Lipid composition and method to deliver payload to extracellular matrix producing cells

Abstract

The object of the present invention is to provide a novel lipid composition for delivering payloads to extracellular matrix-producing cells such as fibroblasts, and a method of delivering payloads to extracellular matrix-producing cells using the above lipid composition. The present invention provides a lipid composition which comprises a payload, an ionizable lipid, a retinoid, a phospholipid, a sterol, and a polymer-conjugated lipid, and does not comprise a constitutively cationic lipid.

Description

LIPID COMPOSITION AND METHOD TO DELIVER PAYLOAD TO EXTRACELLULAR MATRIX PRODUCING CELLS

[0001] The present invention relates to a lipid composition comprising a payload, and a method to deliver payload to extracellular matrix producing cells using the lipid composition.

[0002] Fibrosis, defined by the excess deposition of extracellular matrix proteins in the extracellular space, underlies tissue dysfunction in multiple chronic diseases. Because of its pathologic role in disorders of the liver, heart, kidney, and lung, among others, as much as 45% of mortality in the USA has been estimated to be attributable to fibrosis. Among fibrosis, nonalcoholic fatty liver disease (NAFLD) is a very common condition, especially in developed countries. According to recent studies, the worldwide prevalence of NAFLD is estimated to be around 25%, but the prevalence can be as high as 75% in some high-risk groups such as people with obesity, type 2 diabetes, or metabolic syndrome. Nonalcoholic steatohepatitis (NASH), a more severe form of NAFLD, is estimated to affect around 3-5% of the general population globally. However, in high-risk groups, such as people with obesity or type 2 diabetes, the prevalence can be much higher, up to 20-30%. NASH can progress to cirrhosis and hepatocellular carcinoma, and it is becoming a leading cause of liver disease worldwide, and it is projected to become the leading indication for liver transplantation in the coming years.

[0003] Hepatic stellate cells (HSCs) are a type of liver cell, which produce extracellular matrix (ECM) proteins. When the liver is exposed to excess fat and inflammation, HSCs become activated and transform into myofibroblast-like cells that produce excessive amounts of ECM proteins, leading to fibrosis of the liver. siRNA-based therapies targeting HSCs hold great promise as a potential treatment strategy for NASH by inhibiting ECM production in HSCs, but further research and development are needed to deliver RNA to HSCs.

[0004] Patent Literatures 1-3 describe lipid compositions for drug delivery which comprise ionizable lipids. Non-Patent Literature 1 and Patent Literatures 4 and 5 describe lipid compositions for drug delivery which comprises retinol and lipids.

[0005] WO2019 / 235635WO2022 / 054955WO2022 / 230964WO2006 / 068232US2017 / 0081663

[0006] Nature Biotechnology volume 26, pages431-442 (2008)（https: / / doi.org / 10.1038 / nbt1396）

[0007] The object of the present invention is to provide a novel lipid composition for delivering payloads to extracellular matrix-producing cells such as fibroblasts. A further object of the present invention is to provide a novel lipid composition which can increase the delivery of payloads to extracellular matrix-producing cells, such as fibroblasts, while decreasing off-target payload delivery to hepatocytes. A further object of the present invention is to provide a method of delivering payloads to extracellular matrix-producing cells using the above lipid composition.

[0008] Retinol has been used deliver siRNA to fibroblasts by combining with constitutively cationic lipid such as DC-6-14 or HEDC. However, constitutively cationic lipids are known to cause undesirable effect such as cytotoxicity, oxidative stress, platelet activation, or coagulation factor activation or depletion. Also, lipid compositions without constitutively cationic lipid showed lower encapsulation efficiency with higher retinol ratio. It was found that this low encapsulation can be recovered by using unsaturated phospholipid. The present inventors have found that the payload can be efficiently delivered to cells by administering, to cells, a lipid composition which comprises a payload, an ionizable lipid, a retinoid, a phospholipid, a sterols, and a polymer-conjugated lipids but does comprise a constitutively cationic lipid. The present invention was completed based on the above findings. According to the present invention, the following invention is provided.

[0009] <1> A lipid composition which comprises a payload, an ionizable lipid, a retinoid, a phospholipid, a sterol, and a polymer-conjugated lipid, and does not comprise a constitutively cationic lipid. <2> The lipidcomposition of <1>, wherein the retinoid doesn’t comprise polyethylene glycol in its structure. <3> The lipid composition of <1>, wherein the retinoid is retinol, all-trans-retinal, retinyl acetate, or all-trans retinoic acid, 9-cis retinoic acid, 13-cis retinoic acid, or retinyl palmitate. <4> The lipid composition of <1>, wherein the retinoid is retinol, retinyl acetate, all-trans retinoic acid, 9-cis retinoic acid, or 13-cis retinoic acid. <5> The lipidcomposition of <1>, which comprises the retinoid at amount of 1 to 40 mol% with respect to the total lipids. <6> The lipidcomposition of <1>, which comprises the retinoid at amount of 2 to 5 mol% with respect to the total lipids. <7> The lipidcomposition of <1>, which further comprises an unsaturated phospholipid. <8> The lipidcomposition of <7>, wherein the unsaturated phospholipid is selected from 18:1 bis(monooleoylglycerol)phosphate or dioleoyl phosphatidylethanolamine or dioleoyl phosphatidylcholine. <9> The lipid composition of <1>, wherein the ionizable lipid is a compound represented by formula (4): In the formula, X represents -NR1- or -O-, R1represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R21-L1-R22-, where R21represents a hydrocarbon group having 1 to 24 carbon atoms, L1represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R22represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R2and R3each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R31-L2-R32-, where R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R4, R5, R6, R7, R8, R9, R10, R11, and R12each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted, groups in any one or more pairs among R4and R5, R10and R5, R5and R12, R4and R6, R5and R6, R6and R7, R6and R10, R12and R7, and R7and R8may be linked to each other to form a 4- to 7-membered ring which may contain an O atom, a substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the substituent on the substituted or unsubstituted aryl group and on the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and a, b, c, and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more. <10> The lipid composition of <1>, wherein the ionizable lipid is a compound represented by formula (5): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), -N(R58)(R59) or -G20-R60, G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R58and R59each independently represent a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, the substituent B is-N(R61)(R62), R61and R62each independently represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R60represents a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), a represents 0 or 1, L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, L20represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65or-O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group. <11> The lipid composition of <1>, wherein the ionizable lipid is a compound represented by formula (2): wherein R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with one or more substituents selected from -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, and -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, R107represents -R110-L102-R111-L103-R112, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, or -O-R166. R161and R162each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R163, R164, R165, and R166each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R163, R164, R165, and R166may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R168, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, -O-R166, or -(hydrocarbon group having 1 to 12 carbon atoms), R168represents a hydrocarbon group having 1 to 12 carbon atoms, and L101, L102, and L103each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, R110represents a hydrocarbon group having 1 to 8 carbon atoms, R111represents a hydrocarbon group having 1 to 24 carbon atoms, R112represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R109and R112may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, where definitions of R153, R154, R155, and R158are as described above, and the hydrocarbon group represented by R111may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where the definitions of R153, R154, and R155are as described above. <12> The lipid composition of <1>, wherein the ionizable lipid is a compound represented by formula (1): wherein R201, R202, R203, and R204each independently represent a hydrogen, a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, the substituted or unsubstituted hydrocarbon groups represented by R201, R202, R203, and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014-, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014-, R2015, and R2016each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R2017, and R2017represents a hydrocarbon group having 1 to 12 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon groups having 1 to 18 carbon atoms represented by R205and R206each represent -OH, -COOH, -NR2021R2022, -OC(O)O-R2023, -C(O)O-R2024, -OC(O)-R2025, -O-R2026, -C(O)NR2027R2028, -NR2029C(O)R2030, -N(R2031)S(O)2R2032, -N(R2033)C(O)N(R2034)R2035, -N(R2036)C(S)N(R2037)R2038, -OC(O)N(R2039)R2040, or -N(R2041)C(O)OR2042, R2021and R2022each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042each independently represent a hyrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, where the substituted groups on the substituted hydrocarbon group having 1 to 24 carbon atoms represented by R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042represent an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or NR2051R2052, and R2051and R2052each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R207, R208, and R209each independently represent a hydrocarbon group having 2 to 8 carbon atoms, R205and R206, or R205and R207may form a 4 to 7-membered ring together. <13> The lipid composition of <1>, wherein the sterol is selected from cholesterol, phytosterol, or cationic cholesterol. <14> The lipid composition of <1>, wherein the payload is a nucleic acid molecule. <15> The lipid composition of <14>, wherein the nucleic acid molecule is RNA. <16> A method for delivering payload to extracellular matrix producing cells, which comprises administering the lipid composition of <1> to a subject. <17> The method of <16>, wherein the extracellular matrix-producing cells are fibroblasts. <18> The method of <16>, wherein the extracellular matrix-producing cells are hepatic stellate cells. <19> The method of <16>, wherein the payload is a nucleic acid molecule. <20> The method of <19>, wherein the nucleic acid molecule is RNA.

[0010] The lipid compositions and methods of the invention allow for delivery of payloads to extracellular matrix-producing cells, such as fibroblasts.

[0011] Figure 1 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 2 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 3 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 4 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 5 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 6 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 7 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 8 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 9 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 10 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 11 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 12 shows the results of in vivo evaluation of Reln and Fvii gene silencing.Figure 13 shows the results of in vivo evaluation of Reln and Fvii gene silencing.

[0012] Hereinafter, the present invention will be described in detail. In this specification, "~" denotes a range including a numerical value described before and after it as a minimum value and a maximum value, respectively.

[0013] The present invention relates to a lipid composition which comprises a payload, an ionizable lipid, a retinoid, a phospholipid, a sterol, and a polymer-conjugated lipid, and does not comprise a constitutively cationic lipid. The invention further relates to a method of delivering a payload to extracellular matrix-producing cells, which comprises administering the lipid composition of the invention described above to a subject.

[0014] <Retinoids> The lipid composition comprises a retinoid. The retinoids are not limited, but it is preferable that the retinoid does not contain polyethylene glycol in the structure. Especially in the lipid composition of the present invention which does not comprise a constitutively cationic lipid., it is preferable to use a retinoid which does not contain polyethylene glycol in the structure.

[0015] Examples of retinoids include retinol, all-trans-retinal, an ester of retinol and carboxylic acid (for example, retinol acetate), or all-trans retinoic acid, Adapalene, Tazarotene, Trifarotene, isotretinoin (13-cis-retinoic acid), alitretinoin (9-cis-retinoic acid), etretinate, acitretin, bexarotene, and Fenretinide and the like. Preferably, the retinoid is retinol, all-trans-retinal, retinyl acetate, or all-trans retinoic acid, 9-cis retinoic acid, 13-cis retinoic acid, or retinyl palmitate. More preferably, the retinoid is retinol, retinyl acetate, all-trans retinoic acid, 9-cis retinoic acid, or 13-cis retinoic acid. The retinoid is especially preferably all-trans retinoic acid. The retinoids may be precursors of retinoids. The precursors of retinoids are carotenoids. Examples of carotenoids are β-carotene, Α-carotene, Δ-carotene, Ε-carotene, Γ-carotene, Ζ-carotene, astaxanthin, apocarotenal, apocarotenoids, anthelaxanthin, isorenieratene, capsanthin, carotene, Canthaxanthin, xanthoxin, xanthophyll, xanthophyll cycle, crocin, crocetin, diadinoxanthin, diatoxanthin, citranaxanthin, dinoxanthin, zeaxanthin, neurosporin, neoxanthin, violaxanthin, bixin, phytoene, phytofluene, fucoxanthin, flavoxanthin, peridinin, helenien, mezerein, lycopene, lutein, rubixanthin, renieratene, and lodoxanthin.

[0016] The content of the retinoids with respect to the total lipids is preferably 1 mol% to 40 mol%, more preferably 1 mol% to 30 mol%, still more preferably 2 mol% to 20 mol%, and further preferably 2 mol% to 5 mol%.

[0017] <Sterols> The lipid composition contains a sterol. In the present invention, since a sterol is contained, the membrane fluidity can be reduced and the effect to stabilize the lipid particles can be obtained. The sterols are not particularly limited, and examples thereof include cholesterol, phytosterol (sitosterol, stigmasterol, campesterol, fucosterol, spinasterol, brassicasterol, beta-sitosterol, and the like), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2’-hydroxyethyl ether, cholesteryl-4’-hydroxybutyl ether, and the like. Cholesterol derivatives can also be used as sterols, and cholesterol derivatives include cholesterol having a cationic substituent. Among these, cholesterol is preferred.

[0018] Cholesterol

[0019] The content of the sterols with respect to the total lipids is preferably 5 mol% to 80 mol%, more preferably 10 mol% to 80 mol%, still more preferably 10 mol% to 60 mol%, and further preferably 30 mol% to 50 mol%.

[0020] <Ionizable Lipids> The lipid composition comprises an ionizable lipid. The ionizable lipid may be a lipid having at least one biodegradable group. The ionizable lipid may be a lipid having at least one ionizable amino group and at least one biodegradable group. Examples of the above-mentioned biodegradable group include groups represented by-O (CO) O-, -O (CO)-, or -(CO) O-.

[0021] <<Lipid represented by Formula (4) or salt thereof>> For example, a lipid represented by Formula (4) or a salt thereof may be used as the ionizable lipid.

[0022] In the formula, X represents -NR1- or -O-, R1represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R21-L1-R22-, where R21represents a hydrocarbon group having 1 to 24 carbon atoms, L1represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R22represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R2and R3each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R31-L2-R32-, where R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R4, R5, R6, R7, R8, R9, R10, R11, and R12each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted, groups in any one or more pairs among R4and R5, R10and R5, R5and R12, R4and R6, R5and R6, R6and R7, R6and R10, R12and R7, and R7and R8may be linked to each other to form a 4- to 7-membered ring which may contain an O atom, a substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the substituent on the substituted or unsubstituted aryl group and on the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and a, b, c, and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more.

[0023] As the hydrocarbon group having 6 to 24 carbon atoms that is represented by R1and the hydrocarbon group having 3 to 24 carbon atoms that is represented by R2and R3, an alkyl group, an alkenyl group, or an alkynyl group is preferable, and an alkyl group or an alkenyl group is more preferable. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and the alkyl group having 3 to 24 carbon atoms is more preferably an alkyl group having 6 to 20 carbon atoms. Specifically, examples thereof include a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, and the like. The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms is more preferably an alkenyl group having 6 to 20 carbon atoms. Specifically, examples thereof include a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), a nonadecenyl group, an icosenyl group (preferably a (Z)-icos-11-enyl group), an icosadienyl group (preferably a (11Z,14Z)-icosa-11,14-dienyl group), and the like. The alkynyl group having 6 to 24 carbon atoms is preferably an alkynyl group having 6 to 20 carbon atoms, and the alkynyl group having 3 to 24 carbon atoms is more preferably an alkynyl group having 6 to 20 carbon atoms. Specifically, examples thereof include a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, an octadecynyl group, and the like. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0024] The hydrocarbon group having 1 to 24 carbon atoms that is represented by R21and R31is preferably an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms, or an alkynyl group having 10 to 24 carbon atoms. The alkyl group having 10 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. The alkyl group having 10 to 24 carbon atoms is preferably an alkyl group having 12 to 24 carbon atoms. Specifically, examples thereof include a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like. The alkenyl group having 10 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. Specifically, examples thereof include a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like. The alkynyl group having 10 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. Specifically, examples thereof include a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, an octadecynyl group, and the like. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0025] The divalent hydrocarbon linking group having 1 to 18 carbon atoms that is represented by R22and R32is preferably an alkylene group having 1 to 18 carbon atoms or an alkenylene group having 2 to 18 carbon atoms. The alkylene group having 1 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkylene group is preferably 1 to 12, more preferably 1 to 10, and still more preferably 2 to 10. Specifically, examples thereof include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, an undecamethylene group, a dodecamethylene group, and the like. The alkenylene group having 2 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkenylene group is preferably 1 to 12, and more preferably 2 to 10.

[0026] -O(CO)O-, -O(CO)-, and -(CO)O- are in a preferred range of L1, and -O(CO)- and -(CO)O- are in a more preferred range of L1. -O(CO)O-, -O(CO)-, and -(CO)O- are in a preferred range of L2, and -O(CO)- and -(CO)O- are in a more preferred range of L2.

[0027] The alkyl group having 1 to 18 carbon atoms which may be substituted and which represented by R4, R6, R9, R10, R11, and R12may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12. Specifically, examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, and the like. In a case where the alkyl group has a substituent, as the substituent, a hydroxyl group, a carboxyl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a group represented by -O(CO)-R42or -(CO)O-R43is more preferable.

[0028] The alkyl group having 1 to 18 carbon atoms which may be substituted and which represented by R5, R7, and R8may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12, and more preferably 1 to 8. Specifically, examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, and the like. In a case where the alkyl group has a substituent, as the substituent, a hydroxyl group, a carboxyl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a group represented by -O(CO)-R42, or -(CO)O-R43is more preferable.

[0029] Examples of the 4- to 7-membered ring which may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring. The 4- to 7-membered ring is preferably a 6-membered ring and is preferably a piperidine ring or a morpholine ring.

[0030] In a case where the alkyl group having 1 to 18 carbon atoms which is represented by R4, R5, R6, R7, R8, R9, R10, R11, and R12and which may be substituted has a substituted or unsubstituted aryl group as a substituent, the number of carbon atoms in the aryl group is preferably 6 to 22, more preferably 6 to 18, and still more preferably 6 to 10. Specifically, examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, and the like. As the substituent on the aryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a hydroxyl group or a carboxyl group is more preferable. Specifically, examples of the substituted aryl group include a hydroxyphenyl group, a carboxyphenyl group, and the like.

[0031] In a case where the alkyl group having 1 to 18 carbon atoms which is represented by R4, R5, R6, R7, R8, R9, R10, R11, and R12and which may be substituted has a substituted or unsubstituted heteroaryl group as a substituent, the number of carbon atoms in the heteroaryl group is preferably 1 to 12, and more preferably 1 to 6. Specifically, examples of the heteroaryl group include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, an oxazolyl group, and the like. As the substituent on the heteroaryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a hydroxyl group or a carboxyl group is more preferable. Specifically, examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, a pyridonyl group, and the like.

[0032] As hydrocarbon group having 1 to 18 carbon atoms that is represented by R41, R42, R43, R44, R45, and R46, an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or an alkynyl group having 2 to 18 carbon atoms is preferable, and an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms is more preferable. The alkyl group having 1 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, and the like. The alkenyl group having 2 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkenyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like. The alkynyl group having 2 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkynyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, an octadecynyl group, and the like.

[0033] In a case where X represents -NR1-, R1preferably represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R21-L1-R22-. In this case, it is preferable that one of R2and R3represent a hydrogen atom and the other represent a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R31-L2-R32-.

[0034] In a case where X represents -O-, it is preferable that R2and R3each independently represent a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R31-L2-R32-.

[0035] It is preferable that R4, R6, R9, R10, R11, and R12each represent a hydrogen atom.

[0036] R5is preferably a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43, an alkyl group having 1 to 18 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 18 carbon atoms which may be substituted with a hydroxyl group. In a case where R5is an alkyl group, R5may be linked to R4, R6, R10, and R12to form a ring which may contain an O atom. Particularly, R5is preferably an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43, an alkyl group having 1 to 12 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43.

[0037] R7and R8preferably each independently represent a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43, an alkyl group having 1 to 8 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group. Alternatively, it is preferable that R7and R8be linked to each other to form a 4- to 7-membered ring which may contain an O atom.

[0038] R5is not linked to R7or R8and does not form a ring with R7or R8.

[0039] a + b is preferably 1 or 2, and more preferably 1. c + d is preferably 1 or 2, and more preferably 1.

[0040] In preferable embodiment, the lipid resented by Formula (4) is preferably a compound represented by Formula (21). In the formula, R2and R3each independently represent a hydrocarbon group containing one or more unsaturated bond and having 3 to 24 carbon atoms, or R2and R3each independently represent a group represented by R31-L2-R32-, or one of R2and R3represents a group represented by R31-L2-R32- and the other represents a hydrocarbon group having 3 to 24 carbon atoms, R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R5represents an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43where R42and R43each independently represent a hydrocarbon group having 1 to 18 carbon atoms, R7and R8each independently represent an alkyl group having 1 to 4 carbon atoms e represents 2 or 3.

[0041] In formula (21), preferably one of R2and R3is a group represented by R31-L2-R32-, and the other is a hydrocarbon group having 3 to 24 carbon atoms. In formula (21), L2 preferably represents -O (CO)- or - (CO) O-.

[0042] The compound represented by Formula (4) may form a salt. Examples of the salt in a basic group include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Examples of the salt in an acidic group include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N’-dibenzylethylenediamine, and the like. Among the above salts, for example, pharmacologically acceptable salts are preferable.

[0043] The lipid represented by the formula (4) and a method for producing the same are described in WO2019 / 235635A and WO2021 / 095876A.

[0044] <<Llipid represented by the formula (5) or a salt thereof>> For example, a lipid represented by Formula (5) or a salt thereof may be used as the ionizable lipid. wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), -N(R58)(R59) or -G20-R60, G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R58and R59each independently represent a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, the substituent B is-N(R61)(R62), R61and R62each independently represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R60represents a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), a represents 0 or 1, L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, L20represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65or-O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group.

[0045] The compound represneted by Formula (5) may be a compound represneted by Formula (5A): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, or -(CO)O-, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0046] The compound represneted by Formula (5) may preferably be a compound represneted by Formula (5B): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)O-, L20represents a hydrocarbon group having 1 to 6 carbon atoms, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by -O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent an alkoxy group having 1 to 10 carbon atoms.

[0047] The compound represneted by Formula (5) may preferably be a compound represneted by Formula (5C): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms L10represents a hydrocarbon group having 1 to 18 carbon atoms, G10represents -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65, R65represents a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms.

[0048] The compound represneted by Formula (5) may preferably be a compound represneted by Formula (5D): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G20represents -(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -(CO)O-, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0049] The hydrocarbon group having 1 to 21 carbon atoms is preferably an alkyl group having 1 to 21 carbon atoms, an alkenyl group having 2 to 21 carbon atoms, or an alkynyl group having 2 to 21 carbon atoms, more preferably an alkyl group having 1 to 21 carbon atoms, or an alkenyl group having 2 to 21 carbon atoms. The alkyl group having 1 to 21 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21 carbon atoms. Examples include propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, tert-butyl group, cyclobutyl group, pentyl group, cyclopentyl group, hexyl group, cyclohexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group and octadecyl group. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Examples include allyl group, prenyl group, pentanyl group, hexenyl group, heptenyl group, octenyl group, nonenyl group (preferably (Z) -2-nonenyl group or (E) -2-nonenyl group), decenyl group, undecenyl group, dodecenyl group, dodecadienyl group, tridecenyl group (preferably (Z) -trideca-8-enyl group), tetradecenyl group (preferably tetradeca-9-enyl group), pentadecenyl group (preferably (Z)-pentadeca-8-enyl group), hexadecenyl group (preferably (Z)-hexadeca-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably (Z)-heptadeca-8-enyl group), heptadecadienyl group (preferably (8Z, 11Z)-heptadeca-8,11-dienyl group), octadecenyl group (preferably (Z)-octadeca-9-enyl group), octadecadienyl Groups (preferably (9Z, 12Z)-octadeca-9,12-dienyl group). The alkynyl group having 2 to 21 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21 carbon atoms. Examples include propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, nonynyl group, decynyl group, undecynyl group, dodecynyl group, tetradecynyl group, pentadecynyl group, hexadecynyl group, heptadecynyl group, octadecynyl group and the like. Examples of the hydrocarbon group having 1 to 18 carbon atoms include those having 1 to 18 carbon atoms among the hydrocarbon groups having 1 to 21 carbon atoms.

[0050] As the cyclic hydrocarbon group, a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkenyl group having 3 to 10 carbon atoms, a cycloalkynyl group having 3 to 10 carbon atoms, and an aryl group having 6 to 10 carbon atoms are preferable.

[0051] The hydrocarbon group having 1 to 6 carbon atoms is preferably an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms or an alkynyl group having 2 to 6 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms. The alkyl group having 1 to 6 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, tert-butyl group, cyclobutyl group, pentyl group, cyclopentyl group and hexyl group. The alkenyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include allyl group, prenyl group, pentenyl group, and hexenyl group. The alkynyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include propargyl group, butynyl group, pentynyl group, and hexynyl group.

[0052] The hydrocarbon group having 1 to 10 carbon atoms is preferably an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and preferably an alkyl group having 1 to 10 carbon atoms or an alkenyl group having 2 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10 carbon atoms. Specific example include propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, tert-butyl group, cyclobutyl group, pentyl group, cyclopentyl group, hexyl group,cyclohexyl group, heptyl group, octyl group, nonyl group, and decyl group. The alkenyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10. Specific examples include allyl group, prenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group, a nonenyl group (preferably (Z)-2-nonenyl group or (E)-2-nonenyl group), and decenyl group. The alkynyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10 carbon atoms. Specific examples thereof include propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, noninyl group and a decynyl group.

[0053] The compound represented by Formula (5) may form a salt. Examples of the salt in a basic group include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Among the above salts, for example, pharmacologically acceptable salts are preferable.

[0054] <<Lipid represented by Formula (2) or salt thereof>> As another example of ionizable lipids, a lipid represented by Formula (2) or a salt thereof may be used as the ionizable lipid. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with one or more substituents selected from -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, and -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, R107represents -R110-L102-R111-L103-R112, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, or -O-R166. R161and R162each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R163, R164, R165, and R166each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R163, R164, R165, and R166may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R168, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, -O-R166, or -(hydrocarbon group having 1 to 12 carbon atoms), R168represents a hydrocarbon group having 1 to 12 carbon atoms, and L101, L102, and L103each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, R110represents a hydrocarbon group having 1 to 8 carbon atoms, R111represents a hydrocarbon group having 1 to 24 carbon atoms, R112represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R109and R112may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, where definitions of R153, R154, R155, and R158are as described above, and the hydrocarbon group represented by R111may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where the definitions of R153, R154, and R155are as described above.

[0055] A hydrocarbon group having 1 to 24 carbon atoms, a hydrocarbon group having 1 to 18 carbon atoms, a hydrocarbon group having 1 to 12 carbon atoms, a hydrocarbon group having 2 to 8 carbon atoms, and a hydrocarbon group having 1 to 8 carbon atoms are each preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0056] The alkyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0057] The alkenyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of the alkenyl group include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like.

[0058] The alkynyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of alkynyl group include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, an octadecynyl group, and the like. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0059] The hydrocarbon group having 1 to 12 carbon atoms in -(hydrocarbon group having 1 to 12 carbon atoms)-R67is preferably an alkylene group having 1 to 12 carbon atoms or an alkenylene group having 2 to 12 carbon atoms. The alkylene group having 1 to 12 carbon atoms and the alkenylene group having 2 to 12 carbon atoms may be linear or branched, or may be chainlike or cyclic. Specifically, examples thereof include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, an undecamethylene group, and the like.

[0060] The aryl group preferably has 6 to 20 carbon atoms, more preferably has 6 to 18 carbon atoms, and even more preferably 6 to 10 carbon atoms. Specifically, examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, and the like.

[0061] R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms. The hydrocarbon groups represented by R101, R102, and R103may be preferably substituted with -OH.

[0062] L101and L103each independently preferably represent -C(O)O- or -OC(O)-. L102preferably represents -OC(O)O-, -C(O)O-, or -OC(O)-.

[0063] R108preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms. R109preferably represents a hydrocarbon group having 1 to 20 carbon atoms and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms. R111preferably represents a hydrocarbon group having 1 to 16 carbon atoms and more preferably represents a hydrocarbon group having 1 to 9 carbon atoms. R112preferably represents a hydrocarbon group having 1 to 20 carbon atoms and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms. The hydrocarbon groups represented by R109and R112may be preferably substituted with an aryl group or -S-R158. Here, R158preferably represents a hydrocarbon group having 1 to 8 carbon atoms. The hydrocarbon group represented by R111may be preferably substituted with -C(O)O-R155or -OC(O)-R156, where R155and R156each independently represent a hydrocarbon group having 1 to 16 carbon atoms. The hydrocarbon groups represented by R155and R156may be preferably substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, and the definition of R158is as described above.

[0064] The compound represented by Formula (2) is preferably a compound represented by Formula (2-1) as a first example. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, L101represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, where the hydrocarbon group represented by R109may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156. R113represents a hydrocarbon group having 1 to 8 carbon atoms, R114represents -R115-L105-R116, where R115represents a hydrocarbon group having 1 to 24 carbon atoms, L105represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, and R116represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon group having 1 to 24 carbon atoms represented by R115may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where definitions of R153, R154, and R155are as described above, and the hydrocarbon group having 1 to 24 carbon atoms represented by R116may be substituted with an aryl group having 6 to 20 carbon atoms, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155or -S-R158, where the definitions of R153, R154, R155, and R158are as described above. 　　　L104represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-.

[0065] In Formula (2-1), R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms. The hydrocarbon groups represented by R101, R102, and R103may be preferably substituted with -OH.

[0066] L101preferably represents -C(O)O- or -OC(O)-. R108preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms. R109preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and the hydrocarbon group represented by R109may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R58. R114preferably represents -R115-L105-R116, where R115represents a hydrocarbon group having 1 to 18 carbon atoms, L115represents -OC(O)O-, and R116represents a hydrocarbon group having 1 to 18 carbon atoms. The hydrocarbon group having 1 to 18 carbon atoms represented by R115may be preferably substituted with -C(O)O-R154or -OC(O)-R155. R154and R155each independently represent a hydrocarbon group having 1 to 16 carbon atoms, and the hydrocarbon groups represented by R154and R155may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, where the definition of R158is as described above. The hydrocarbon group having 1 to 18 carbon atoms represented by R116may be preferably substituted with an aryl group or -S-R158, where the definition of R158is as described above.

[0067] The compound represented by Formula (2) is preferably a compound represented by Formula (2-2) as a second example. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156, R104and R108each independently represent a hydrocarbon having 1 to 8 carbon atoms, R121and R122each independently represent a hydrocarbon group having 1 to 18 carbon atoms, R123and R124each independently represent a hydrocarbon group having 1 to 12 carbon atoms, R125and R126each independently represent a hydrocarbon group having 1 to 24 carbon atoms, L121and L122each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, the hydrocarbon groups represented by R125and R126may be substituted with an aryl group having 6 to 20 carbon atoms, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the above-described aryl group having 6 to 20 carbon atoms may be substituted with OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, and R157represents -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156. R158represents a hydrocarbon group having 1 to 12 carbon atoms.

[0068] In Formula (2-2), R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. The hydrocarbon groups represented by R101and R102may be preferably substituted with -OH, but has more preferably a hydrocarbon having no substituent.

[0069] R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0070] R121and R122each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 6 carbon atoms. R123and R124each independently preferably represent a hydrocarbon group having 1 to 10 carbon atoms and more preferably represent a hydrocarbon group having 1 to 8 carbon atoms. R125and R126each independently preferably represent a hydrocarbon group having 1 to 20 carbon atoms, more preferably represent a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 12 carbon atoms. L121and L122each independently preferably represent -C(O)O- or -OC(O)-.

[0071] The compound represented by Formula (2) is preferably a compound represented by Formula (2-3) as a third example. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156, R104and R108each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R131, R132, R133, and R134each independently represent a hydrocarbon group having 1 to 12 carbon atoms, R135, R136, R137, and R138each independently represent a hydrocarbon group having 1 to 24 carbon atoms, L131, L132, L133, and L134each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, the hydrocarbon groups represented by R135, R136, R137, and R138may be substituted with an aryl group having 6 to 20 carbon atoms, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or S-R158, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the above-described aryl group having 6 to 20 carbon atoms may be substituted with OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, and R157represents -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156. R158represents a hydrocarbon group having 1 to 12 carbon atoms.

[0072] In Formula (2-3), R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. The hydrocarbon groups represented by R101and R102may be preferably substituted with -OH, but has more preferably a hydrocarbon having no substituent.

[0073] R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0074] R131, R132, R133, and R134each independently preferably represent a hydrocarbon group having 1 to 10 carbon atoms, more preferably represent a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms.

[0075] R135, R136, R137, and R138each independently preferably represent a hydrocarbon group having 1 to 20 carbon atoms, more preferably represent a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 12 carbon atoms. The hydrocarbon groups represented by R135, R136, R137, and R138may be preferably substituted with an aryl group having 6 to 20 carbon atoms or S-R158. More preferably, these may be substituted with -S-R158. R135, R136, R137, and R138each independently particularly preferably represent a hydrocarbon group having 1 to 12 carbon atoms substituted with -S-R158, or a hydrocarbon group having 1 to 12 carbon atoms.

[0076] L131, L132, L133, and L134each independently preferably represent -C(O)O-, or -OC(O)-.

[0077] R158preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0078] The compound according to the embodiment of the present invention may form a salt. Examples of the salt in a basic group include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Examples of the salt in an acidic group include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N’-dibenzylethylenediamine; and the like. Among the above-described salts, for example, pharmacologically acceptable salts are preferable.

[0079] Preferred specific examples of compounds represented by formula (2) include those listed in Example below, but the present invention should not be interpreted as limited thereto.

[0080] The compound represented by the formula (2) is described in WO2022 / 230964A, and can be made by the production method shown in WO2022 / 230964A .

[0081] <<Lipid represented by Formula (1) or salt thereof>> As another example of ionizable lipids, a lipid represented by Formula (1) or a salt thereof may be used. In the formula, R201, R202, R203, and R204each independently represent a hydrogen, a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, the substituted or unsubstituted hydrocarbon groups represented by R201, R202, R203, and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014-, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014-, R2015, and R2016each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R2017, and R2017represents a hydrocarbon group having 1 to 12 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon groups having 1 to 18 carbon atoms represented by R205and R206each represent -OH, -COOH, -NR2021R2022, -OC(O)O-R2023, -C(O)O-R2024, -OC(O)-R2025, -O-R2026, -C(O)NR2027R2028, -NR2029C(O)R2030, -N(R2031)S(O)2R2032, -N(R2033)C(O)N(R2034)R2035, -N(R2036)C(S)N(R2037)R2038, -OC(O)N(R2039)R2040, or -N(R2041)C(O)OR2042, R2021and R2022each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042each independently represent a hyrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, where the substituted groups on the substituted hydrocarbon group having 1 to 24 carbon atoms represented by R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042represent an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or NR2051R2052, and R2051and R2052each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R207, R208, and R209each independently represent a hydrocarbon group having 2 to 8 carbon atoms, R205and R206, or R205and R207may form a 4 to 7-membered ring together.

[0082] The hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon group having 1 to 18 carbon atoms, the hydrocarbon group having 1 to 12 carbon atoms, and the hydrocarbon group having 1 to 8 carbon atoms are each preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0083] The alkyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0084] The alkenyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of the alkenyl group include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like.

[0085] The alkynyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of alkynyl group include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, an octadecynyl group, and the like.

[0086] All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0087] The hydrocarbon group having 2 to 8 carbon atoms represented by R207, R208, and R209is preferably an alkylene group, an alkenylene group, or an alkynylene group. The alkylene group having 2 to 8 carbon atoms, the alkenylene group having 2 to 8 carbon atoms or the alkynylene group having 2 to 8 carbon atoms may be linear or branched, or may be chainlike or cyclic. Specifically, examples thereof include an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, and the like.

[0088] The aryl group having 6 to 20 carbon atoms preferably has 6 to 18 carbon atoms, and more preferably 6 to 10 carbon atoms. Specifically, examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, and the like.

[0089] A heterocyclic group means a heteroaryl group or a heteroaliphatic ring group.

[0090] A heteroaryl group means an aromatic heterocyclic group, may be an aromatic heterocyclic group fused with an aromatic hetero ring, an aromatic hydrocarbon ring, a heteroaliphatic ring or an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing heteroaryl group, a monocyclic oxygen-containing heteroaryl group, a monocyclic sulfur-containing heteroaryl group, a monocyclic nitrogen and oxygen-containing heteroaryl group, a monocyclic nitrogen and sulfur-containing heteroaryl group, a bicyclic nitrogen-containing heteroaryl group, a bicyclic oxygen heteroaryl group, a bicyclic sulfur heteroaryl group, a bicyclic nitrogen and oxygen-containing heteroaryl group or a bicyclic nitrogen and sulfur-containing heteroaryl group. A 5 membered ring heteroaryl group is a monocyclic heteroaryl group with five atoms constituting ring thereof.

[0091] An aromatic heterocyclic ring means an aromatic ring having heteroatoms as ring members thereof, may be fused with an aromatic heterocyclic ring, an aromatic hydrocarbon ring, a heteroaliphatic ring, or an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing aromatic heterocyclic ring, a monocyclic oxygen-containing aromatic heterocyclic ring, a monocyclic sulfur-containing aromatic heterocyclic ring, a monocyclic nitrogen and oxygen-containing aromatic heterocyclic ring, a monocyclic nitrogen and sulfur-containing aromatic heterocyclic ring, a bicyclic nitrogen-containing aromatic heterocyclic ring, a bicyclic oxygen-containing aromatic heterocyclic ring, a bicyclic sulfur-containing aromatic heterocyclic ring, a bicyclic nitrogen-containing sulfur-containing aromatic heterocyclic ring, a bicyclic nitrogen and oxygen-containing aromatic heterocyclic ring or a bicyclic nitrogen and sulfur-containing aromatic heterocyclic ring.

[0092] A monocyclic nitrogen-containing heteroaryl group means a heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one nitrogen atom, such as a pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl and tetrazolyl groups, is aromatic. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring. A monocyclic oxygen-containing heteroaryl group means a heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one oxygen atom, such as a furanyl or pyranyl group, is aromatic. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring. A monocyclic nitrogen and oxygen-containing heteroaryl group means an oxazolyl, isoxazolyl or oxadiazolyl group, and the like. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring. A monocyclic nitrogen and sulfur-containing heteroaryl group means a thiazolyl, isothiazolyl or thiadiazolyl group, and the like. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring.

[0093] A bicyclic nitrogen-containing heteroaryl group means a bicyclic heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one nitrogen atom, such as an indolyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pyrrolopyridyl, imidazopyridyl, pyrazolopyridyl, pyridopyrazyl, purinyl, pteridinyl, 5,6,7,8-tetrahydrophthalazinyl, 5,6,7,8-tetrahydrocinnolinyl, 1,2,3,4-tetrahydropyrido[2,3-d]pyridazinyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, 5,6,7,8-tetrahydropyrido[3,4-d]pyridazinyl, 5,6,7,8-tetrahydropyrido[3,2-c]pyridazinyl, 5,6,7,8-tetrahydropyrido[4,3-c]pyridazinyl, 6,7-dihydro-5H-cyclopenta[d]pyridazinyl, 6,7-dihydro-5H-cyclopenta[c]pyridazinyl, 2,3-dihydro-1H-pyrrolo[2,3-d]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,2-c]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-c]pyridazinyl and 6,7-dihydro-5H-pyrrolo[2,3-c]pyridazinyl groups, is aromatic.

[0094] A bicyclic oxygen-containing heteroaryl group means a bicyclic heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one oxygen atom, such as a benzofuranyl, isobenzofuranyl and chromenyl groups, is aromatic.

[0095] A bicyclic nitrogen and oxygen-containing heteroaryl group means a bicyclic heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one nitorogen atom and at least one oxygen atom, such as a benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, dihydropyranopyridyl, dihydrodioxinopyridyl, dihydropyrido oxazienyl, 3,4-dihydro-2H-pyrano[2,3-d]pyridazinyl, 7,8-dihydro-5H-pyrano[3,4-d]pyridazinyl, 7,8-dihydro-6H-pyrano[3,2-c]pyridazinyl, 7,8-dihydro-5H-pyrano[4,3-c]pyridazinyl, 2,3-dihydrofuro[2,3-d]pyridazinyl, 5,7-dihydrofuro[3,4-d]pyridazinyl, 6,7-dihydrofuro[3,2-c]pyridazinyl, 5,7-dihydrofuro[3,4-c]pyridazinyl and 5,6-dihydrofluoro[2,3-c]pyridazinyl groups, is aromatic.

[0096] A heteroaliphatic ring group means a nitrogen-containing heteroaliphatic ring group, an oxygen-containing heteroaliphatic ring group, a sulfur-containing heteroaliphatic ring group, a nitrogen and oxygen-containing heteroaliphatic ring group, a nitrogen and sulfur-containingheteroaliphatic ring group, a hetero cross-linked ring group or a heterospiro ring group. A heteroaliphatic ring also means an aliphatic ring having heteroatoms as ring members thereof, and includes a nitrogen-containing heteroaliphatic ring, an oxygen-containing heteroaliphatic ring, a sulfur-containing heteroaliphatic ring, a nitrogen and oxygen-containing heteroaliphatic ring, a nitrogen and sulfur-containing heteroaliphatic ring, a hetero cross-linked ring, and heterospiro ring as a preferred example.

[0097] A nitrogen-containing heteroaliphatic ring group means a heteroaliphatic ring group in which the ring containing at least one nitrogen atom, such as an azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl and homopiperazinyl groups, is not aromatic. This nitrogen-containing heteroaliphatic ring group may be further fused with other aromatic ring or aliphatic ring. An oxygen-containing heteroaliphatic ring group means a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl or 1,3-dioxanyl group, and the like. The oxygen-containing heteroaliphatic ring group may be further fused with other aromatic ring or aliphatic ring. A nitrogen and oxygen-containingheteroaliphatic ring group means a morpholinyl or 1,4-oxazepanyl group, and the like. The nitrogen and oxygen-containing heteroaliphatic ring group may be further fused with other aromatic ring or aliphatic ring.

[0098] A heteroaliphatic ring C1-8alkyl group means a linear, branched-chain or cyclic C1-8alkyl group to which a heteroaliphatic ring group such as a pyrrolidinyl methyl group, pyrrolidinyl ethyl group, pyrrolidinyl propyl group, pyrrolidinyl octyl group, piperidinyl methyl group and tetrahydrofuranyl methyl group is attached.

[0099] In Formula (1), preferably, R201represents -R201a-L201-R201b, R201arepresents a hydrocarbon group having 1 to 18 carbon atoms, L201represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-, R201brepresents a hydrocarbon group having 1 to 18 carbon atoms, R203represents -R203a-L203-R203b, R203arepresents a hydrocarbon group having 1 to 18 carbon atoms, L203represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-, R203brepresents a hydrocarbon group having 1 to 18 carbon atoms, R202and R204each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 18 carbon atoms represented by R202and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014, R2015, and R2016each independently represent a hydrocarbon group having 1 to 18 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 12 carbon atoms represented by R205and R206each independently represent -OH, -O-R2026, -C(O)NR2027R2028, or -NR2029C(O)R2030, R2026, R2027, R2028, R2029, and R2030each independently represent a hydrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 12 carbon atoms represented by R2026, R2027, R2028, R2029, and R2030each independently represent an aryl group having 6 to 10 carbon atoms, or a heterocyclic group, and R207, R208, and R209each independently represent -(CH2)n-, where n represents an integer of 2 to 8.

[0100] In Formula (1), more preferably, R201represents -R201a-L201-R201b, R201arepresents a hydrocarbon group having 1 to 18 carbon atoms, L201represents -C(O)O-, or -OC(O)-, R201brepresents a hydrocarbon group having 1 to 18 carbon atoms, R203represents -R203a-L203-R203b, R203arepresents a hydrocarbon group having 1 to 18 carbon atoms, L203represents -C(O)O-, or -OC(O)-, R203brepresents a hydrocarbon group having 1 to 18 carbon atoms, R202and R204each independently represent a hydrocarbon group having 1 to 10 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 6 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 6 carbon atoms represented by R205and R206each independently represent -OH, -O-R2026, -C(O)NR2027R2028, or -NR2029C(O)R2030, R2026, R2027, R2028, R2029, and R2030each independently represent a hydrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 12 carbon atoms represented by R2026, R2027, R2028, R2029, and R2030each independently represent an aryl group having 6 to 10 carbon atoms, and R207, R208, and R209each independently represent -(CH2)n-, where n represents an integer of 2 to 8.

[0101] In Formula (1), most preferably, R201represents -R201a-L201-R201b, R201arepresents a hydrocarbon group having 1 to 5 carbon atoms, L201represents -C(O)O-, R201brepresents a hydrocarbon group having 7 to 14 carbon atoms, R203represents -R203a-L203-R203b, R203arepresents a hydrocarbon group having 1 to 5 carbon atoms, L203represents -C(O)O-, R203brepresents a hydrocarbon group having 7 to 14 carbon atoms, R202and R204each independently represent a hydrocarbon group having 3 to 8 carbon atoms, R205and R206each independently represent a hydrocarbon group having 2 carbon atoms, R207, R208, and R209each independently represent -(CH2)n-, where n represents an integer of 2 to 4.

[0102] The compound of the present invention may form a salt. Examples of the salt in a basic group include, for example, salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts with sulfonic acids such as methane sulfonic acid, benzenesulfonic acid, p-toluene sulfonic acid, mesitylene sulfonic acid and naphthalene sulfonic acid. Examples of the salt in an acidic group include, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine and N,N'-dibenzylethylenediamine. Among the above salts, preferred salts include pharmacologically acceptable salts. 　　　The ionizable lipid is preferably a compound represented by formula (4), formula (2) or formula (1).

[0103] <Production method> The compound represented by the formula (1) is described in PCT / JP2024 / 002320, and can be made by the production method shown in PCT / JP2024 / 002320.

[0104] <<Examples of ionizable lipids>> Examples of ionizable lipids include the following lipids. FL-A FL-B FL-C FL-D FL-E FL-F FL-G FL-H FL-I MC3 ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate) WO2010 / 054405 L-319; (bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) WO2011 / 153493, WO2013 / 086354, WO2013 / 086322 ALC-0315; (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) WO2017 / 075331 SM-102; (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) WO2017 / 099823 WO2017 / 099823 Lipid 5; (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) WO2017 / 099823 Lipid 29; (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) Adv. Funct. Mater. 2021, 2106727, DOI: 10.1002 / adfm.202106727 ATX-100; (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) WO2019 / 191780 Lipid A9; (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) WO2017 / 004143 Lp01; ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate) WO2015 / 09534, WO2020 / 219876 TCL053; ([2-[4-(dimethylamino)butanoyloxymethyl]-3-[(Z)-tetradec-9-enoyl]oxy-2-[[(Z)-tetradec-9-enoyl]oxymethyl]propyl] (Z)-tetradec-9-enoate) WO2020 / 032184 TCL065; ([2-[5-(dimethylamino)pentanoyloxymethyl]-3-(3-pentyloctanoyloxy)-2-(3-pentyloctanoyloxymethyl)propyl] 3-pentyloctanoate) WO2020 / 032184 GCL1; ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate) WO2020 / 219941 CL4H6; ([7-[4-(dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] (Z)-octadec-9-enoate)

[0105] In the lipid composition of the present invention, the content of the ionizable lipid or a salt thereof with respect to the total lipids is preferably 10 mol% to 80 mol%, more preferably 20 mol% to 80 mol%, still more preferably 30 mol% to 70 mol%, further more preferably 40 mol% to 60 mol%.

[0106] <Phospholipid> The lipid composition contain a phospholipid. The phospholipid is preferably Zwitterionic phospholipid. Examples of the zwitterionic phospholipid include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, bis(monoacylglycero)phosphate and the like. As the phospholipid, a phospholipid having a choline group such as phosphatidylcholine is preferable. The zwitterionic lipid may be used alone or in combination of a plurality of different phospholipids.

[0107] The phosphatidylcholine is not particularly limited, and examples thereof include soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and the like. Among these, distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC) are preferable.

[0108] DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine

[0109] The phosphatidylethanolamine is not particularly limited, and examples thereof include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, diphytanylphosphatidylethanolamine, and the like.

[0110] The sphingomyelin (SM) is not particularly limited, and examples thereof include egg yolk-derived sphingomyelin, milk-derived sphingomyelin, and the like.

[0111] Bis(monoacylglycero)phosphate (BMP) is not particularly limited, and examples thereof include bis(monooleoylglycero)phosphate (18:1 BMP), bis(dioleoylglycero)phosphate (18:1 BDP, sn-[2,3-dioleoyl]-glycerol-1- phospho-sn-1'-[2',3'-dioleoyl]-glycerol), 18:1 hemi-BMP (sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho sn-3'-(1',2'-dioleoyl)-glycerol), and the like. As the phospholipids, unsaturated phospholipids are preferred. Unsaturated phospholipids can be selected from 18:1 bis(monooleoylglycerol)phosphate, dioleoylphosphatidylethanolamine or dioleoylphosphatidylcholine, but are not limited thereto. The use of unsaturated phospholipids can increase the inclusion rate of nucleic acids and can reduce particle size.

[0112] In the lipid composition of the present invention, the amount of the phospholipid is preferably 1 to 30 mol%, more preferably 5 to 25 mol%, still more preferably 7 to 23 mol% with respect to the total amount of the lipids.

[0113] <Polymer-conjugated lipid > The lipid composition of the present invention contains a polymer-conjugated lipid. The polymer-conjugated lipid is preferably a lipid having nonionic hydrophilic polymer. The lipid having nonionic hydrophilic polymer preferably contains an acyl group, and the carbon chain length of the acyl group is preferably 8 to 26.

[0114] The polymer is not particularly limited, and examples thereof include a nonionic vinyl-based polymer, a nonionic polyamino acid, a nonionic polyester, a nonionic polyether, a nonionic natural polymer, a nonionic modified natural polymer, and a block polymer or a graft copolymer having two or more kinds of these polymers as constitutional units. Among these polymers, a nonionic polyether, a nonionic polyester, a nonionic polyamino acid, or a nonionic synthetic polypeptide is preferable, a nonionic polyether or a nonionic polyester is more preferable, a nonionic polyether or a nonionic monoalkoxy polyether is even more preferable, and polyethylene glycol (hereinafter, polyethylene glycol will be also called PEG) is particularly preferable. That is, preferably, the lipid composition may contain a PEG-conjugated lipid.

[0115] The polymer-conjugated lipid is not particularly limited, and examples thereof include PEG-modified phosphoethanolamine, a diacylglycerol PEG derivative, monoacylglycerol PEG derivative, a dialkylglycerol PEG derivative, a cholesterol PEG derivative, a ceramide PEG derivative, and the like. Among these, a monoacylglycerol PEG and a diacylglycerol PEG is preferable.

[0116] The alkyl chain of the polymer-conjugated lipid preferably has 8 to 26 carbon atoms, and more preferably 10 to 22 carbon atoms. The weight average molecular weight of the polymer is preferably 100 to 10000, more preferably 500 to 5000, and even more preferably 750 to 3000. The polymer chain may be branched or may have a substituent such as a hydroxymethyl group.

[0117] Preferred examples of the polymer-conjugated lipid include the following lipids. DMG-mPEG2000: 1,2-dimiristyl-rac-glycero-3-methoxypolyethylene glycol-2000 DPG-mPEG2000: 1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DSG-mPEG2000: 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000

[0118]

[0119] In the lipid composition of the present invention, the amount of the polymer-conjugated lipid with respect to the total amount of lipids is preferably 0.1 mol% to 10 mol%, more preferably 0.3 mol% to 8 mol%, further preferably 0.5 mol% to 5 mol% and even more preferably 1 mol% to 3 mol%.

[0120] <Payload> The lipid composition contains a payload. The payload may be a therapeutic agent or other drug, preferably a nucleic acid molecule such as a polynucleotide. The nucleic acid molecule such as a polynucleotide may be either DNA or RNA, and may be plasmid (including nanoplasmid), single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (micro RNA), mRNA, antisense oligonucleotide (also known as ASO), ribozyme, aptamer, decoy nucleic acid, guide RNA (gRNA) used in genome editing and the like. The mRNA may encode DNA binding proteins. Examples of the DNA binding protein includes TALEN, ZFN, CRISPR-Cas system or their derivatives. RNA is the preferred nucleic acid molecule. It may also contain modified nucleic acids. In the lipid composition of the present invention, the weight ratio of the lipid to the payload is preferably 5 to 100, more preferably 5 to 70, still more preferably 5 to 40, and particularly preferably 5 to 35.

[0121] <Method for manufacturing lipid composition> The method for manufacturing the lipid composition of the present invention will be described. The method for manufacturing the lipid composition is not limited. For example, the lipid composition can be manufactured by a method in which all of the constituent components of the lipid particles or some of oil-soluble components of the lipid particles are dissolved in an organic solvent or the like such that an oil phase is formed, water-soluble components of the lipid particles are dissolved in water such that a water phase is formed, and the oil phase and the water phase are mixed together. A micromixer may be used for mixing, or an emulsifying machine such as a homogenizer, an ultrasonic emulsifying machine, or a high-pressure injection emulsifying machine may be used for emulsification.

[0122] Alternatively, the lipid composition can also be manufactured by a method in which a lipid-containing solution is subjected to evaporation to dryness using an evaporator under reduced pressure or subjected to spray drying using a spray drier such that a dried mixture containing a lipid is prepared, and the mixture is added to an aqueous solvent and further emulsified using the aforementioned emulsifying machine or the like.

[0123] One of the examples of the method for manufacturing the lipid composition containing is a method including a step (a) of dissolving the lipid components in an organic solvent so as to obtain an oil phase; a step (b) of mixing the oil phase obtained in the step (a) with a water phase containing a payload; a step (c) of diluting the mixed solution containing the oil phase and the water phase obtained in step (b) so as to obtain a dispersion liquid of payload-containing lipid composition; and a step (d) of removing the organic solvent from the dispersion liquid of the lipid composition obtained in the step (c).

[0124] In the step (a), the lipid components are dissolved in an organic solvent (an alcohol such as ethanol, an ester, or the like). The total lipid concentration is not particularly limited, but is generally 1 mmol / L to 100 mmol / L, preferably 3 mmol / L to 50 mmol / L, and more preferably 5 mmol / L to 30 mmol / L.

[0125] In the step (b), the water phase can be obtained by dissolving a payload (for example, nucleic acid molecule or the like) in water or a buffer. If necessary, a component such as an antioxidant can be added. The mixing ratio (volume ratio) of water phase:oil phase is preferably 5:1 to 1:1 and more preferably 4:1 to 2:1.

[0126] In the step (b), the mixed solution can be diluted with water or a buffer (for example, phosphate buffered saline (PBS) or the like).

[0127] In the step (c), as the method of removing the organic solvent from the dispersion liquid of the lipid composition, a general method can be used without particular limitation. For example, by dialyzing the dispersion liquid with the phosphate buffered saline, the organic solvent can be removed.

[0128] If necessary, the lipid composition can be subjected to sizing. Although the sizing method is not particularly limited, an extruder or the like can be used to reduce the particle size.

[0129] <Composition> The lipid composition of the present invention may be lipid particle. The lipid particle means a particle composed of a lipid, and includes a composition having any structure selected from a lipid aggregate in which the lipid is aggregated, a micelle, a liposome, a lipid nanoparticle (LNP), and lipoplex. However, the structure of the lipid particles is not limited to these as long as the composition contains lipids. The lipid composition of the present invention is preferably lipid nanoparticles (LNPs).

[0130] The form of the lipid particles can be checked by electron microscopy, structural analysis using X-rays, and the like. For example, by a method using Cryo transmission electron microscopy (CryoTEM method), it is possible to check, for example, whether a lipid particle such as a liposome has a structure composed of a bimolecular lipid membrane structure (lamella structure) and an inner water layer or a structure composed of an inner core with a high electron density and packed with constituent components including a lipid. The X-ray small angle scattering (SAXS) analysis also makes it possible to check whether or not a lipid particle has a bimolecular lipid membrane structure (lamella structure).

[0131] When the lipid composition of the present invention is a particle, the particle size is not particularly limited, but is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and even more preferably 50 to 250 nm. The particle size of the lipid particles can be measured by a general method (for example, a dynamic light scattering method, a laser diffraction method, or the like).

[0132] When the lipid composition of the present invention is a particle, the zeta potential of the particle is not particularly limited, but is preferably -20 to +20 mV, and more preferably -10 to 10 mV. The zeta potential in the present invention is a value measured by the electrophoresis method obtained by diluting the lipid composition in a phosphate buffer solution, but the method is not limited thereto.

[0133] The pKa of the lipid composition of the present invention is not particularly limited, but is preferably 9 to 4, more preferably 8 to 5, and even more preferably 7.5 to 6. The pKa of the lipid composition in the present invention adopts the value measured by the TNS assay, but is not limited to this.

[0134] <Use of lipid composition> In one example of the use of lipid compositions in the present invention, a therapeutic agent (e.g., a nucleic acid) can be introduced into the cell by introducing the lipid composition containing an acid into a cell. That is, the lipid composition of the present invention can be used as a composition for introducing nucleic acids into cells. The lipid compositions of the invention can also be used as pharmaceutical composition for in vivo nucleic acid delivery.

[0135] In the present invention, the payload can be delivered to extracellular matrix-producing cells. Extracellular matrix-producing cells are cells that secrete extracellular matrix such as collagen. Examples of the extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, and are preferably fibroblasts. Retinoid storage cells (astrocytes) are examples of extracellular matrix-producing cells. Examples of astrocytes include hepatic stellate cells, pancreatic stellate cells, kidney stellate cells, lung stellate cells, spleen stellate cells, adrenal stellate cells, intestinal stellate cells, seminal duct stellate cells, and vocal cord stellate cells.

[0136] When the lipid composition of the present invention comprises a nucleic acid having medical use, the lipid composition can be administered to a living body as a nucleic acid medicine. When the lipid composition of the present invention is used as a nucleic acid drug, the lipid composition of the present invention alone may be administered to a living body, or the lipid composition may be mixed with a pharmaceutically acceptable carrier (eg, an administration medium such as saline or phosphate buffer) and administered to a living body. That is, the lipid composition of the present invention may further contain a pharmaceutically acceptable carrier.

[0137] The concentration of the lipid composition in the mixture with the pharmaceutically acceptable carrier is not particularly limited and can generally be 0.05% by weight to 90% by weight. Further, other pharmaceutically acceptable additives such as a pH adjustment buffer and an osmotic pressure adjustment agent may be added to the nucleic acid drug containing the lipid composition of the present invention.

[0138] The route of administration for administering the lipid composition of the present invention is not particularly limited. The lipid composition can be administered by any method. Examples of the administration method include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intracutaneous administration, intravitreal administration, intraperitoneal administration, intramuscular administration, intravaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colonic administration, buccal administration, nasal administration, intracisternal administration, inhalation, and the like). Among these, parenteral administration is preferable. As the method of administration, intravenous injection, subcutaneous injection, intracutaneous injection, or intramuscular injection is preferable. Intravenous injection or intramuscular injection is particularly preferable. As the administration, nucleic acid delivery can also be performed by local administration in vivo. The lipid composition of the present invention can also be administered by direct injection into the diseased site.

[0139] The dosage form of the lipid composition according to the present invention is not particularly limited. For oral administration, the lipid composition of the present invention can be used in the form of tablets, troches, capsules, pills, suspension, syrup, and the like by being combined with an appropriate excipient. In addition, additives such as an antioxidant, a buffer, a bacteriostat, an isotonic sterile injection, a suspending agent, a solubilizer, a thickener, a stabilizer, and a preservative can be appropriately incorporated into formulations suitable for parenteral administration.

[0140] <Use of lipid composition as nucleic acid delivery carrier> The lipid composition of the present invention can retain a nucleic acid at a high encapsulation rate. Therefore, the lipid composition are extremely useful as a nucleic acid delivery carrier. According to the nucleic acid delivery carrier using the present invention, for example, by mixing the obtained composition with a nucleic acid or the like and performing transfection in vitro, ex vivo or in vivo, the nucleic acid and the like can be introduced into cells. Furthermore, the nucleic acid delivery carrier using the present invention is also useful as a nucleic acid delivery carrier in nucleic acid drugs. That is, the lipid composition of the present invention are useful as a composition for in vitro, ex vivo or in vivo (preferably in vivo) delivery of a nucleic acid.

[0141] Next, the present invention will be described based on examples, but the present invention is not limited thereto. Examples

[0142] Example 1: Materials and methods <Materials> All chemicals obtained from commercial sources were stored following the manufacturer's instruction and used without further purification. The entirety of each publication is hereby incorporated by reference.

[0143] <siRNA> One of the most common evaluation systems for extrahepatic delivery is bioluminescence imaging (BLI) using Firefly luciferase (FLuc) mRNA. Upon administering a nontoxic, stable substrate, FLuc emits luminescence at tissue-penetrating wavelengths that can be imaged in vivo and ex vivo, making it useful to identify tissues where mRNA is successfully delivered and translated. Although these BLI techniques are simple and convenient, quantification of bioluminescence from bulk tissue may overlook RNA delivery to cell types that are underrepresented in the tissue. This is especially true for extracellular matrix producing cells such as fibroblasts and stellate cells. siRNA is a useful therapeutic modality as well as a powerful tool for LNP in vivo screening. For example, early efforts to improve the potency of ionizable lipids were supported by a screening system using siRNA against Factor VII (Fvii gene), a blood clotting factor secreted by hepatocytes. Since Fvii expression is strictly limited to hepatocytes, RNA delivery efficiency to hepatocytes can be easily evaluated by quantifying serum Factor VII protein level or Fvii mRNA in the liver. Regarding extracellular matrix producing cells, using siRNAs against genes specifically expressed in extracellular matrix producing cells such as Col1a1 can be effective. Also, Reelin (Reln gene) is another example of cell-type specific expression in hepatic stellate cells. By quantifying Col1a1 or Reln mRNA remaining in the tissues after siCol1a1 or siReln administration, RNA delivery efficiency to extracelluar matrix producing cells or hepatic stellate cells can be evaluated, respectively.

[0144] The following custom siRNA was manufactured by Horizon. siFvii Sense (5' to 3') GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT Antisense(5' to 3') GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT

[0145] siCol1a1 Sense (5' to 3') GmUmCmUAGAmCAmUGmUmUmCAGmCmUmUdTsdT Antisense(5' to 3') AAGCUGAAmCAUGUCmUAGACdTsdT

[0146] siReln Sense (5' to 3') GGmUmCmUmCAAGmCmCAmCmUmCGmUmUmUdTsdT Antisense(5' to 3') AAACGAGUGGCUUGAGACCdTsdT

[0147] Abbreviation Nucleotide monomer abbreviations used in the nucleic acid sequences. These monomers, when present in oligonucleotide, are linked by 5'-3'-phosphate ester bonds. When phosphorothioate is present, they are linked by phosphorothioate bond. A Adenosine-3’-phosphate C Cytidine-3’-phosphate G Guanonsine-3’-phosphate U Uridine-3’-phosphate mA 2’-O-methyladenosine-3’-phosphate mC 2’-O-methylcytidine-3’-phosphate mG 2’-O-methylguanonsine-3’-phosphate mU 2’-O-methyluridine-3’-phosphate fA 2'-Fluoroadenosine-3'-phosphate fC 2'-Fluoromethylcytidine-3'-phosphate fG 2'-Fluoromethylguanosine-3'-phosphate fU 2'-Fluoromethyluridine-3'-phosphate dT 2’-deoxythymidine-3’-phosphate dTs 2’-deoxythymidine-3’-phosphate-phosphorothioate

[0148] <Ionizable lipids> The methods for the production of FL-A, FL-B and FL-G corresponding to ionizable lipids represented by formula (4) are described in WO2019 / 235635. The methods for the production of FL-C, FL-D, FL-E and FL-F corresponding to ionizable lipids represented by formula (2) are described in WO2022 / 230964. The methods for the production of FL-H and FL-I corresponding to ionizable lipids represented by formula (1) are as described above in PCT / JP2024 / 002320. FL-A FL-B FL-C FL-D FL-E FL-F FL-G FL-H FL-I MC3; ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino) butanoate) WO2010 / 054405 L-319; (bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) WO2011 / 153493, WO2013 / 086354, WO2013 / 086322 ALC-0315; (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) WO2017 / 075331 SM-102; (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) WO2017 / 099823 Lipid 5; (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) WO2017 / 099823 Lipid 29; (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) Adv. Funct. Mater. 2021, 2106727, DOI: 10.1002 / adfm.202106727 ATX-100; (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) WO2019 / 191780 Lipid A9; (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) WO2017 / 004143 Lp01; ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate) WO2015 / 09534, WO2020 / 219876 TCL053; ([2-[4-(dimethylamino)butanoyloxymethyl]-3-[(Z)-tetradec-9-enoyl]oxy-2-[[(Z)-tetradec-9-enoyl]oxymethyl]propyl] (Z)-tetradec-9-enoate) WO2020 / 032184 TCL065; ([2-[5-(dimethylamino)pentanoyloxymethyl]-3-(3-pentyloctanoyloxy)-2-(3-pentyloctanoyloxymethyl)propyl] 3-pentyloctanoate) WO2020 / 032184 GCL1; ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate) WO2020 / 219941 CL4H6; ([7-[4-(dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] (Z)-octadec-9-enoate)

[0149] <Lipid nanoparticle formulation> Lipid Nanoparticle Formulation: Lipid nanoparticles were synthesized using a microfluidics chip device as previously described (Chen, D., J. Am. Chem. Soc. (2012). Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation.). Briefly, a lipid-containing ethanol phase was mixed with a siRNA-containing aqueous phase through microfluidic channel in the PDMS (poly-dimethyl-siloxane) chip. siRNA was diluted in 10 mM citrate buffer, pH 3.0, (aqueous phase) while the appropriate amounts of lipids were co-dissolved in 200 proof ethanol (ethanol phase). Syringe pumps were used to mix the ethanol and aqueous phases together at a 1:3 volume ratio (total flow rate 1.2 mL / min). The resulting LNPs were dialyzed against phosphate-buffered saline (PBS) in a 20 kDa molecular weight cut-off (MWCO) cassette at 4 °C or room temperature overnight and stored at 4 °C until use. In some experiments, LNP solutions were further concentrated using a 100 kDa MWCO Amicon ultra centrifugal filters (Millipore Sigma). For animal experiments, LNP solutions were sterilized through a 0.22 um filter (Millipore Sigma).

[0150] <Particle size measurement> LNP particle size, PDI(polydispersity index), and ζ potential (i.e., zeta potential) were obtained using a Zetasizer (Malvern). For size measurement, LNPs were diluted in PBS at a 1 / 200 v / v ratio and z-average values were reported. For zeta potential measurement, LNPs were diluted in 0.1X PBS at a 1 / 200 v / v ratio.

[0151] <Quantification of siRNA concentration and encapsulation> The siRNA concentration in dialyzed particles was determined via a modified Quant- iT RiboGreen RNA assay (Thermo Fisher). A nanoparticle dilution of ~l ng pL-l siRNA was made in TE buffer (pH 8.5) and siRNA standards were made ranging from 2 ng pL-l to 0.125 ng pL-l. 50 pL of each solution was added to separate wells in a 96-well black polystyrene plate. To each well was added either 50 pL of TE buffer. The plate was incubated at 37°C for 15 minutes with shaking at 350 rpm. Following the incubation, the diluted RiboGreen reagent was added (100 pL per well), and the plate was incubated as before for 3 minutes. RiboGreen fluorescence was measured according to the supplied protocol using a Tecan plate reader, and the siRNA standard was used to determine nanoparticle siRNA concentration. It should be noted that two separate standards were made: one with and without Triton-X. The particles in TE buffer were used to determine un-encapsulated siRNA concentration and TE-TX, and encapsulation efficiency was determined via the following equation:

[0152]

[0153] <pKa Value Measurement by TNS Assay> The apparent pKa values of lipid nanoparticles were determined using 2-(p-toluidinyl)naphthalene-6-sulphonic acid (TNS) assay as described previously (Heyes J. et al. Journal of Controlled Release 107 (2005) 276-287). Briefly, pH buffers ranging from 3.0 to 9.0 in 0.5 increments were prepared by mixing a solution of 20 mM sodium phosphate buffer, 20 mM sodium citrate buffer, 20 mM sodium borate buffer, and 150 mM NaCl. 300 μM TNS solution was added to the above pH buffer in the final concentration of 6 μM. 46 μL of each pH buffer containing 6 μM TNS was added to a black 384-well plate. Lastly, 4 μL of LNP solutions with 500 ng / μL ionizable lipid were added to each well (Quadruplicate). Fluorescence intensity was measured using a plate reader (Tecan) at an excitation of 320 nm and an emission of 465 nm. The resulting pH-fluorescence sigmoidal curves were evaluated by a curve fit analysis using Prism (Graphpad), and the apparent pKa values were obtained as the inflection points of the sigmoidal curves.

[0154] <Animal experiments> All animal studies were approved by the MIT Institutional Animal Care and Use Committee (CAC) and were consistent with local, state, and federal regulations as applicable. All experimental procedures were performed with ethical compliance and approval under the guidelines for Division of Comparative Medicine by Massachusetts Institute of Technology. Female C57BL / 6 mice (6 weeks) were obtained from Jackson Laboratory, housed in an MIT animal facility, and acclimated for at least 3 days before the initiation of a study. For intravenous administration, RNA-lipid nanoparticles diluted in PBS were injected via the tail vein using 29 g, 3 / 10 cc insulin syringes (BD Biosciences) after gentle warming of the animals using a heat lamp. 48-72 hours after injection, organs or tissues including the heart, liver, spleen, lung, kidneys, muscle (quadriceps), and brain were collected and soaked in RNAlater solution (Thermo Fisher) for 12-48 hours at 4 °C and stored at -20 °C after the removal of RNAlater.

[0155] <Total RNA isolation and cDNA synthesis> An approximately 10 mg piece of tissue was homogenized, and total RNA was isolated from the liver lysate using Zymo Quick-RNA MagBead (Zymo research). cDNA was synthesized using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368814) according to manufacturer instructions. Briefly, 10 μl of a master mix containing 2 μl 10X Buffer, 0.8 μl 25X dNTPs, 2 μl 10X Random primers, 1 μl Reverse Transcriptase, and 4.2 μl of water per reaction was added to 10 μl mRNA solution that was isolated using the above protocol. Plates were sealed, mixed, and incubated on an thermal cycler for 10 minutes at room temperature, followed by 2 hours at 37°C and 5 minutes at 85°C.

[0156] <Tissue mRNA Quantification by RT-qPCR> Total RNA was isolated from tissues using the Quick-RNA MagBead (Zymo Research). Briefly, tissue punches were placed in a deep-well 96 well plate with 4-mm stainless beads and lysed with 350 μL 250 μL DNA / RNA Shield (Zymo Reaearch) using a GenoGrinder2010. Total RNA was further purified using Quick-RNA MagBead (Zymo Research) according to the manufacturer’s protocol.

[0157] cDNA was synthesized using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368814) according to manufacturer instructions. Briefly, 5 μl of a master mix containing 1 μl 10X Buffer, 0.4 μl 25X dNTPs, 1 μl 10X Random primers, 0.5 μl Reverse Transcriptase, and 2.1 μl of water per reaction was added to 5 μl total RNA solution that was isolated using the above protocol. Plates were sealed, mixed, and incubated on a thermal cycler for 10 minutes at 25 °C, followed by 2 hours at 37 °C and 5 minutes at 85 °C. Gene expression was analyzed in qPCR with LunaTMUniversal Probe RT-qPCR Kit (NEB) and TaqMan probes. Samples were amplified using a LightCycler 480 qPCR machine (Roche). Fvii, Reln, and Col1a1 expression was normalized to Gusb. The TaqMan probes used in the research are listed as below;

[0158]

[0159] Example 2: In vivo RNA delivery to hepatic stellate cells with various retinoid ratio in mice To examine the potential of retinoids for hepatic stellate cell delivery, cholesterol was first replaced with retinol in a standard hepatocyte-targeting LNP formulation. The resulting LNP was formulated with siRNA against Fvii Reln to evaluate gene silencing in hepatic stellate cells in vivo. The details of the formulations and their physicochemical properties are given in Table 1.

[0160] The results are shown in Figure 1 and Figure 2. These results indicate that incorporation of the retinoid in hepatocyte-targeting LNPs can change the cell-type tropism of LNPs. Also, LNPs with 20% retinol showed negligible gene silencing in hepatocytes at these doses (0.2 mg kg-1and 0.3 mg kg-1), suggesting that retinol ratio higher than 10% is useful to suppress off-target delivery to hepatocytes. It is worth noting that smaller particle size and loss of encapsulation efficiency was observed as retinol ration increased.

[0161] Table 1: LNP formulations containing different amount of retinol and their physicochemical properties

[0162] Figure 1 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with various retinol ratio in total lipids were intravenously administered to mice at 0.3 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 1 A and Figure 1 B shows the Reln and Fvii gene silencing, respectively. Figure 2 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with various retinol ratio in total lipids were intravenously administered to mice at 0.2 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested.

[0163] Example 3: in vivo RNA delivery to hepatic stellate cells with various retinoids in mice To elucidate the relationship between the chemical structure of retinoid and the efficiency of RNA delivery to hepatic stellate cells, multiple retinoid molecules were evaluated by incorporating to hepatocyte-targeting LNPs encapsulating siFvii and siReln, and the resultant LNPs were intravenously administered at 0.3 mg kg-1each to female CD-1 mice. The details of the formulations and their physicochemical properties are given in Table 2 and Table 3. The results are shown in Figure 3 and Figure 4. PEG-containing retinoid showed slight increase of siRNA delivery efficiency to hepatic stellate cells compared to conventional hepatocyte LNPs without retinoids. However, this increase is not as significant as retinol (Figure 3). All the other retinoids, such as all-trans retinal, retinyl acetate, and all-trans retinoic acid, showed improvement in hepatic stellate cell delivery comparable to retinol. As for hepatocyte off-target delivery, retinol and all-trans retinoic acid showed smallest gene silencing in hepatocytes (Figure 4).

[0164] Retinol All-trans Retinal Retinyl acetate All-trans retinoic acid Retinoic acid PEG1000 (n=22)

[0165] Table 2

[0166] Table 3

[0167] Figure 3 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 2% Retinol or 2% Retinol-PEG1000 were intravenously administered to mice at 0.3 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 3A and Figure 3B shows the Reln and Fvii gene silencing, respectively.

[0168] Figure 4 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 20% of various retinoid in total lipids were intravenously administered to mice at 0.3 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 4A and Figure 4B shows the Reln and Fvii gene silencing, respectively.

[0169] Example 4: In vivo RNA delivery to hepatic stellate cells with different phospholipids in mice (20% retinoid) To improve RNA encapuslation efficiency in 20% retinol LNP formulations, LNPs with different phospholipids were prepared and tested for their hepatoyte and hepatic stellate cell delivery efficiency in vivo using a single 0.2 or 0.3 mg / kg siFvii and siReln (each) intravenous administration. Three days post administration, the tissues were harvested and Fvii and Reln mRNA was quantified by RT-qPCR. The LNP formulations with unsaturated phospholipids such as DOPC, 18:1 BMP, and DOPE showed improvement of RNA encapulation efficiency and smaller paritcle size regardless of their head group strucrture. Also, all the LNPs containing 20% retinol showed significant Reln gene silencing in the liver, indicating that these novel formulations can be used for RNA delivery to the hepatic stellate cell RNA delivery. The details of the formulations are given in Table 4. The results are shown in Figure 5 and Figure 6.

[0170] Table 4

[0171] Figure 5 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 20% retinol and various phospholipids were intravenously administered to mice at 0.3 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 5A and Figure 5B shows the Reln and Fvii gene silencing, respectively.

[0172] Figure 6 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 20% retinol and various phospholipids were intravenously administered to mice at 0.2 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 6A and Figure 6B shows the Reln and Fvii gene silencing, respectively.

[0173] Example 5: In vivo RNA delivery to hepatic stellate cells with different sterols in mice (20% retinoid) To improve RNA encapuslation efficiency in 20% retinol LNP formulations, LNPs with different sterols were prepared and tested for their hepatoyte and hepatic stellate cell delivery efficiency in vivo using a single 0.3 mg / kg siFvii and siReln (each) intravenous administration. Three days post administration, the tissues were harvested and Fvii and Reln mRNA was quantified by RT-qPCR. The LNP formulations with ionizable cationic sterol such asHACP-cholesterol showed improvement of RNA encapulation efficiency and smaller paritcle size. Also, all the LNPs containing different sterols showed significant Reln gene silencing in the liver, indicating that these novel formulations can be used for RNA delivery to the hepatic stellate cell RNA delivery. The details of the formulations are given in Table 5. The results are shown in Figure 7.

[0174] Table 5

[0175] Figure 7 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 20% retinol and various sterols were intravenously administered to mice at 0.2 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 7A and Figure 7B shows the Reln and Fvii gene silencing, respectively.

[0176] Example 6: In vivo RNA delivery to hepatic stellate cells with different phospholipids in mice (2% retinoid) To improve RNA improve hepatic stellate cell delivery efficiency and suppress hepatocyte off-targete deliver in 2% retinol LNP formulations, LNPs with different phospholipids were prepared and tested for their hepatoyte and hepatic stellate cell delivery efficiency in vivo using a single 0.2 mg / kg siFvii and siReln (each) intravenous administration. Three days post administration, the tissues were harvested and Fvii and Reln mRNA was quantified by RT-qPCR. The LNP formulations with unsaturated phospholipids such as DOPC, 18:1 BMP, and DOPE showed smaller paritcle size regardless of their head group strucrture. Also, all the LNPs containing unsaturated phospholipid showed smaller Fvii gene silencing in the liver, indicating that combination of retinoids and unsaturated phospholipids can be used for more selective RNA delivery to the hepatic stellate cells. The details of the formulations are given in Table 6. The results are shown in Figure 8.

[0177] Table 6

[0178] Figure 8 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 2% retinol and various phospholipids were intravenously administered to mice at 0.2 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 8A and Figure 8B shows the Reln and Fvii gene silencing, respectively.

[0179] Example 7: In vivo RNA delivery to hepatic stellate cells with different sterols in mice (2% retinoid) To improve RNA improve hepatic stellate cell delivery efficiency and suppress hepatocyte off-targete deliver in 2% retinol LNP formulations, LNPs with different sterols were prepared and tested for their hepatoyte and hepatic stellate cell delivery efficiency in vivo using a single 0.2 mg / kg siFvii and siReln (each) intravenous administration. Three days post administration, the tissues were harvested and Fvii and Reln mRNA was quantified by RT-qPCR. The LNP formulations with ionizable cationic sterol such as HAPC-cholesterol showed higher RNA encaplulation efficiency and smaller paritcle size. Also, the LNPs containing ionizable cationic sterol showed smaller Fvii gene silencing in the liver, indicating that combination of retinoids and ionizable cationic sterols can be used for more selective RNA delivery to the hepatic stellate cells. The details of the formulations are given in Table 7. The results are shown in Figure 9.

[0180] Table 7

[0181] Figure 9 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with 2% retinol and various sterols were intravenously administered to mice at 0.2 mg / kg each and livers were harvested 72 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 9A and Figure 9B shows the Reln and Fvii gene silencing, respectively.

[0182] Example 8: In vivo RNA delivery to hepatic stellate cells with different ionizable lipids in mice To test whether retinoid-mediated hepatic stellate cell delivery is generalizable to other ionizable lipids, 2% retionol LNPs with various ionizable lipids were prepared and tested for their hepatoyte and hepatic stellate cell delivery efficiency in vivo using a single 0.15 or 0.2 mg / kg siFvii and siReln (each) intravenous administration. Two days post administration, the tissues were harvested and Fvii and Reln mRNA was quantified by RT-qPCR. The details of the formulations and their physicochemical properties are given in Table 8, Table 9, and Table 10.

[0183] The results are shown in Figure 10, Figure 11, and Figure 12. These results indicate that incorporation of the retinoid in hepatocyte-targeting LNPs can change the cell-type tropism of LNPs regardless of ionizable lipid structure.

[0184] The methods for the production of FL-A, FL-B and FL-G corresponding to ionizable lipids represented by formula (4) are described in WO2019 / 235635. The methods for the production of FL-C, FL-D, FL-E and FL-F corresponding to ionizable lipids represented by formula (2) are described in WO2022 / 230964. The methods for the production of FL-H and FL-I corresponding to ionizable lipids represented by formula (1) are as described above in PCT / JP2024 / 002320. FL-A FL-B FL-C FL-D FL-E FL-F FL-G FL-H FL-I

[0185] Table8

[0186] Table 9

[0187] Table 10

[0188] Figure 10 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with different retinol ratio were intravenously administered to mice at 0.15 mg / kg each and livers were harvested 48 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 10 A and Figure 10 B show the Reln and Fvii gene silencing, respectively.

[0189] Figure 11 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with various ionizable lipid and 2% retionl were intravenously administered to mice at 0.15 mg / kg each and livers were harvested 48 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 11 A and Figure 11 B show the Reln and Fvii gene silencing, respectively.

[0190] Figure 12 shows the results of in vivo evaluation of Reln and Fvii gene silencing. Formulations encapsulating siRNAs against Reln and Fvii with various ionizable lipid and 2% retionl were intravenously administered to mice at 0.2 mg / kg each and livers were harvested 48 hours after injection. After isolation and purification of total RNA from organs, Reln and Fvii mRNA was quantified relative to a housekeeping gene, GusB. A PBS control was also tested. Figure 12 A and Figure 21 B show the Reln and Fvii gene silencing, respectively.

[0191] Example 9: in vivo RNA delivery to hepatic stellate cells with various retinoids in mice (2% retinoid) To elucidate the relationship between the chemical structure of retinoid and the efficiency of RNA delivery to hepatic stellate cells, multiple retinoid molecules were evaluated by incorporating to hepatocyte-targeting LNPs encapsulating siFvii and siReln, and the resultant LNPs were intravenously administered at 0.1 mg kg-1each to female CD-1 mice. The details of the formulations and their physicochemical properties are given in Table 11. The results are shown in Figure 13. All LNPs containing retinoids demonstrated significant Reln gene silencing in hepatic stellate cells. Furthermore, except for retinyl palmitate, these LNPs exhibited greater gene silencing efficiency in hepatic stellate cells compared to hepatocytes at the same dose of 0.1 mg / kg, indicating a selective preference for hepatic stellate cells. (Figure 13).

[0192] Table 11

Claims

1. A lipid composition which comprises a payload, an ionizable lipid, a retinoid, a phospholipid, a sterol, and a polymer-conjugated lipid, and does not comprise a constitutively cationic lipid.

2. The lipid composition of claim 1, wherein the retinoid doesn’t comprise polyethylene glycol in its structure.

3. The lipid composition of claim 1, wherein the retinoid is retinol, all-trans-retinal, retinyl acetate, or all-trans retinoic acid, 9-cis retinoic acid, 13-cis retinoic acid, or retinyl palmitate.

4. The lipid composition of claim 1, wherein the retinoid is retinol, retinyl acetate, all-trans retinoic acid, 9-cis retinoic acid, or 13-cis retinoic acid.

5. The lipid composition of claim 1, which comprises the retinoid at amount of 1 to 40 mol% with respect to the total lipids.

6. The lipid composition of claim 1, which comprises the retinoid at amount of 2 to 5 mol% with respect to the total lipids.

7. The lipid composition of claim 1, which further comprises an unsaturated phospholipid.

8. The lipid composition of claim 7, wherein the unsaturated phospholipid is selected from 18:1 bis(monooleoylglycerol)phosphate or dioleoyl phosphatidylethanolamine or dioleoyl phosphatidylcholine.

9. The lipid composition of claim 1, wherein the ionizable lipid is a compound represented by formula (4): In the formula, X represents -NR1- or -O-, R1represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R21-L1-R22-, where R21represents a hydrocarbon group having 1 to 24 carbon atoms, L1represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R22represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R2and R3each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R31-L2-R32-, where R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R4, R5, R6, R7, R8, R9, R10, R11, and R12each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted, groups in any one or more pairs among R4and R5, R10and R5, R5and R12, R4and R6, R5and R6, R6and R7, R6and R10, R12and R7, and R7and R8may be linked to each other to form a 4- to 7-membered ring which may contain an O atom, a substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the substituent on the substituted or unsubstituted aryl group and on the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and a, b, c, and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more.

10. The lipid composition of claim 1, wherein the ionizable lipid is a compound represented by formula (5): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), -N(R58)(R59) or -G20-R60, G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R58and R59each independently represent a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, the substituent B is-N(R61)(R62), R61and R62each independently represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R60represents a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), a represents 0 or 1, L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, L20represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65or-O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group.

11. The lipid composition of claim 1, wherein the ionizable lipid is a compound represented by formula (2): wherein R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with one or more substituents selected from -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, and -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, R107represents -R110-L102-R111-L103-R112, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, or -O-R166. R161and R162each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R163, R164, R165, and R166each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R163, R164, R165, and R166may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R168, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, -O-R166, or -(hydrocarbon group having 1 to 12 carbon atoms), R168represents a hydrocarbon group having 1 to 12 carbon atoms, and L101, L102, and L103each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, R110represents a hydrocarbon group having 1 to 8 carbon atoms, R111represents a hydrocarbon group having 1 to 24 carbon atoms, R112represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R109and R112may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, where definitions of R153, R154, R155, and R158are as described above, and the hydrocarbon group represented by R111may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where the definitions of R153, R154, and R155are as described above.

12. The lipid composition of claim 1, wherein the ionizable lipid is a compound represented by formula (1): wherein R201, R202, R203, and R204each independently represent a hydrogen, a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, the substituted or unsubstituted hydrocarbon groups represented by R201, R202, R203, and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014-, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014-, R2015, and R2016each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R2017, and R2017represents a hydrocarbon group having 1 to 12 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon groups having 1 to 18 carbon atoms represented by R205and R206each represent -OH, -COOH, -NR2021R2022, -OC(O)O-R2023, -C(O)O-R2024, -OC(O)-R2025, -O-R2026, -C(O)NR2027R2028, -NR2029C(O)R2030, -N(R2031)S(O)2R2032, -N(R2033)C(O)N(R2034)R2035, -N(R2036)C(S)N(R2037)R2038, -OC(O)N(R2039)R2040, or -N(R2041)C(O)OR2042, R2021and R2022each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042each independently represent a hyrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, where the substituted groups on the substituted hydrocarbon group having 1 to 24 carbon atoms represented by R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030,R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042represent an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or NR2051R2052, and R2051and R2052each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R207, R208, and R209each independently represent a hydrocarbon group having 2 to 8 carbon atoms, R205and R206, or R205and R207may form a 4 to 7-membered ring together.

13. The lipid composition of claim 1, wherein the sterol is selected from cholesterol, phytosterol, or cationic cholesterol.

14. The lipid composition of claim 1, wherein the payload is a nucleic acid molecule.

15. The lipid composition of claim 14, wherein the nucleic acid molecule is RNA.

16. A method for delivering payload to extracellular matrix producing cells, which comprises administering the lipid composition of claim 1 to a subject.

17. The method of claim 16, wherein the extracellular matrix-producing cells are fibroblasts.

18. The method of claim 16, wherein the extracellular matrix-producing cells are hepatic stellate cells.

19. The method of claim 16, wherein the payload is a nucleic acid molecule.

20. The method of claim 19, wherein the nucleic acid molecule is RNA.

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