that lipid nanoparticles containing a pH-sensitive cationic lipid with a branched-chain scaffold structure are more selective for the liver and useful as a delivery carrier that specifically delivers to the liver than lipid nanoparticles containing a pH-sensitive cationic lipid with a linear scaffold structure when encapsulating mRNA.

[0107] [Example 3] Lipid nanoparticles loaded with pDNA instead of siRNA were prepared, and the in vivo gene expression activity was examined. For pDNA, a plasmid (pFluc) that expresses firefly luciferase (Fluc) under the CMV promoter was used.

[0108] <In vitro gene expression activity> First, lipid nanoparticles equipped with pFluc (pFluc-equipped lipid nanoparticles) were prepared by alcohol dilution using a composition of pH-sensitive cationic lipid, DSPC, cholesterol, and PEG-DMG in a molar ratio of 50:10:40:1.5. CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 were used as the pH-sensitive cationic lipid. The N / P ratio in the microfluidic channel was set to 9.

[0109] [Table 3]

[0110] The average particle size, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were investigated. The measurement results are shown in Table 3. In Table 3, "CL" refers to cationic lipids. The average particle size calculated by dynamic light scattering was 90-150 nm for all samples (Table 3). The pDNA encapsulation rates for CL4H6-LNP and CL15H6-LNP were 70% and 82%, respectively. On the other hand, lipid nanoparticles containing other cationic lipids showed good values ​​of pDNA encapsulation rates of 90% or more.

[0111] We introduced pFluc-containing lipid nanoparticles into cultured cells and investigated their in vitro gene expression activity. Specifically, HeLa-GFP cells cultured in 96-well plates were transfected with pFluc-containing lipid nanoparticles at 0.0625 μg pDNA / well, and Fluc activity was measured after 24 hours. As a positive control, pFluc was introduced into HeLa-GFP cells using the transduction reagent "Lipofectamine 3000" (Thermo Fisher Scientific). Fluc activity was measured using a luminometer (RLU) and corrected for protein levels quantified by the BCA method.

[0112] The measurement results of the Fluc activity of HeLa-GFP cells transfected with each pFluc-loaded lipid nanoparticle are shown in Fig. 4. In the figure, "Lipo3K" means a positive control transfected using Lipofectamine 3000. As shown in Fig. 4, the cells transfected with CL15F6-LNP showed higher activity than the cells transfected with CL4H6-LNP, CL15H6-LNP, and the positive control.

[0113] <in vitro gene expression activity> pFluc-loaded lipid nanoparticles were prepared in the same manner as described above, except that the N / P ratio in the microchannel was set to 6.

[0114]

Table 4

[0115] The average particle size, PDI, zeta potential, and mRNA encapsulation efficiency of the prepared lipid nanoparticles were examined. The measurement results are shown in Table 4. In Table 4, "CL" means a cationic lipid. The average particle sizes calculated by the dynamic light scattering method were all 70 - 125 nm (Table 4). For the pDNA encapsulation efficiency, CL4F6-LNP, CL4G6-LNP, CL15F6-LNP, and CL15G6-LNP all showed good values of 90% or more.

[0116] Next, each prepared pFluc-loaded lipid nanoparticle was administered to ICR mice (4 weeks old, female) to examine the in vivo gene expression activity. Specifically, each pFluc-loaded lipid nanoparticle was intravenously administered to ICR mice at 0.5 mg mRNA / kg, and the Fluc activity in the liver and spleen 6 hours later was measured. The Fluc activity was measured using a luminometer (RLU) and corrected by the protein amount quantified by the BCA method.

[0117] The measurement results of Fluc activity (RLU / mg protein) in the liver and spleen of mice administered with each pFluc-loaded lipid nanoparticle are shown in Fig. 5. Fig. 5(A) shows the measurement results of Fluc activity in the liver, and Fig. 5(B) shows the measurement results of Fluc 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. Fig. 5(C) is a diagram showing the calculation results of [Fluc activity in the liver] / [Fluc activity in the spleen] of mice administered with each pFluc-loaded lipid nanoparticle. As shown in Fig. 5(A), the Fluc activity in the liver was better in mice administered with CL4F6-LNP and CL15F6-LNP than in mice administered with CL4H6-LNP and CL15H6-LNP. Also, as shown in Fig. 5(C), mice administered with CL4F6-LNP and CL4G6-LNP showed higher liver selectivity compared to mice administered with CL4H6-LNP. Similarly, mice administered with CL15F6-LNP and CL15G6-LNP showed higher liver selectivity compared to mice administered with CL15H6-LNP. From these results, when comparing lipids having the same hydrophilic moiety, even when encapsulating pDNA, lipid nanoparticles containing a pH-sensitive cationic lipid with a branched-chain scaffold structure have higher selectivity for the liver than lipid nanoparticles containing a pH-sensitive cationic lipid with a linear scaffold structure, and it was found that they are useful as delivery carriers that specifically deliver to the liver.

[0118] III. Synthesis of CL4F6 derivative and CL15F6 derivative <Synthesis of CL4F6 and CL4F6 derivative> The synthesis of CL4F6 and CL4F6 derivatives, namely 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, CL4F 10-2, 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 CL4F 16-1 was carried out as follows. 7-(4-(Dipropylamino)butyl)tridecane-1,7,13-triol (1.0 mmol) synthesized by the method described in Patent Document 1 was dissolved in 5 mL of dichloromethane. Subsequently, 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) were added, and the reaction was allowed to proceed overnight at room temperature. After distilling off the solvent using a rotary evaporator, the residue was suspended in ethyl acetate and then washed by liquid separation with a 0.5 N aqueous sodium hydroxide solution and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by subjecting it 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 CL4F6 or a CL4F6 derivative.

[0119] <Synthesis of CL15F6 and CL15F6 Derivatives> The synthesis of CL15F6 and its derivatives, 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, was carried out as follows. 5,11-dihydroxy-5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate (1.00 mmol), synthesized by the method described in Patent Document 1, was dissolved in 10 mL of dichloromethane. Subsequently, 2-hexyldecanoic acid (2.40 mmol), DMAP (0.10 mmol), and EDCI (3.0 mmol) were added, and the mixture was reacted overnight at room temperature. After removing the solvent using a rotary evaporator, the mixture was suspended in ethyl acetate and then separated and washed with 0.5 N aqueous sodium hydroxide solution and saturated saline solution. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtration, the solvent was removed using a rotary evaporator to obtain the crude product. The crude product was purified by ODS-mediated silica gel chromatography [eluent: acetonitrile / isopropanol (50:50):water (0.1% TFA) (continuous gradient)] and silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to obtain CL15F6 or CL15F6 derivatives.

[0120] The branched fatty acids mentioned above were synthesized using straight-chain fatty acids or dimethyl malonate as raw materials, as follows. <Synthesis of branched fatty acids from straight-chain fatty acids> Linear fatty acid (10.28 mmol) was dissolved in 36 mL of THF, and lithium diisopropylamide (24 mmol) was added dropwise at -20°C or below, and the mixture was stirred at 0°C for 30 minutes. Subsequently, DMPU (18 mL) was added and the mixture was stirred at 0°C for 60 minutes. Next, iodoalkane (23.2 mmol) was added and the mixture was reacted overnight at 10°C. After quenching with 2N hydrochloric acid, the mixture was diluted with diethyl ether and separated by liquid-liquid washing with saturated saline. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtration, the solvent was removed using a rotary evaporator to obtain the crude product. The crude product was purified by ODS-mediated silica gel chromatography [eluent: acetonitrile / isopropanol (50:50): water (10 mM ammonium acetate) (continuous gradient)] to obtain branched fatty acid.

[0121] <Synthesis of branched fatty acids using dimethyl malonate as a raw material> 7.56 mmol of NaH was dissolved in 18 mL of THF and stirred at 0°C for 10 minutes. Next, 7.56 mmol of dimethyl malonate was added and stirred at 0°C for 10 minutes. Next, an arbitrary iodoalkane (7.56 mmol) was added and the mixture was reacted overnight at room temperature. 11.34 mmol of NaH was added and stirred at 0°C for 10 minutes. Next, an arbitrary iodoalkane (11.34 mmol) was added and the mixture was reacted overnight at room temperature. After quenching with acetic acid, the mixture was diluted with ethyl acetate and separated by liquid-liquid washing with saturated saline. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtering, the solvent was removed using a rotary evaporator. The solvent removal component was dissolved in 16 mL of ethanol, 5 mL of 8N sodium hydroxide aqueous solution was added and the mixture was reacted overnight at 60°C. After neutralization with 6N hydrochloric acid, the mixture was diluted with ethyl acetate and separated by liquid-liquid washing with saturated saline. Anhydrous sodium sulfate was added to the organic layer to dehydrate it. After filtering, the solvent was removed using a rotary evaporator, and the mixture was heated at 160°C for 2 hours to obtain the crude product. The crude product was purified by ODS silica gel chromatography [eluent: acetonitrile / isopropanol (50:50): water (10 mM ammonium acetate) (continuous gradient)] to obtain branched fatty acids.

[0122] In the branched fatty acids used for the synthesis of CL4F 7-4, CL4F 8-5, CL4F 9-6, and CL4F 10-7, the method using linear fatty acids as raw materials gave branched fatty acids in higher yields than the method using dimethyl malonate as a raw material. In the branched fatty acids used for 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, CL4F6, 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, CL15F6, 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 as a raw material gave branched fatty acids in higher yields than the method using linear fatty acids as raw materials.

[0123] IV. Preparation and evaluation of lipid nanoparticles using CL4F6 derivatives and CL15F6 derivatives 1. Preparation and evaluation of mRNA-loaded lipid nanoparticles <Preparation of mRNA-loaded lipid nanoparticles (mRNA-LNP)> Lipid nanoparticles were prepared by the alcohol dilution method using a flow path. As the flow path, a microfluidic device "NanoAssemblr" (manufactured by Precision NanoSystems) with a built-in mixer was used. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into a microfluidic channel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0124] <Constituent lipids of lipid nanoparticles> Lipid nanoparticles loaded with FlucmRNA (FlucmRNA-loaded lipid nanoparticles) were prepared using pH-sensitive cationic lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:38.5:1.5, by alcohol dilution. The FlucmRNA used was CleanCap® FLuc mRNA (5 moU) from TriLink Biotechnologies.

[0125] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in PBS(-) were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering.

[0126] <Measurement of pKa of lipid nanoparticles> The pKa of lipid nanoparticles was measured using p-toluidino-2-naphthalenesulfonic acid (TNS). First, TNS (final concentration: 0.75 μM) and lipid nanoparticles (final concentration: 60 μM) were mixed in buffer solutions adjusted to various pH levels. The fluorescence intensity of the prepared mixtures was measured using a microplate reader. For the excitation wavelength, the measurement at pH 3.5 was taken as the 100% charge value, and the measurement at pH 9.5 was taken as the 0% charge value. The pH at which the 50% charge value was observed was used as the pKa.

[0127] <Nucleic acid encapsulation rate of lipid nanoparticles> The encapsulation rates of siRNA and mRNA in lipid nanoparticles were measured using Ribogreen reagent. A solution for measuring the nucleic acid concentration on the surface of the nanoparticles was prepared by diluting the lipid nanoparticles with TE buffer to a nucleic acid concentration of 8 μg / mL. In addition, a solution for measuring the total nucleic acid concentration was prepared by adding 1% (w / w) X-triton100 to a lipid nanoparticle concentration of 1.2 μg / mL. Ribogreen (registered trademark) reagent (Quant-iT) was added to 100 μL of each solution. TM Ribo Green TM 100 μL of RNA Reagent (Thermofisher Scientific) was thoroughly mixed in the wells of a 96-well microplate (black, polystyrene, flat-bottom, Corning), and the fluorescence intensity at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm was measured using a microplate reader. Nucleic acid concentration was calculated using a calibration curve created by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0-2.5 μg / mL) in the same manner as described above. The nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula. Inclusion rate % = (Nucleic acid concentration of total nucleic acid concentration measurement solution (μg / mL) - Nucleic acid concentration of nanoparticle surface nucleic acid concentration measurement solution (μg / mL)) ÷ Nucleic acid concentration of total nucleic acid concentration measurement solution (μg / mL) × 100

[0128] <Result> The average particle size, PDI, mRNA encapsulation rate, and pKa of the prepared lipid nanoparticles were investigated. The measurement results for CL4F6 derivative nanoparticles are shown in Tables 5 and 6, and the measurement results for CL15F6 derivative nanoparticles are shown in Table 7. As a control, D-Lin-MC3-DMA(MC3) (MedChemExpress), which has the following structure, was used. [ka] The average particle size of the CL4F6 derivative nanoparticles shown in Table 5, calculated by dynamic light scattering, was 60-220 nm for all of them. Except for CL4F 16-0-LNP, the PDI was low and highly uniform particles were formed. The mRNA encapsulation rate for CL4F 6-2-LNP and CL4F 16-1-LNP was below 80%, while the other LNPs containing cationic lipids all showed encapsulation rates of 80% or higher. Table 7 shows that the average particle size of CL15F6 derivative nanoparticles exceeded 300 nm for CL15F 6-4-LNP and CL15F 7-3-LNP, while all other LNPs containing cationic lipids were between 90 and 200 nm. For PDI, small, highly uniform particles were formed for all LNPs except CL15F 6-4-LNP, CL15F 7-3-LNP, CL15F 16-0-LNP, and CL15H6-LNP. The mRNA encapsulation rate was below 80% for CL15F 6-4-LNP and CL15F 7-3-LNP, while all other LNPs containing cationic lipids showed encapsulation rates of 80% or higher.

[0129] Next, each prepared flucmRNA-loaded lipid nanoparticle was administered to Balb / c mice (Charles River, Japan, 7 weeks old, female) and its in vivo gene expression activity was examined. Specifically, each flucmRNA-loaded lipid nanoparticle was intravenously administered to Balb / c mice at a dose of 0.1 mg mRNA / kg, and flu activity in the liver and spleen was measured 6 hours later. Flu activity was measured using an in vivo imaging system (Perkin Elmer, IVIS200) after administering 1.5 mg of VivoGlo Luciferin, In Vivo Grade (Promega, P1041), dissolved in PBS at 15 mg / mL, via tail vein per mouse. The unit of flu activity is the luminescence intensity per unit area (Avg Radiance [p / s / cm2 / sr]) at the maximum emission wavelength of approximately 560 nm.

[0130] Tables 5-7 show the results of measuring Fluc activity (Avg Radiance [p / s / cm2 / sr]) in the liver and spleen of mice administered with each FlucmRNA-carrying lipid nanoparticle. Tables 5 and 6 show that the Fluc activity of CL4F6 derivative nanoparticles in the liver was higher in mice administered with CL4F 8-6, CL4F 9-7, or CL4F 11-6-containing FlucmRNA-laden lipid nanoparticles than in mice administered with CL4F6-containing FlucmRNA lipid nanoparticles. On the other hand, the Fluc activity of CL4F6 derivative nanoparticles in the spleen was higher in mice administered with FlucmRNA 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 FlucmRNA lipid nanoparticles containing CL4F6. Table 7 shows that the Fluc activity of CL15F6 derivative nanoparticles in the liver was higher in mice administered with FlucmRNA-loaded lipid nanoparticles containing CL15F 9-7, 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 FlucmRNA-loaded lipid nanoparticles containing CL15F6. Table 7 shows that CL15F6 showed higher Fluc activity in the spleen in mice administered with FlucmRNA 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 FlucmRNA lipid nanoparticles containing CL15F6.

[0131] [Table 5]

[0132] [Table 6]

[0133]

Table 7

[0134] 2. Preparation and Evaluation of siRNA-Loaded Lipid Nanoparticles <Preparation of siRNA-Loaded Lipid Nanoparticles> Lipid nanoparticles loaded with siRNA instead of mRNA were prepared, and their in vivo F7 knockdown activity was examined. The siRNA-loaded lipid nanoparticles were prepared in the same manner as described above, except that the N / P ratio in the microchannel was set to 6. The nucleotide sequences of siRNAs against F7 are shown in Table 8. In the table, uppercase letters represent native RNA (only T represents native DNA), lowercase letters represent 2'-fluoro modified forms, * and phosphorothioate bonds are represented respectively.

Table 8

[0135] First, pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG were used at a molar ratio of 50:10:38.5:1.5, and lipid nanoparticles loaded with siRNA against F7 (F7 siRNA-loaded lipid nanoparticles) were prepared by the alcohol dilution method.

[0136] <Results> The average particle size, PDI, siRNA encapsulation efficiency, and pKa of the prepared lipid nanoparticles were examined. The measurement results of CL4F6 derivative nanoparticles are shown in Tables 9 and 10, and the measurement results of CL15F6 derivative nanoparticles are shown in Table 11. The average particle size of CL4F6 derivative nanoparticles was 60 - 280 nm. The siRNA encapsulation efficiency of CL4F6-2-LNP exceeded 90%, while the LNPs containing other cationic lipids all showed more than 90%. Particles with a small PDI and high uniformity were formed except for CL4F16-2-LNP. The average particle size of CL15F6 derivative nanoparticles was over 300 nm for CL15F 6-2-LNP, but all other LNPs containing cationic lipids were between 85 and 290 nm. siRNA encapsulation rates were over 90% for all LNPs. Except for CL15F 6-2-LNP, CL15F 6-4-LNP, CL15F 7-3-LNP, and CL15F 16-0-LNP, particles with low PDI and high uniformity were formed. Next, each prepared F7siRNA-laden lipid nanoparticle was administered to Balb / c mice (Charles River, Japan, 5 weeks old, female) and its in vivo F7 knockdown activity was investigated. Specifically, BALB / c mice were intravenously administered F7siRNA-laden lipid nanoparticles containing the lipids listed in Table 9 at a dose of 0.025 mg siRNA / kg, and F7siRNA-laden lipid nanoparticles containing the lipids listed in Tables 10 and 11 at a dose of 0.025 mg siRNA / kg. Plasma F7 enzyme activity was measured 24 hours later using BIOPHEN FVII (Biophen, A221304). The relative plasma F7 enzyme activity (%) of mice administered with each F7siRNA-laden lipid nanoparticle was calculated, with the plasma F7 enzyme activity of the untreated group of mice set to 100%. Furthermore, the ratio was calculated by setting the relative plasma F7 enzyme activity (%) of mice administered with MC3-containing F7siRNA-laden lipid nanoparticles to 1. The measurement results for CL4F6 derivative nanoparticles are shown in Tables 9 and 10, and the measurement results for CL15F6 derivative nanoparticles are shown in Table 11. F7siRNA-equipped lipid nanoparticles containing CL4F 10-4, CL4F 8-5, CL4F 9-5, or CL4F 12-6, as shown in Tables 9 and 10, exhibited higher knockdown activity than F7siRNA-equipped lipid nanoparticles containing MC3. F7siRNA-equipped lipid nanoparticles 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, as shown in Table 11, exhibited higher knockdown activity than F7siRNA-equipped lipid nanoparticles containing MC3.

[0137] [Table 9]

[0138]

Table 10

[0139]

Table 11

[0140] 3. Preparation and Evaluation of Lipid Nanoparticles Encapsulating pDNA <Preparation of Lipid Nanoparticles Encapsulating pDNA><00…​​​​​​​​​​​​​​​​​​The encapsulation rate of pDNA in lipid nanoparticles was measured using Picogreen® reagent (Quant-iT™ dsDNA Assay Kit, broad range, Thermofisher Scientific). For pDNA encapsulation rate measurement, the nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles was 1.6 μg / mL, the nucleic acid concentration of the solution for measuring the total nucleic acid concentration was 24 ng / mL, and the nucleic acid concentration used for the calibration curve was 0-0.5 μg / mL. Picogreen® reagent (Quant-iT™) was used as the measurement reagent. TM The procedure was the same as for measuring siRNA and mRNA inclusion rates, except that a dsDNA Assay Kit (broad range, Thermofisher Scientific) was used. Inclusion rate % = (Nucleic acid concentration of total nucleic acid concentration measurement solution (μg / mL) - Nucleic acid concentration of nanoparticle surface nucleic acid concentration measurement solution (μg / mL)) ÷ (Nucleic acid concentration of total nucleic acid concentration measurement solution (μg / mL)) × 100

[0143] <Result> The results are shown in the table below. The prepared lipid nanoparticles were left standing at 40°C, and after each storage period, their quality was evaluated according to the following criteria: Good (〇): Particle size is within ±20 nm of the particle size immediately after preparation, PDI is 0.2 or less, and encapsulation rate is 80% or higher; Poor (×): Particle size is greater than ±20 nm of the particle size immediately after preparation, or the encapsulation rate is less than 80%. Lipid nanoparticles that maintain good quality for more than a week when left standing at 40°C can be evaluated as having excellent stability. [Table 12]

[0144] 4. Evaluation of storage stability of lipid nanoparticles <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device, "NanoAssemblr" (Precision NanoSystems), was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into a microfluidic channel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0145] <Constituent lipids of lipid nanoparticles> Lipid nanoparticles loaded with FlucmRNA (FlucmRNA-loaded lipid nanoparticles) were prepared 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, by alcohol dilution. The FlucmRNA used was CleanCap® FLuc mRNA (5 moU) from TriLink Biotechnologies. <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in PBS(-) were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering.

[0146] <Nucleic acid encapsulation rate of lipid nanoparticles> The mRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (Life Technologies). <Storage Stability Evaluation> Lipid nanoparticles were left to stand at temperatures of -80°C, 5°C, 25°C, and 40°C. After storage for each period, the average particle size, PDI, and nucleic acid encapsulation rate were measured. Lipid nanoparticles that met all three conditions—maintaining an average particle size within ±20 nm of the LNP preparation date, maintaining high uniformity with a PDI of 0.2 or less, and maintaining a nucleic acid encapsulation rate of 80% or more—were judged to have maintained good quality.

[0147] <Result> Each flucmRNA-loaded lipid nanoparticle was left to stand at -80°C, 5°C, 25°C, and 40°C, respectively. After storage for each period, the average particle size, PDI, and nucleic acid encapsulation rate were measured. Tables 13 and 14 show the periods during which the quality of the lipid nanoparticles was judged to be well maintained. Lipid nanoparticles that maintained good quality for more than one week at 5°C can be evaluated as having excellent stability.

[0148] [Table 13]

[0149] [Table 14]

[0150] 5. Preparation and evaluation of lipid nanoparticles with various compositions <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device, "NanoAssemblr" (Precision NanoSystems), was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a nucleic acid concentration of 46.1 μg / mL were delivered into a microfluidic channel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0151] <Constituent lipids of lipid nanoparticles> Lipids were prepared with the compositions listed in Tables 15-18. CL4F 10-5, CL4F 9-7, CL4F 8-4, CL4F6, CL15 10-5, and CL15F6 were used as pH-sensitive cationic lipids. Other lipids include cholesterol (Nacalai Tesque), β-sitosterol (22,23-Dihydrostigmasterol, beta-Sitosterol, 5-Stigmasten-3β-ol, α-Dihydrofucosterol, 24α-Ethylcholesterol, Sigma-Aldrich), DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine, COATSOME MC-8080, Yuka Sangyo Co., Ltd.), DMG-PEG2K (1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylen, SUNBRIGHT GM-020, Yuka Sangyo Co., Ltd.), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine, COATSOME MC-8181, Yuka Sangyo Co., Ltd.), and DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, COATSOME ME-8181 (Yuka Sangyo Co., Ltd.) and DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, COATSOME MC-6060, Yuka Sangyo Co., Ltd.) were used. Fluc expression mRNA (CleanCap Fluc mRNA (5 moU), TriLink Biotechnologies Co., Ltd.) and mCherry expression mRNA (CleanCap mCherry mRNA (5 moU), TriLink Biotechnologies Co., Ltd.) were used as mRNA.

[0152] <Storage Stability Evaluation> The prepared lipid nanoparticles were left standing at 40°C, and after each storage period, the average particle size, PDI, and nucleic acid encapsulation rate were measured. The quality was evaluated according to the following criteria: Good (〇): The particle size is within ±20 nm of the particle size immediately after preparation, the PDI is 0.2 or less, and the encapsulation efficiency is 80% or more; Not good (×): The particle size exceeds ±20 nm of the particle size immediately after preparation, or the encapsulation efficiency is less than 80%. For lipid nanoparticles whose quality is maintained well for one week or longer by standing at 40°C, it can be evaluated that they have excellent stability.

[0153] <Measurement of luciferase expression activity in vitro> As a cell culture medium, E-MEM medium (containing L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, 1,500 mg / L sodium bicarbonate) (product code 055-08975, Wako Pure Chemical Industries, Ltd.) was used with 10% fetal bovine serum (Fetal Bovine Serum Characterized, Corning) and 1% antibiotic (Penicillin-Streptomycin (10,000 U / mL), Thermo Fisher). Human fetal kidney cells 293 (HEK293 cells) were added to a 96-well white plate (SIGMA) at 2.0×10 4 cells / well and cultured overnight at 37°C in a 5% CO2 atmosphere. Then, lipid nanoparticles were added at 100 ng / well in terms of mRNA content. After culturing for 24 hours under the same conditions, 100 μg / well of a 300 μg / mL luciferin solution (Beetle luciferin, Promega) was added, and the luminescence intensity was measured with a multiplate reader (EnSight multimode plate reader, Perkin Elmer). At that time, the luciferin solution was added to the wells with only the medium added and the cell wells without lipid nanoparticles added, and the measurement was performed in the same way. The value obtained by subtracting the luminescence intensity of the medium well as the background was adopted. The luminescence intensity of the cell wells without lipid nanoparticles added was below the background.

[0154] <Measurement of mCherry expression activity in vitro> As a cell culture medium, E-MEM medium (containing L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, and 1,500 mg / L sodium bicarbonate) (product code 055-08975, Wako Pure Chemical Industries) was used, supplemented with 10% Fetal Bovine Serum Characterized (Corning Corporation) and 1% antibiotic (Penicillin-Streptomycin (10,000 U / mL), Thermo Fisher). Human fetal kidney cells 293 (HEK293 cells) were cultured in 2.0 × 10⁶ cells. 4 Cells were added to a 96-well black plate (SIGMA) at a rate of cells / well and cultured overnight at 37°C in a 5% CO2 atmosphere. Then, lipid nanoparticles were added at a rate equivalent to 1000 ng / well of mRNA content. After culturing under the same conditions for 24 hours, fluorescence intensity (excitation wavelength 587 nm / fluorescence wavelength 610 nm) was measured using a multi-plate reader (Ensight multimode plate reader, Perkin Elmer). Wells with only culture medium and cell wells without lipid nanoparticles were also prepared and measured similarly. The fluorescence intensity of the culture medium wells was subtracted as background and the resulting values ​​were used. The fluorescence intensity of the cell wells without lipid nanoparticles was below the background level.

[0155] [Table 15]

[0156] [Table 16]

[0157] [Table 17]

[0158] [Table 18]

[0159] <Result> The results are shown in Tables 19-23 below. [Table 19]

[0160] [Table 20]

[0161] [Table 21]

[0162] [Table 22]

[0163] [Table 23]

Claims

1. The following equation (I): 【Chemistry 1】 [In formula (I), a represents an integer from 3 to 5; b represents 0 or 1; R １ and R ２ Each of these independently corresponds to the following general formula (A): 【Chemistry 2】 (In formula (A), R １１ and R １２ Each of these independently represents a linear or branched C2-15 alkyl group; c represents 1; and v represents an integer from 4 to 12. This indicates the group represented by; If b is 0, then X is given by the following general formula (B): 【Transformation 3】 (In formula (B), d represents an integer between 0 and 3; R ３ and R ４ Each of them independently, C １～４ Alkyl alkyl group or C ２～４ It shows a group represented by an alkenyl group. When b is 1, X is a 5- to 7-member non-aromatic heterocyclic group (provided that the group is bonded to (O-CO)b- by a carbon atom, and one or two hydrogen atoms of the ring are optionally substituted with a C １～４ alkyl group or a C ２～４ alkenyl group), where the 5- to 7-member non-aromatic heterocyclic group contains a nitrogen atom as a heteroatom) [Indicates] A pH-sensitive cationic lipid, its stereoisomer, or a mixture of stereoisomers, represented by the terms "pH-sensitive cationic lipid," "stereoisomer," or "mixture of stereoisomers." (However, excluding pH-sensitive cationic lipids as shown below: 【Chemistry 4】 ）。

2. The pH-sensitive cationic lipid is the pH-sensitive cationic lipid according to claim 1, represented by the following formula, a stereoisomer thereof, or a mixture of stereoisomers: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 【Transformation 5-5】 [Chemistry 5-6] [Transformation 5-7] [Transformation 5-8] 【Chemistry 5-9】 【Chemistry 5-10】 【Chemistry 5-11】

3. The following formula (I): 【Transformation 6】 [In formula (I), a represents an integer between 3 and 5; b represents 0 or 1; and R1 and R2 are independently expressed in the following general formula (A): 【Transformation 7】 [In formula (A), R11 and R12 each independently represent a linear or branched C2-C15 alkyl group; c represents 1; and v represents an integer from 4 to 12.] This indicates the group represented by; If b is 0, then X is given by the following general formula (B): 【Transformation 8】 (In formula (B), d represents an integer between 0 and 3; R3 and R4 each independently represent a group (which is a C1-4 alkyl group or a C2-4 alkenyl group), When b is 1, X is a 5-7 member non-aromatic heterocyclic group (where the group is bonded to a carbon atom by (O-CO)b-, and one or two hydrogen atoms of the ring may be substituted with a C1-4 alkyl group or a C2-4 alkenyl group), where the 5-7 member non-aromatic heterocyclic group includes a nitrogen atom as a heteroatom). [Indicates] Lipid nanoparticles comprising a pH-sensitive cationic lipid, a stereoisomer thereof or a mixture of stereoisomers, and nucleic acids, represented by The nucleic acid is mRNA or plasmid DNA. Lipid nanoparticles.

4. The pH-sensitive cationic lipid is the lipid nanoparticle according to claim 3, excluding the pH-sensitive cationic lipid of the following formula: 【Chemistry 9】 。

5. The lipid nanoparticles according to claim 3, wherein the pH-sensitive cationic lipid is represented by the following formula: 【Chemistry 10-1】 【Chemistry 10-2】 【Chemistry 10-3】 [Chemistry 10-4] 【Transformation 10-5】 【Chemistry 10-6】 【Chemistry 10-7】 [Transformation 10-8] 【Chemistry 10-9】 【Chemistry 10-10】 【Chemistry 10-11】

6. Furthermore, the lipid nanoparticles according to claim 3 contain sterols and polyalkylene glycol-modified lipids.

7. The lipid nanoparticle according to claim 3, wherein the nucleic acid is a gene expressed in liver cells.

8. A pharmaceutical composition comprising the lipid nanoparticles described in claim 3 as an active ingredient.

9. A pharmaceutical composition according to claim 8, used in gene therapy.

10. A method for expressing an exogenous gene, comprising administering lipid nanoparticles, as described in claim 3, which contain an exogenous gene intended to be expressed in liver cells, to a test animal (excluding humans) and expressing the exogenous gene in the liver of the test animal.

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