Methods for delivering therapeutic agents and lipid compositions

Abstract

An object of the present invention is to provide a method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, which can achieve excellent delivery efficiency even to organs other than the liver, and a composition comprising a therapeutic agent and lipid nanoparticles, which can achieve excellent delivery efficiency even to organs other than the liver.According to the present invention, there is provided a method for delivering a therapeutic agent to endothelial cells, which comprises administering a lipid composition to a subject, wherein the lipid composition comprises the therapeutic agent and lipid nanoparticles, and the lipid nanoparticles comprise an ionizable lipid and a compound represented by formula (1) or a salt thereof. JPEG2025539924000152.jpg61170In the formula, G1 represents -C(O)-, -OC(O)-, -O(CO)O-, or -C(O)O-, and L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, X represents a basic functional group.

Description

[Technical Field]

[0001] The present invention relates to a method for delivering a therapeutic agent to endothelial cells, mesenchymal cells or cancer cells, and to a lipid composition comprising the therapeutic agent and a lipid nanoparticle (LNP). [Background technology]

[0002] LNPs are materials that can deliver therapeutic agents, such as nucleic acids, to the liver. One example of an LNP is the small interfering RNA (siRNA) LNP therapy for transthyretin-mediated amyloidosis, called Onpattro®, which was recently approved by the U.S. Food and Drug Administration. Despite these advances, it has not been possible to predictably and rationally design nanoparticles for delivery to target tissues other than the liver.

[0003] Traditionally, effective intracellular delivery agents have relied on an optimal balance of ionizable amines for binding and releasing RNA (pKa between 6.0 and 6.5) and hydrophobic moieties for stabilizing the nanoparticles. Focusing on ionizable cationic lipids has produced highly effective carriers for hepatocytes, but not for delivery to other organs.

[0004] Lipid nanoparticles (LNPs) are self-assembled nanostructures capable of encapsulating, protecting, and delivering nucleic acids. Conventional LNPs are composed of ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. Screening and designing novel ionizable lipids has produced highly effective RNA delivery carriers for hepatocytes and mRNA vaccines. However, these studies have not yielded effective carriers capable of reaching organs other than the liver.

[0005] The present inventors have demonstrated that incorporation of permanently cationic lipids (Non-Patent Documents 1-3) or surface modification of LNPs with ligands enables RNA delivery to lung endothelial cells (Non-Patent Documents 4-7). However, cationic lipids are known to be toxic, and ligand modification of LNPs is laborious and potentially heterogeneous. Furthermore, even with these techniques, RNA delivery remains limited to lung endothelial cells. Therefore, there is a need for lipid nanoparticle compositions that do not constitutively use cationic lipids or ligands.

[0006] There have also been several studies into replacing cholesterol in LNPs with cholesterol analogs. For example, oxidized cholesterol and cholesterol esters have been used to enhance RNA delivery efficiency to liver endothelial cells and all cell types in the liver (Non-Patent Documents 8 and 9). Furthermore, naturally occurring cholesterol analogs such as phytosterols have been tested for improving endosomal escape efficiency in vitro (Non-Patent Document 10).

[0007] Several synthetic cholesterol analogs with basic functional groups have been developed for use as nucleic acid delivery carriers. DC-cholesterol is a cholesterol derivative with a tertiary amine group and a pKa value of 7.8 (Non-Patent Documents 11 and 12). Conventionally, DC-cholesterol has been formulated with phospholipids, such as DOPE, to encapsulate nucleic acids as liposomes. The present inventors tested the combination of non-biodegradable ionizable lipids (C12-200 and cKK-E12) with DC-cholesterol in a subcutaneous mRNA vaccine to enhance mRNA delivery to dendritic cells in lymph nodes, but no advantage over cholesterol was observed, and in fact, delivery efficiency was reduced (Non-Patent Document 13). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] “ Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing,” Nat. Nanotechnol., vol. 15, no. April,

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[0009] In view of the above problems in the background art, the present invention aims to provide a lipid composition that enables the delivery of a therapeutic agent to endothelial cells, mesenchymal cells or cancer cells in organs other than the liver.

[0010] An object of the present invention is to provide a method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, which can achieve excellent delivery efficiency even to organs other than the liver. Another object of the present invention is to provide a composition containing a therapeutic agent and lipid nanoparticles, which can achieve excellent delivery efficiency even to organs other than the liver. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the present inventors have found that by using an ionizable lipid and a cholesterol derivative having a specific structure, it is possible to achieve excellent delivery efficiency of a therapeutic agent to organs other than the liver. The present invention was completed based on the above findings. According to the present invention, the following inventions are provided:

[0012] <1> 1. A method for delivering a therapeutic agent to an endothelial cell, a mesenchymal cell, or a cancer cell, comprising administering a lipid composition to a subject, the lipid composition comprises the therapeutic agent and lipid nanoparticles; The lipid nanoparticles comprise an ionizable lipid and a compound represented by formula (1) or a salt thereof. [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, X represents a basic functional group. <2> the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5- or 6-membered heterocyclic alkyl group, or a 5- or 6-membered heterocyclic aryl group; <1> The method described below. <3> The compound represented by formula (1) is a compound represented by formula (2). <1> or <2> The method described below. [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, R 2 , R3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH)=NH, and R 2 , R 3 and R 4 One of them may not be present. <4> The compound represented by formula (2) is a compound represented by formula (3): <3> The method described below. [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms; R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms; G2 represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group; R 2 , R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH)=NH; R 2 , R 3 and R 4 One of them may not be present. <5> The compound represented by formula (3) is a compound represented by formula (3A). <4> The method described below. [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms; R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms; G2 represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group; R 2 , R 3 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH2)=NH2. <6> G1 represents -C(O)- or -C(O)O-; <1> from <5> 1. The method according to claim 1 , <7> R1 represents a hydrogen atom or an aminoalkyl group having 1 to 4 carbon atoms; <5> The method described below. <8> R1 represents a hydrogen atom; <5> The method described below. <9> G2 represents a single bond or -C(O); <5> The method described below. <10> G2 represents a single bond; <5> The method described below. <11> L2 represents an alkylene group having 1 to 3 carbon atoms, and R 2 and R 3 each independently represents a hydrocarbon group having 1 to 2 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, <5> The method described below. <12> The compound represented by formula (1) or a salt thereof is any one of the following: <1> or <2> The method described below. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] <13> The content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol% based on the total lipids. <1> from <12> 1. The method according to claim 1 , wherein <14> the therapeutic agent is a nucleic acid; <1> from <13> 1. The method according to claim 1 , wherein <15> the therapeutic agent is DNA or RNA; <1> from <14> 1. The method according to claim 1 , wherein <16> the therapeutic agent is mRNA or siRNA; <1> from <15> 1. The method according to claim 1 , wherein <17> The ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and the biodegradable group is represented by -O(CO)O-, -O(CO)-, or -(CO)O-; <1> from <16> 1. The method according to claim 1 , wherein <18> The ionizable lipid is a compound represented by formula (4): <1> from <17> 1. The method according to claim 1 , wherein [ka] In the formula, X is -NR 1 - or -O-, R 1 is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 -, and R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 22 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R 31 -L2 -R 32 -, and R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 32 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and R 8 any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR 45 R 46 an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 and R41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 The amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c, and d each independently represent an integer of 0 to 3, provided that a+b is 1 or more, and c+d is 1 or more. <19> The ionizable lipid is a compound represented by formula (1): <1> from <17> 1. The method according to claim 1 , wherein [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , and OR 56 and optionally substituted with one or more substituents selected from R 4represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R 8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, R 7 -R 10 -L 2 -R 11 -L 3 -R 12 indicates, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R 57 -OH, COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , or -OR66 Shows. R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 63 ,R 64 ,R 65 , and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 63 ,R 64 ,R 65 , and R 66 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 68 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , -OR 66 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 67 may be substituted with R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, L 1 , L 2 , and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R 8 represents a hydrocarbon group having 1 to 12 carbon atoms, R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, R 10 represents a hydrocarbon group having 1 to 8 carbon atoms, R 11 represents a hydrocarbon group having 1 to 24 carbon atoms, R 12 represents a hydrocarbon group having 1 to 24 carbon atoms, R 9 , and R 12 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54, -OC(O)-R 55 , or SR 58 and R 53 , R 54 , R 55 , and R 58 is defined as above, R 11 The hydrocarbon group represented by is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 The definition of is as above. <20> The ionizable lipid is a compound represented by the following formula (5): <1> from <17> 1. The method according to claim 1 , wherein [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The substituent A is a hydroxyl group, —G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 represents a group represented by G 20 represents -O(CO)- or -(CO)O-, R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 58 and R 59 each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, Substituent B is -N(R 61 )(R 62 ) and R 61 and R 62each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R 60 represents a hydrocarbon group having 1 to 18 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 30 is -S-(CO)-NR 64 - indicates R 64 L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G 10 is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- indicates R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is -(CO)OR 65 , or -O(CO)-R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms. <21> The lipid nanoparticles further comprise a lipid having a non-ionic hydrophilic polymer; <1> from <20> 1. The method according to claim 1 , wherein <22> the lipid nanoparticles further comprise a phospholipid; <1> from <21> 1. The method according to claim 1 , wherein <23> The lipid composition is administered to the subject by intravenous or intramuscular injection; <1> from <22> 1. The method according to claim 1 , wherein <24> The mesenchymal cells are muscle cells, <1> from <23> 1. The method according to claim 1 , wherein <25> Mesenchymal cells are extracellular matrix-producing cells; <1> from <23> 1. The method according to claim 1 , wherein <26> the extracellular matrix cells are astrocytes or fibroblasts; <1> from <23> 1. The method according to claim 1 , wherein <27> the stellate cells are hepatic stellate cells, pancreatic stellate cells, or colonic stellate cells; <1> from <23> 10. The method according to any one of the preceding claims. <28> 1. A lipid composition comprising a therapeutic agent and lipid nanoparticles, The lipid nanoparticles comprise a compound represented by formula (1) or a salt thereof and an ionizable lipid having a biodegradable group. Lipid composition. [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L Y represents a single bond or an alkylene group having 1 to 14 carbon atoms, a substituted alkylene, a heteroalkylene, or a substituted heteroalkylene; X represents a basic functional group. <29> the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5- or 6-membered heterocyclic alkyl group, or a 5- or 6-membered heterocyclic aryl group; <28> The lipid composition described in <30> The compound represented by formula (1) is a compound represented by formula (2). <28> or <29> The lipid composition described in [ka] During the ceremony, G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-; L Y represents a single bond or an alkylene group, substituted alkylene group, heteroalkylene group, or substituted heteroalkylene group having 1 to 14 carbon atoms, R 2 , R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH)=NH, and R 2 , R 3 and R 4 One of them may not be present. <31> The compound represented by formula (2) is a compound represented by formula (3): <30> The lipid composition described in [ka] During the ceremony, G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-; L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms; R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms; G2 represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group; R 2 , R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH)=NH; R 2 , R 3 and R 4 One of them may not be present. <32> The ionizable lipid having a biodegradable group is a compound represented by formula (4): <28> from <31> The lipid composition described in any one of the above. [ka] In the formula, X is -NR 1 - or -O-, R 1 is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 -, and R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 22 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R 31 -L 2 -R 32 -, and R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 32 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and R 8 any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR 45 R 46 an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 The amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 and R 41 , R 42 , R 43 , R 44 , R 45 and R 46each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c, and d each independently represent an integer of 0 to 3, provided that a+b is 1 or more, and c+d is 1 or more. <33> The ionizable lipid is a compound represented by formula (1): <28> from <31> The lipid composition described in any one of the above. [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , and OR 56 and optionally substituted with one or more substituents selected from R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R 8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, R 7 -R 10 -L 2 -R 11 -L 3 -R 12 indicates, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 ,R 54 ,R 55, and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R 57 -OH, COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , or -OR 66 Shows. R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 63 ,R 64 ,R 65 , and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 63 ,R 64 ,R 65 , and R 66 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 68 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64, -OC(O)-R 65 , -OR 66 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 67 may be substituted with R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, L 1 , L 2 , and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R 8 represents a hydrocarbon group having 1 to 12 carbon atoms, R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, R 10 represents a hydrocarbon group having 1 to 8 carbon atoms, R 11 represents a hydrocarbon group having 1 to 24 carbon atoms, R 12 represents a hydrocarbon group having 1 to 24 carbon atoms, R 9 , and R 12 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or SR 58 and R 53 , R 54 , R 55 , and R 58 is defined as above, R 11 The hydrocarbon group represented by is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 The definition of is as above. <34> The ionizable lipid having a biodegradable group is a compound represented by formula (5): <28> from <31> The lipid composition described in any one of the above. [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The substituent A is a hydroxyl group, -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R 60 represents a group represented by G 20 represents -O(CO)- or -(CO)O-, R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 58 and R 59 each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, Substituent B is -N(R 61 )(R 62 ) and R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R 60 represents a hydrocarbon group having 1 to 18 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 30 is -S-(CO)-NR 64 - indicates R 64 L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G 10is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- indicates R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is -(CO)OR 65 , or -O(CO)-R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms. <35> The content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol% based on the total lipids. <28> from <34> The lipid composition according to any one of the preceding claims. <36> the therapeutic agent is a nucleic acid; <28> from <35> The lipid composition described in any one of the above. <37> the therapeutic agent is DNA or RNA; <28> from <36> The lipid composition described in any one of the above. <38> the therapeutic agent is mRNA or siRNA; <28> from <37> The lipid composition described in any one of the above. [Effects of the Invention]

[0013] The methods and compositions of the present invention can achieve excellent delivery efficiency of therapeutic agents to organs other than the liver. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the results of measuring the uptake of lipid compositions into various tissues. [Figure 2] FIG. 2 shows the results of measuring the uptake of lipid compositions into various tissues. [Figure 3] FIG. 3 shows the results of measuring the uptake of lipid compositions into various tissues. [Figure 4] FIG. 4 shows the results of measuring the uptake of lipid compositions into various tissues. [Figure 5] FIG. 5 shows the results of measuring the uptake of lipid compositions into various tissues. [Figure 6] FIG. 6 shows the results of measuring the uptake of lipid compositions into various tissues. [Figure 7] FIG. 7 shows the results of RNA delivery to endothelial cells in vivo using various ionizable lipids. [Figure 8] FIG. 8 shows the results of RNA delivery to endothelial cells in vivo using various ionizable cholesterol ratios (1) HAPC-cholesterol. [Figure 9] FIG. 9 shows the results of RNA delivery to endothelial cells in vivo using DC-cholesterol with various ionizable cholesterol ratios (2). [Figure 10] FIG. 10 shows the results of RNA delivery to endothelial cells in vivo using HAPC-cholesterol with various ionizable cholesterol ratios (3). [Figure 11] FIG. 11 shows the results of serum protein-independent RNA delivery to endothelial cells. [Figure 12] FIG. 12 shows the results of an in vitro LNP-mediated endothelial cell injury assay. [Figure 13] FIG. 13 shows the results of an in vitro blood compatibility analysis using human primary red blood cells. [Figure 14] FIG. 14 shows the results of in vivo hepatic stellate cell delivery. [Figure 15] FIG. 15 shows the results of in vivo RNA delivery to extrahepatic extracellular matrix-producing cells. [Figure 16] FIG. 16 shows the results of in vivo RNA delivery to extrahepatic extracellular matrix-producing cells. [Figure 17] FIG. 17 shows the results of intramuscular administration and delivery to muscle cells and endothelial cells. [Figure 18] FIG. 18 shows the results of intramuscular administration and delivery to muscle cells and endothelial cells. [Figure 19] FIG. 19 shows the results of in vitro delivery to cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0016] The present invention relates to a method for delivering a therapeutic agent to endothelial cells, mesenchymal cells, or cancer cells, comprising administering to a subject a lipid composition comprising the therapeutic agent and lipid nanoparticles, the lipid nanoparticles comprising an ionizable lipid and a compound represented by formula (1) or a salt thereof.

[0017] The present invention further relates to a lipid composition comprising a therapeutic agent and lipid nanoparticles, wherein the lipid nanoparticles comprise a compound represented by formula (1) or a salt thereof and an ionizable lipid having a biodegradable group. In the present invention, it has been found that by replacing cholesterol with a cholesterol analog in hepatocyte-targeted LNPs, the cell type and tissue type targeting can be changed from hepatocytes to endothelial cells, mesenchymal cells, or cancer cells in various organs. The combination of an ionizable lipid and a cholesterol analog, which is a compound of formula (1), is useful for safe and efficient systemic and local delivery of therapeutic agents to endothelial cells, mesenchymal cells, or cancer cells.

[0018] Examples of mesenchymal cells include bone cells (osteoblasts), chondrocytes (chondrocytes), muscle cells (myocytes, skeletal muscle cells, cardiac muscle cells), connective tissue cells (fibroblasts, myofibroblasts, astrocytes), bone marrow stromal cells, tenocytes, and adipocytes (adipocytes). Fibroblasts are ubiquitous mesenchymal cells that are normally present in the matrix of many tissues. Myofibroblasts are an activated form of fibroblasts that have contractile capacity due to the presence of cytoskeletal proteins (especially α-smooth muscle actin) that are normally present in smooth muscle cells. Stellate cells are retinoid-storing fibroblasts present in various organs, including the liver, pancreas, lungs, kidneys, intestine, spleen, adrenal glands, vas deferens, and vocal cords.

[0019] <Compound represented by formula (1), formula (2) or formula (3)> In the present invention, a compound represented by the following formula (1) or a salt thereof is used. [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, X represents a basic functional group.

[0020] G1 is preferably -C(O)- or -OC(O)-. L Y The alkylene group having 1 to 14 carbon atoms represented by may be linear or branched, chain or cyclic, and preferably has 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 2 to 10 carbon atoms. Specific examples include a methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, decamethylene group, undecamethylene group, and dodecamethylene group.

[0021] L Y Examples of the alkylene group having 1 to 14 carbon atoms in the substituted alkylene group having 1 to 14 carbon atoms represented by the formula (I) are as described above. Examples of the substituent on the alkylene group having 1 to 14 carbon atoms include a hydrocarbon group having 1 to 4 carbon atoms, an aminoalkyl group having 1 to 4 carbon atoms, and an amino group. L Y Examples of heteroalkylene groups having 1 to 14 carbon atoms represented by the formula (I) include groups in which one or more heteroatoms selected from an oxygen atom, a nitrogen atom, or a sulfur atom are linked to an alkylene group having 1 to 14 carbon atoms, or groups in which one or more heteroatoms selected from an oxygen atom, a nitrogen atom, or a sulfur atom are present in the alkyl chain of an alkylene group having 1 to 14 carbon atoms. Examples of heteroalkylene groups include, but are not limited to, -O-, -S-, -NH-, -NR-, -C(O)-, -CN-, -NR-C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, etc. in the alkyl group. In addition, two heteroatoms may be consecutive, such as -SS-.

[0022] L Y In the substituted heteroalkylene group having 1 to 14 carbon atoms represented by the formula (I), examples of the substituent on the alkylene group having 1 to 14 carbon atoms include a hydrocarbon group having 1 to 4 carbon atoms, an aminoalkyl group having 1 to 4 carbon atoms, and an amino group. L Y is preferably a heteroalkylene group having 1 to 14 carbon atoms or a substituted heteroalkylene group having 1 to 14 carbon atoms.

[0023] Examples of the basic functional group represented by X include an amino group, a substituted amino group, a guanidino group, a 5- or 6-membered heterocyclic alkyl group, and a 5- or 6-membered heterocyclic aryl group. Examples of the substituent in the substituted amino group include an optionally substituted alkyl group having 1 to 4 carbon atoms, or -C(NH)=NH. Heterocyclic alkyl groups include cyclic functional groups containing carbon atoms and one or more heteroatoms selected from oxygen, nitrogen, and sulfur atoms. Examples include, but are not limited to, pyrrolidine, pyrazolidine, imidazolidine, pyrroline, pyrazoline, imizoline, piperidine, piperazine, and morpholine groups. Heterocyclic aryl groups include aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen, or sulfur atoms and carbon atoms. Examples include, but are not limited to, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, pyridine, pyrazine, pyrimidine, pyridazine, triazine, tetrazine, and pentazine groups.

[0024] The compound represented by formula (1) may preferably be a compound represented by formula (2). [ka] In the formula, G1 represents —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L Y represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, R 2 , R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH)=NH, and R 2 , R 3 and R 4 One of them may not be present.

[0025] G1 and L in Equation (2) Y The definition of is the same as that in formula (1). R 2 , R 3 or R 4Examples of the hydrocarbon group having 1 to 4 carbon atoms in the hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a tert-butyl group. R 2 , R 3 and R 4 is preferably a hydrogen atom, a methyl group, a 1-hydroxyethyl group, or -C(NH2)=NH2.

[0026] The compound represented by formula (2) may preferably be a compound represented by formula (3). [ka] During the ceremony, G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-; L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms; R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms; G2 represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group; R 2 , R 3 and R 4 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH)=NH; R 2 , R 3 and R 4 One of them may not be present.

[0027] G1 and R in equation (3) 2 , R 3 and R 4 The definition of is the same as that in formula (1) and formula (2). Examples of the alkylene group having 1 to 6 carbon atoms represented by L1 include methylene, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, etc. L1 is preferably a single bond or hexane-1,6-diyl.

[0028] The compound represented by formula (3) is preferably a compound represented by formula (3A). [ka] During the ceremony, G1 represents -C(O)-, -OC(O)-, -O(CO)O- or -C(O)O-; L1 represents a single bond or an alkylene group having 1 to 6 carbon atoms; R1 represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms; G2 represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—; L2 represents an alkylene group having 1 to 6 carbon atoms which may have an amino group; R 2 , R 3 each independently represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or -C(NH2)=NH2.

[0029] Examples of the hydrocarbon group having 1 to 4 carbon atoms represented by R1 include a methyl group, an ethyl group, a propyl group, and a butyl group. Examples of the aminoalkyl group having 1 to 4 carbon atoms represented by R1 include an aminomethyl group, an aminoethyl group, an aminopropyl group, and an aminobutyl group. R1 is preferably a hydrogen atom or an aminoalkyl group having 1 to 4 carbon atoms. G2 preferably represents a single bond or -C(O).

[0030] Examples of the alkylene group having 1 to 6 carbon atoms in the alkylene group having 1 to 6 carbon atoms which may have an amino group and is represented by L2 include methylene, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, and hexane-1,6-diyl. L2 is preferably ethylene, propane-1,3-diyl, or butane-1,4-diyl having an amino group.

[0031] Salts of the compound represented by formula (1), formula (2), formula (3), or formula (3A) include salts at basic groups, such as 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. Specific examples of the compound represented by formula (1) or a salt thereof include the following compounds.

[0032] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0033] Among the above, the following are preferred examples: [ka] [ka] [ka] [ka] [ka]

[0034] Particularly preferred examples are as follows: [ka] [ka] [ka] [ka]

[0035] The compound represented by the above formula (1) can be obtained as a commercial product from, for example, Cayman Chemical Company and Avanti Polar Lipids Company. The content of the compound represented by formula (1) is preferably 5 to 80 mol%, more preferably 10 to 80 mol%, more preferably 10 mol% to 60 mol%, and even more preferably 30 mol% to 50 mol%, based on the total lipids.

[0036] <Ionizable lipids> In the present invention, an ionizable lipid is used. 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 biodegradable group include groups represented by -O(CO)O-, -O(CO)-, and -(CO)O-.

[0037] <<Lipid represented by formula (4) or a salt thereof>> As the ionizable lipid, for example, a lipid represented by the following formula (4) or a salt thereof may be used. [ka] During the ceremony, X is -NR 1 - or -O-, R 1 is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 -, and R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 22 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 2 and R 3 are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R 31 -L 2 -R 32 -, and R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates R 32represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and R 8 any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR 45 R 46 an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 The amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c, and d each independently represent an integer of 0 to 3, provided that a+b is 1 or more, and c+d is 1 or more.

[0038] R 1 and R 2 and R 3The hydrocarbon group having 3 to 24 carbon atoms in the formula (I) is preferably an alkyl group, an alkenyl group, or an alkynyl group, and more preferably an alkyl group or an alkenyl group. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and more preferably an alkyl group having 3 to 24 carbon atoms. Specific examples include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, nonadecyl, and icosyl. The alkenyl groups having 6 to 24 carbon atoms and the alkenyl groups having 3 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. The alkenyl groups having 6 to 24 carbon atoms are preferably alkenyl groups having 6 to 20 carbon atoms, and the alkenyl groups having 3 to 24 carbon atoms are more preferably alkenyl groups having 6 to 20 carbon atoms. Specifically, 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)-heptadeca-8-enyl group), a heptadecadienyl group (preferably a (8Z, (11Z)-heptadeca-8,11-dienyl group), octadecenyl group (preferably, (Z)-octadec-9-enyl group), octadecadienyl group (preferably, (9Z,12Z)-octadeca-9,12-dienyl group), nonadecenyl group, icosenyl group (preferably, (Z)-icos-11-enyl group), icosadienyl group (preferably, (11,14)-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. Specific examples 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, and an octadecynyl group. Each of the above alkenyl groups preferably has one or two double bonds, and each of the alkynyl groups preferably has one or two triple bonds.

[0039] R 21 and R 31The hydrocarbon group having 1 to 24 carbon atoms is 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, and may be linear 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, Examples include 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, and a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group. The alkenyl group having 10 to 24 carbon atoms may be linear or branched, open-chain or cyclic.Specifically, 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)-pentadecen-8-enyl group), a hexadecenyl group (preferably, a (Z)-hexadec-9 ... Examples of the alkynyl group include a dienyl group, a heptadecenyl group (preferably a (Z)-heptadecen-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecen-9-enyl group), and an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group). The alkynyl group having 10 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. Specific examples include a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group. Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds.

[0040] R 22 and R 32 Regarding the above, the divalent linking group and hydrocarbon linking group having 1 to 18 carbon atoms is 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, and may be chain-like or cyclic. It preferably has 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 2 to 10 carbon atoms. Specific examples include a methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, decamethylene group, undecamethylene group, and dodecamethylene group. The alkenylene group having 2 to 18 carbon atoms may be linear or branched, and may be chain-like or cyclic. It preferably has 1 to 12 carbon atoms, and more preferably 2 to 10 carbon atoms.

[0041] L1 The preferred range of is -O(CO)O-, -O(CO)-, or -(CO)O-, and -O(CO)- or -(CO)O- is more preferred. L 2 The preferred range of is -O(CO)O-, -O(CO)-, or -(CO)O-, and -O(CO)- or -(CO)O- is more preferred.

[0042] R 4 , R 6 , R 9 , R 10 , R 11 , and R 12 The alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 1 to 12. Specific examples include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl groups. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: -O(CO)-R 42 OR-(CO)OR 43 A group represented by the following formula is more preferred.

[0043] R 5 , R 7 , and R 8The alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms may be linear or branched, chain-like or cyclic. The number of carbon atoms is preferably 1 to 12, and more preferably 1 to 8. Specific examples 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, and a dodecyl group. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: -O(CO)-R 42 , -(CO)OR 43 A group represented by the following formula is more preferred.

[0044] 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, and a 6-membered ring is preferred, with a piperidine ring and a morpholine ring being more preferred.

[0045] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 Regarding the above, when the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a substituted or unsubstituted aryl group, the aryl group preferably has 6 to 22 carbon atoms, more preferably 6 to 18 carbon atoms, and even more preferably 6 to 10 carbon atoms. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group. Examples of the substituent on the aryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 The amino group represented by -O(CO)OR41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specific examples of the substituted aryl group include a hydroxyphenyl group and a carboxyphenyl group.

[0046] R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 In the case where the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a substituted or unsubstituted heteroaryl group, the heteroaryl group preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Specific examples include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, and an oxazolyl group. Examples of the substituent on the heteroaryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR 45 R 46 The amino group represented by -O(CO)OR 41 , -O(CO)-R 42 , -(CO)OR 43 , or -OR 44 A group represented by the formula: is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specific examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, and a pyridonyl group.

[0047] R 41 , R 42 , R 43 , R 44 , R 45 and R 46The hydrocarbon group having 1 to 18 carbon atoms in the above formula is preferably 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, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, open-chain, or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specifically, 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)-trideca-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadeca-8-enyl group), Examples of the alkynyl group include a hexadecenyl group (preferably a (Z)-hexadecan-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadecan-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadecan-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecan-9-enyl group), and an octadecadienyl group (preferably a (9Z,12Z)-octadecan-9,12-dienyl group). The alkynyl group having 2 to 18 carbon atoms may be linear or branched, open-chain or cyclic. The alkynyl group preferably has 3 to 18 carbon atoms, and more preferably has 5 to 18 carbon atoms.Specific examples include a 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, and octadecynyl group.

[0048] X is -NR 1 - When indicating R 1 is a hydrocarbon group having 6 to 24 carbon atoms, or R 21 -L 1 -R 22 In this case, R 2 and R 3 is a hydrogen atom; 2 and R 3 The other is a hydrocarbon group having 6 to 24 carbon atoms, or R 31 -L 2 -R 32 A group represented by - is preferred. When X represents -O-, R 2 and R 3 are each independently a hydrocarbon group having 6 to 24 carbon atoms, or R 31 -L 2 -R 32 A group represented by - is preferred.

[0049] R 4 , R 6 , R 9 , R 10 , R 11 , and R 12 is preferably a hydrogen atom. R 5 represents a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, -O(CO)-R 42 OR-(CO)OR 43 Preferably, R is an alkyl group having 1 to 18 carbon atoms which may be substituted with 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. When R is an alkyl group, 4 , R 6 , R 10 and R 12and may be linked together to form a ring which may contain an O atom. Among these, an alkyl group having 1 to 18 carbon atoms, -O(CO)-R 42 OR-(CO)OR 43 The alkyl group may be substituted with an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 12 carbon atoms, or an alkyl group having 1 to 8 carbon atoms, which may be substituted with a hydroxyl group. 42 OR-(CO)OR 43 It is more preferably an alkyl group having 1 to 18 carbon atoms which may be substituted with.

[0050] R 7 and R 8 are each independently a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, or -O(CO)-R 42 OR-(CO)OR 43 an alkyl group having 1 to 18 carbon atoms which may be substituted with 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, or R 7 and R 8 are preferably linked to each other to form a 4- to 7-membered ring which may contain an O atom. R 5 and R 7 or R 8 are not linked to each other and do not form a ring. a+b is preferably 1 or 2, and more preferably 1. c+d is preferably 1 or 2, and more preferably 1.

[0051] In a preferred embodiment, the lipid represented by formula (4) is a lipid represented by the following formula (21): [ka] During the ceremony, R 2 and R 3 are each independently a hydrocarbon group having 3 to 24 carbon atoms and containing one or more unsaturated bonds, R2 and R 3 are each independently R 31 -L 2 -R 32 -, or R 2 and R 3 One of them is R 31 -L 2 -R 32 -, and the other is a hydrocarbon group having 3 to 24 carbon atoms, R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, L 2 is -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or [ka] indicates, R 32 represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R 5 is -O(CO)-R 42 OR-(CO)OR 43 represents an alkyl group having 1 to 18 carbon atoms which may be substituted by R 42 , and R 43 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, R 7 and R 8 each independently represents an alkyl group having 1 to 4 carbon atoms, e indicates 2 or 3.

[0052] In formula (21), preferably, R 2 and R 3 One of them is R 31 -L 2 -R 32 In formula (21), preferably, L 2 represents -O(CO)- or -(CO)O-. The lipid represented by formula (4) may form a salt.

[0053] Salts of basic groups 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 methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Salts of acidic groups 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. Of the above salts, preferred salts include pharmacologically acceptable salts. The lipid represented by formula (4) and a method for producing the same are described in WO2019 / 235635 and WO2021 / 095876.

[0054] <<Lipid represented by formula (1) or a salt thereof>> As another example, the ionizable lipid may be a lipid represented by the following formula (1) or a salt thereof: [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , and -OR 56 and optionally substituted with one or more substituents selected from R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R 8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, R 7 -R 10 -L 2 -R 11 -L 3 -R 12 indicates, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R 57 -OH, COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , -OR 66 Shows. R 61 and R 62 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 63 ,R 64 ,R 65 , and R 66 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 63 ,R 64 ,R 65 , and R 66 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 68 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 61 R 62 , -OC(O)OR 63 , -C(O)OR 64 , -OC(O)-R 65 , -OR 66 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 67 may be substituted with R 68 represents a hydrocarbon group having 1 to 12 carbon atoms, L 1 , L 2 , and L 3 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R 8 represents a hydrocarbon group having 1 to 12 carbon atoms, R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, R 10 represents a hydrocarbon group having 1 to 8 carbon atoms, R 11 represents a hydrocarbon group having 1 to 24 carbon atoms, R 12 represents a hydrocarbon group having 1 to 24 carbon atoms, R 9 , and R 12 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 and R 53 , R 54 , R 55 , and R 58 is defined as above, R 11 The hydrocarbon group represented by is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 The definition of is as above.

[0055] 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, the hydrocarbon group having 2 to 8 carbon atoms, and the hydrocarbon group having 1 to 8 carbon atoms are preferably an alkyl group, an alkenyl group, or an alkynyl group, respectively.

[0056] The alkyl group may be linear or branched, and may be linear or cyclic. Specific 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, and a 2-butyloctyl group. , 1-pentylhexyl group, 2-pentylheptyl group, 3-pentyloctyl group, 1-hexylheptyl group, 1-hexylnonyl group, 2-hexyloctyl group, 2-hexyldecyl group, 3-hexylnonyl group, 1-heptyloctyl group, 2-heptylnonyl group, 2-heptylundecyl group, 3-heptyldecyl group, 1-octylnonyl group, 2-octyldecyl group, 2-octyldodecyl group, 3-octylundecyl group, 2-nonylundecyl group, 3-nonyldodecyl group, 2-decyldodecyl group, 2-decyltetradecyl group, 3-decyltridecyl group, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0057] The alkenyl group may be linear or branched, linear or cyclic. Specifically, it includes 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)-trideca-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), and a pentadecenyl group (preferably, a (Z)-pentadeca-8-enyl group). , a hexadecenyl group (preferably a (Z)-hexadecan-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadecan-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadecan-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecan-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadecan-9,12-dienyl group), and the like.

[0058] The alkynyl group may be linear or branched, open-chain or cyclic, and specific 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, and an octadecynyl group. Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds.

[0059] -(C1-C12 hydrocarbon group)-R 67 The hydrocarbon group having 1 to 12 carbon atoms is 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, and may be linear or cyclic. Specific examples 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, and an undecamethylene group. The aryl group preferably has 6 to 20 carbon atoms, more preferably 6 to 18, and even more preferably 6 to 10. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group.

[0060] R 1 and R 2 are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 1 , R 2 and R 3 The hydrocarbon group represented by may preferably be substituted with —OH. L 1 , and L 3 are each independently preferably -C(O)O- or -OC(O)-. L 2 preferably represents —OC(O)O—, —C(O)O—, or —OC(O)—. R 8 represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms. R 9 represents a hydrocarbon group preferably having 1 to 20 carbon atoms, and more preferably a hydrocarbon group having 1 to 16 carbon atoms. R 11 represents a hydrocarbon group preferably having 1 to 16 carbon atoms, and more preferably a hydrocarbon group having 1 to 9 carbon atoms. R 12 represents a hydrocarbon group preferably having 1 to 20 carbon atoms, and more preferably a hydrocarbon group having 1 to 16 carbon atoms.

[0061] R 9 , and R 12 The hydrocarbon group represented by is preferably an aryl group or -SR 58 where R 58 represents a hydrocarbon group preferably having 1 to 8 carbon atoms. R 11 The hydrocarbon group represented by is preferably —C(O)OR 55 , or -OC(O)-R 56 where R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 16 carbon atoms; R 55 , and R 56 The hydrocarbon group represented by is preferably an aryl group having 6 to 20 carbon atoms or -SR 58 and R 58 The definition of is as above.

[0062] The compound represented by formula (1) is preferably, as a first example, a compound represented by the following formula (1-1). [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -OR 56 may be substituted with R 4 represents a hydrocarbon group having 1 to 8 carbon atoms, R 5 and R 6 are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R8 -L 1 -R 9 where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms, L 1 represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-; R 8 represents a hydrocarbon group having 1 to 12 carbon atoms, R 9 represents a hydrocarbon group having 1 to 24 carbon atoms, and R 9 The hydrocarbon group represented by is an aryl group, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 may be substituted with R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R 53 ,R 54 ,R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 may be substituted with The above aryl group having 6 to 20 carbon atoms includes -OH, -COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 may be substituted with R 58 represents a hydrocarbon group having 1 to 12 carbon atoms, R 57 -OH, COOH, -NR 51 R52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 Shows. R 13 represents a hydrocarbon group having 1 to 8 carbon atoms, R 14 -R 15 -L 5 -R 16 indicates R 15 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 5 indicates -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, and R 16 represents a hydrocarbon group having 1 to 24 carbon atoms, R 15 The hydrocarbon group having 1 to 24 carbon atoms is -OC(O)OR 53 , -C(O)OR 54 , or -OC(O)-R 55 and R 53 , R 54 , and R 55 is defined as above, R 16 The hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 and R 53 , R 54 , R 55 , and R 58 The definition of is as above.

[0063] In formula (1-1), R 1 and R 2 are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 1 , R 2 and R 3 The hydrocarbon group represented by may preferably be substituted with —OH. L 1 preferably represents —C(O)O— or —OC(O)—. R 8 represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms. R 9 preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and R 9 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, or -SR 58 may be substituted with R 14 is preferably -R 15 -L 5 -R 16 indicates R 15 represents a hydrocarbon group having 1 to 18 carbon atoms, and L 5 indicates -OC(O)O-, and R 16 represents a hydrocarbon group having 1 to 18 carbon atoms. R 15 The hydrocarbon group having 1 to 18 carbon atoms represented by is preferably —C(O)OR 55 , or -OC(O)-R 56 may be substituted with R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 16 carbon atoms; R 55 , and R 56 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -SR 58 and R 58 The definition of is as above. R 16 The hydrocarbon group having 1 to 18 carbon atoms represented by is preferably an aryl group or -SR 58 and R 58 The definition of is as above.

[0064] A second example of the compound represented by formula (1) is preferably a compound represented by the following formula (1-2). [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -OR 56 may be substituted with R 4 and R 8 each independently represents a hydrocarbon having 1 to 8 carbon atoms, R 21 and R 22 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, R 23 and R 24 each independently represents a hydrocarbon group having 1 to 12 carbon atoms, R 25 and R 26 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, L 21 and L 22 each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-; R 25 and R 26 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -SR 58 may be substituted with R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 , R 54 , R55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The above aryl group having 6 to 20 carbon atoms is OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 and optionally substituted by R 57 -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 Shows. R 58 represents a hydrocarbon group having 1 to 12 carbon atoms. In formula (1-2), R 1 and R 2 R each independently represents a hydrocarbon group preferably having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2 The hydrocarbon group represented by may be substituted with -OH, but is more preferably a hydrocarbon group without any substituents. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 21 and R 22 are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably a hydrocarbon group having 1 to 6 carbon atoms. R 23 and R 24 are each independently preferably a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms. R 25and R 26 are each independently preferably a hydrocarbon group having 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably a hydrocarbon group having 1 to 12 carbon atoms. L 21 and L 22 are each independently preferably -C(O)O- or -OC(O)-.

[0065] A third example of the compound represented by formula (1) is preferably a compound represented by the following formula (1-3). [ka] During the ceremony, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R 3 represents a hydrocarbon group having 2 to 8 carbon atoms, and R 1 , R 2 and R 3 The hydrocarbon group represented by is -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or -OR 56 may be substituted with R 4 and R 8 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 31 , R 32 , R 33 , and R 34 each independently represents a hydrocarbon group having 1 to 12 carbon atoms, R 35 , R 36 , R 37 , and R 38 each independently represents a hydrocarbon group having 1 to 24 carbon atoms, L 31 , L 32 , L 33 , and L 34each independently represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-; R 35 , R 36 , R 37 , and R 38 The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , or SR 58 may be substituted with R 51 and R 52 each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R 53 , R 54 , R 55 , and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The above aryl group having 6 to 20 carbon atoms is OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 or -(a hydrocarbon group having 1 to 12 carbon atoms)-R 57 and optionally substituted by R 57 -OH, COOH, -NR 51 R 52 , -OC(O)OR 53 , -C(O)OR 54 , -OC(O)-R 55 , -OR 56 Shows. R 58 represents a hydrocarbon group having 1 to 12 carbon atoms. In formula (1-3), R 1 and R 2 R each independently represents a hydrocarbon group preferably having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. 1 and R 2The hydrocarbon group represented by may be substituted with -OH, but is more preferably a hydrocarbon group without any substituents. R 3 represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. R 31 , R 32 , R 33 , and R 34 are each independently preferably a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. R 35 , R 36 , R 37 , and R 38 R each independently represents a hydrocarbon group having preferably 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably a hydrocarbon group having 1 to 12 carbon atoms. 35 , R 36 , R 37 , and R 38 The hydrocarbon group represented by is preferably an aryl group having 6 to 20 carbon atoms, or SR 58 More preferably, it is substituted with -SR 58 may be substituted with. R 35 , R 36 , R 37 , and R 38 are each independently particularly preferably -SR 58 or represents a hydrocarbon group having 1 to 12 carbon atoms substituted with L 31 , L 32 , L 33 , and L 34 are each independently preferably -C(O)O- or -OC(O)-. R 58 represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms.

[0066] The compound of the present invention may form a salt. Examples of salts of basic groups 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. Salts of acidic groups 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. Of the above salts, preferred salts include pharmacologically acceptable salts. The lipid represented by formula (1) and a method for producing the same are described in WO2022 / 230964, the entire contents of which are incorporated herein by reference.

[0067] <<Lipid represented by formula (5) or a salt thereof>> As the ionizable lipid, for example, a lipid represented by the following formula (5) or a salt thereof may be used. [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The substituent A is a hydroxyl group, -G 20 -CH(R 55 )(R 56 ), -N(R 58 )(R 59 ), or -G 20 -R60 represents a group represented by G 20 represents -O(CO)- or -(CO)O-, R 55 and R 56 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 58 and R 59 each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, Substituent B is -N(R 61 )(R 62 ) and R 61 and R 62 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R 60 represents a hydrocarbon group having 1 to 18 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 30 is -S-(CO)-NR 64 - indicates R 64 -L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by a represents 0 or 1, L 30 represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G 10 is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R 63 )- indicates R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R 53 , R 54 and R 57each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is -(CO)OR 65 , or -O(CO)-R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms.

[0068] The compound of formula (5) may be a compound of formula (5A). [ka] In the formula, R 51 and R 52 each independently represents a C1 to C21 group optionally having a substituent A; represents a hydrocarbon group of the formula The substituent A is a hydroxyl group or —G 20 -CH(R 55 )(R 56 ) represents a group represented by G 20 represents -O(CO)- or -(CO)O-, R 55 and R 56 each independently represents a hydrocarbon group having 1 to 18 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 10 represents -O(CO)- or -(CO)O-, R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, R 53 , R 54 and R 57each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0069] The compound represented by formula (5) may be a compound represented by formula (5B). [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 10 represents -O(CO)O-, L 20 represents a hydrocarbon group having 1 to 6 carbon atoms, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is —O(CO) —R 65 represents a group represented by R 65 is a hydrocarbon group having 1 to 18 carbon atoms or -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents an alkoxy group having 1 to 10 carbon atoms.

[0070] The compound of formula (5) may be a compound of formula (5C). [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G10 is -N(C(O)R 63 )- indicates R 63 represents a hydrocarbon group having 1 to 18 carbon atoms, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is —(CO)O —R 65 represents a group represented by R 65 -L 40 -CH(R 66 )(R 67 ) represents a group represented by L 40 represents a hydrocarbon group having 1 to 6 carbon atoms, R 66 and R 67 represents a hydrocarbon group having 1 to 10 carbon atoms.

[0071] The compound of formula (5) may be a compound of formula (5D). [ka] In the formula, R 51 and R 52 each independently represents a hydrocarbon group having 1 to 21 carbon atoms, L 10 represents a hydrocarbon group having 1 to 18 carbon atoms, G 30 is -S-(CO)-NR 64 - indicates R 64 L 30 -G 20 -CH(R 55 )(R 56 ) represents a group represented by L 30 represents a hydrocarbon group having 1 to 18 carbon atoms, G 20 represents -(CO)O-, R 55 and R 56each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, G 10 represents -(CO)O-, R 53 , R 54 and R 57 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0072] 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, and 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 linear or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, open-chain, or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specifically, 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)-trideca-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadeca-8-enyl group), Examples of the alkynyl group include a hexadecenyl group (preferably a (Z)-hexadecanyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadecanyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadecanyl-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadecanyl group), and an octadecadienyl group (preferably a (9Z,12Z)-octadecanyl-9,12-dienyl group). The alkynyl group having 2 to 21 carbon atoms may be linear or branched, open-chain or cyclic. The alkynyl group preferably has 3 to 21 carbon atoms, and more preferably has 5 to 21 carbon atoms.Specific examples include a 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, and octadecynyl group. Examples of hydrocarbon groups having 1 to 18 carbon atoms include hydrocarbon groups having 1 to 21 carbon atoms and having 1 to 18 carbon atoms.

[0073] The cyclic hydrocarbon group is preferably 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, or an aryl group having 6 to 10 carbon atoms. 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-like or cyclic. Specific examples 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, and a hexyl group. The alkenyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain-like or cyclic. Specific examples include an allyl group, a prenyl group, a pentenyl group, and a hexenyl group. The alkynyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain-like or cyclic. Specific examples include a propargyl group, a butynyl group, a pentynyl group, and a hexynyl group.

[0074] 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 more 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-like or cyclic. It preferably has 3 to 10 carbon atoms, and more preferably has 5 to 10 carbon atoms. Specific examples 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, and a decyl group. The alkenyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain-like or cyclic. It preferably has 3 to 10 carbon atoms, and more preferably has 5 to 10 carbon atoms. Specific examples include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably (Z)-2-nonenyl or (E)-2-nonenyl), and decenyl. The alkynyl group having 2 to 10 carbon atoms may be linear or branched, and may be linear or cyclic. It preferably has 3 to 10 carbon atoms, and more preferably has 5 to 10 carbon atoms. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl.

[0075] The compound represented by formula (5) may form a salt. Salts of basic groups 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 methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Of the above salts, preferred salts include pharmacologically acceptable salts.

[0076] <<Examples of ionizable lipids>> Specific examples of ionizable lipids include the following lipids: Note that cKK-E12 (MD-1) and C12-200 are compounds not included in the above formula (5).

[0077] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0078] In the lipid composition of the present invention, the content of the ionizable lipid or a salt thereof is preferably 10 mol% to 80 mol% relative to the total lipid amount, more preferably 20 mol% to 80 mol%, even more preferably 30 mol% to 70 mol%, and particularly preferably 40 mol% to 60 mol%.

[0079] <Neutral lipids> The lipid composition of the present invention may comprise a neutral lipid. The neutral lipid is preferably a zwitterionic lipid. The zwitterionic lipid is preferably a phospholipid, specifically, phosphatidylcholine, phosphatidylethanolamine, or sphingomyelin. The phospholipid is preferably a phospholipid having a choline group, such as phosphatidylcholine. The zwitterionic lipid may be a single lipid or a combination of multiple different neutral lipids.

[0080] Phosphatidylcholines include, but are not limited to, 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), etc. Among these, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), and dilauroylphosphatidylcholine (DLPC) are preferred, with distearoylphosphatidylcholine (DSPC) being particularly preferred. DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine [ka]

[0081] Examples of phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, and diphytanylphosphatidylethanolamine. Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin and milk-derived sphingomyelin.

[0082] In the lipid composition of the present invention, the content of neutral lipids is preferably 1 to 30 mol %, more preferably 5 to 25 mol %, and even more preferably 7 to 23 mol %, based on the total lipids.

[0083] <Lipids with non-ionic hydrophilic polymers> The lipid composition of the present invention may contain a lipid having a nonionic hydrophilic polymer. The lipid having a nonionic hydrophilic polymer preferably contains an acyl group, and the carbon chain length of the acyl group is preferably 8 to 26. Examples of nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers having two or more of these polymers as constituent units.

[0084] Of these nonionic hydrophilic polymers, nonionic polyethers, nonionic polyesters, nonionic polyamino acids, or nonionic synthetic polypeptides are preferred, nonionic polyethers or nonionic polyesters are more preferred, nonionic polyethers or nonionic monoalkoxy polyethers are even more preferred, and polyethylene glycol (polyethylene glycol will also be referred to as PEG hereinafter) is particularly preferred.

[0085] The lipid having a nonionic hydrophilic polymer is not particularly limited, but includes PEG-modified phosphoethanolamine, diacylglycerol PEG derivative, monoacylglycerol PEG derivative, dialkylglycerol PEG derivative, cholesterol PEG derivative, ceramide PEG derivative, etc. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred. The alkyl chain of the lipid having a nonionic hydrophilic polymer preferably has 8 to 26 carbon atoms, more preferably 10 to 22 carbon atoms.

[0086] The weight average molecular weight of the nonionic hydrophilic polymer is preferably from 100 to 10,000, more preferably from 500 to 5,000, and even more preferably from 750 to 3,000. The non-ionic hydrophilic polymer may be branched and may have a substituent such as a hydroxymethyl group.

[0087] Preferred examples of lipids having a nonionic hydrophilic polymer include the following lipids: DMG-mPEG2000: 1,2-dimyristoyl-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 [ka]

[0088] In the lipid composition of the present invention, the content of lipids having nonionic hydrophilic polymers is preferably 0.1 to 10 mol % relative to the total lipids, more preferably 0.3 to 8 mol %, even more preferably 0.5 to 5 mol %, and particularly preferably 1 to 3 mol %.

[0089] <Nucleic acid> The lipid composition of the present invention contains a therapeutic agent. The therapeutic agent is preferably a nucleic acid. The nucleic acid may be either DNA or RNA, and examples thereof include a plasmid, a single-stranded DNA, a double-stranded DNA, a small interfering RNA (siRNA), a microRNA (miRNA), an mRNA, an antisense oligonucleotide (also known as an ASO), a ribozyme, an aptamer, a decoy nucleic acid, and a gRNA used in genome editing. The lipid composition may also contain a modified nucleic acid.

[0090] In the lipid composition of the present invention, the weight ratio of lipid to nucleic acid is preferably 5-100, more preferably 5-70, even more preferably 5-40, and particularly preferably 5-35.

[0091] <Method of producing the composition> A method for producing the lipid composition of the present invention will now be described. The method for producing the lipid composition is not limited, but the lipid composition can be produced by dissolving all or some of the oil-soluble components of the lipid composition in an organic solvent or the like to form an oil phase, dissolving the water-soluble components in water to form an aqueous phase, and mixing the oil and aqueous phases. A micromixer may be used for mixing, or the lipid composition may be emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure injection emulsifier, or the like.

[0092] Alternatively, a lipid-containing solution can be subjected to vacuum drying using an evaporator or the like, or spray drying using a spray dryer or the like to prepare a dried mixture containing lipids, and this mixture can then be added to an aqueous solvent and further emulsified using the aforementioned emulsifier or the like to produce the composition.

[0093] An example of a method for producing a lipid composition is (a) a step of dissolving a lipid component in an organic solvent to obtain an oil phase; a step (b) of mixing the oil phase obtained in the step (a) with an aqueous phase containing nucleic acids; (c) diluting the mixture containing the oil phase and the aqueous phase obtained in step (b) to obtain a dispersion of a lipid composition containing nucleic acids; Step (d) of removing the organic solvent from the dispersion of the lipid composition obtained in step (c); The method includes the steps of:

[0094] In step (a), lipid components are dissolved in an organic solvent (an alcohol such as ethanol, or an ester). 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.

[0095] In step (b), the aqueous phase can be obtained by dissolving nucleic acids (e.g., siRNA, antisense oligonucleotides, mRNA, etc.) in water or a buffer solution. Components such as antioxidants can be added as needed. The mixing ratio (volume ratio) of the aqueous phase to the oil phase is preferably 5:1 to 1:1, more preferably 4:1 to 2:1. In step (b), the mixture can be diluted with water or a buffer solution (such as phosphate buffered saline (PBS)).

[0096] In step (c), the method for removing the organic solvent from the dispersion of the lipid composition is not particularly limited, and a general method can be used. For example, the organic solvent can be removed by dialysis using phosphate-buffered saline. The lipid composition may be subjected to sizing as needed. The sizing method is not particularly limited, but the particle size can be reduced using an extruder or the like.

[0097] <Composition> The lipid composition of the present invention may be a lipid particle. The lipid particle means a particle composed of lipids, and includes compositions having any structure selected from lipid aggregates (e.g., lipid nanoparticles), micelles, and liposomes, but the structure of the lipid particle is not limited thereto as long as it is a composition containing lipids. The morphology of the lipid composition can be confirmed by electron microscopy or structural analysis using X-rays. For example, cryo-transmission electron microscopy (CryoTEM) can be used to determine whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer, like liposomes, or whether the particles have a core with high electron density and are packed with lipids and other components. Small-angle X-ray scattering (SAXS) measurements can also be used to determine whether the lipid particles have a lipid bilayer structure (lamellar structure).

[0098] When the lipid composition of the present invention is in the form of particles, the particle size of the particles is not particularly limited, but is preferably 10 to 1000 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, dynamic light scattering, laser diffraction, etc.). When the lipid composition of the present invention is in the form of particles, the zeta potential of the particles is not particularly limited, but is preferably −20 to +20 mV, more preferably −10 to 10 mV. The zeta potential in the present invention is a value measured by electrophoresis after diluting the lipid composition in a phosphate buffer solution, but is not limited thereto. 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 of the present invention is a value measured by TNS assay, but is not limited to this.

[0099] <Use of lipid composition> As an example of the use of the lipid composition of the present invention, a therapeutic agent (e.g., nucleic acid) can be introduced into a cell by introducing a lipid composition containing nucleic acid into the cell. That is, the lipid composition of the present invention can be used as a composition for introducing nucleic acid into a cell. The lipid composition of the present invention can also be used as a pharmaceutical composition for in vivo nucleic acid delivery. In the present invention, therapeutic agents can be delivered to endothelial cells, mesenchymal cells, or cancer cells in particular. Therefore, therapeutic agents can be delivered to organs other than the liver. Organs other than the liver include the spleen, kidneys, lungs, heart, muscle, and brain. Organs other than the liver to which the lipid composition of the present invention delivers therapeutic agents are preferably the spleen, kidneys, lungs, heart, muscle, and brain, more preferably the kidneys, lungs, heart, muscle, and brain, and even more preferably the lungs and heart.

[0100] In addition, when the lipid composition of the present invention contains a nucleic acid having pharmaceutical uses, the lipid composition can be administered to a living body as a nucleic acid drug.When the lipid composition of the present invention is used as a nucleic acid drug, the lipid composition of the present invention can be administered to a living body alone or mixed with a pharmaceutically acceptable carrier (e.g., an administration medium such as physiological saline or phosphate buffer).That is, the lipid composition of the present invention may further contain a pharmaceutically acceptable carrier. The concentration of the lipid composition in the mixture with a pharmaceutically acceptable carrier is not particularly limited and can generally be 0.05% by mass to 90% by mass. In addition, other pharmaceutically acceptable additives, such as pH adjusting buffers and osmotic pressure adjusting agents, may be added to the nucleic acid drug containing the lipid composition of the present invention.

[0101] The administration route of the lipid composition of the present invention is not particularly limited, and can be administered by any method. Examples of administration methods include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intradermal 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, etc.). Parenteral administration is preferred, and intravenous injection, subcutaneous injection, intradermal injection, or intramuscular injection is preferred, with intravenous injection or intramuscular injection being particularly preferred. Nucleic acid delivery can also be achieved by local administration in vivo. The lipid composition of the present invention can also be administered by direct injection to the diseased site.

[0102] The dosage form of the lipid composition of the present invention is not particularly limited, but when administered orally, the lipid composition of the present invention can be combined with an appropriate excipient and used in the form of tablets, troches, capsules, pills, suspensions, syrups, etc. Furthermore, formulations suitable for parenteral administration can contain additives such as antioxidants, buffers, bacteriostatic agents, and isotonic sterile injections, suspending agents, solubilizers, thickeners, stabilizers, or preservatives, as appropriate.

[0103] <Use of lipid nanoparticles as nucleic acid delivery carriers> The lipid nanoparticles of the present invention are highly useful as nucleic acid delivery carriers because they are capable of retaining therapeutic agents such as nucleic acids at a high encapsulation rate. Using a nucleic acid delivery carrier utilizing the present invention, for example, the resulting composition can be mixed with nucleic acids and transfected in vitro or in vivo to introduce nucleic acids into cells. Furthermore, nucleic acid delivery carriers utilizing the present invention are also useful as nucleic acid delivery carriers for nucleic acid medicines. That is, the lipid nanoparticles of the present invention are useful as compositions for nucleic acid delivery in vitro or in vivo (preferably in vivo). The present invention will now be described with reference to examples, but the present invention is not limited to these examples. [Example]

[0104] Materials and Methods <sirna> The following custom siRNAs were manufactured by Horizon. siRNA against rabbit VE-cadherin (siVEcad, siCdh5) Sense: 5'-mCmCAAAAGAGAGAmCmUGGAmUmUdTsdT-3' Antisense: 5'-AAUCmCAGUCUCUCUUUUGGdTsdT-3' Abbreviation A. Adenosine 3'-phosphate C cytidine-3'-phosphate G Guanosine-3'-phosphate U Uridine-3'-phosphate mA 2'-O-methyladenosine-3'-phosphate mC 2'-O-methylcytidine-3'-phosphate mG 2'-O-methylguanosine-3'-phosphate mU 2'-O-methyluridine-3'-phosphate dT 2'-deoxythymidine-3'-phosphate dTs 2'-deoxythymidine-5'-phosphate-phosphorothioate

[0105] Cadherin 5 (Cdh5), also known as vascular endothelial cadherin (VE-cadherin), is a junctional protein whose expression is restricted to endothelial cells. Quantifying the residual Cdh5 mRNA after siCdh5 administration can be used to assess the efficiency of siCdh5 delivery to endothelial cells.

[0106] <Lipid nanoparticle formulation> All chemicals obtained from commercial sources were stored according to the manufacturer's instructions and used without further purification. Lipid nanoparticles were synthesized using a microfluidic chip device as previously described. Lipid nanoparticles were formed by mixing a lipid-containing ethanol phase with an siRNA-containing aqueous phase and pumping them through microfluidic channels in a PDMS (polydimethylsiloxane) chip.

[0107] Ionizable lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, NOF), cholesterol (Sigma) or the compound represented by formula (1), and 1,2-dimyristoyl-lactoyl-glycero-3-methylpolyoxyethylene (DMG-mPEG2000, NOF) were dissolved in ethanol and mixed at a predetermined molar ratio and total lipid: siRNA weight ratio. The aqueous phase was prepared in 10 mM citrate buffer (pH 3.0, Fisher) containing siRNA. The ethanol and aqueous phases were mixed in a 3:1 ratio in a microfluidic chip device using a syringe pump as previously described. The total flow rate was 1.2 mL / min. Lipid nanoparticles were dialyzed against 1x PBS in 20,000 MWCO cassettes (Invitrogen) overnight at 4°C or room temperature. If necessary, preparations were concentrated using Amicon ultracentrifugal filters (Millipore Sigma), sterilized through 0.22 μm filters (Millipore Sigma), and stored at 4°C.

[0108] <Characteristics of lipid nanoparticles> The nucleic acid encapsulation efficiency of the LNP was calculated using the modified QUANT-IT™ RIBOGREEN® RNA Assay (Invitrogen Corporation, Carlsbad, Calif) according to the method described in Walsh C. et al. Methods Mol Biol. 2014;1141:109-20. Briefly, the sample was diluted to a concentration of approximately 1.5 μg / ml in TE buffer. 50 μl of the diluted sample was transferred to a black U-bottom 96-well plate, and either 50 μl of TE buffer or 50 μl of TE buffer containing 2% Triton X-100 was added to the wells. The plate was incubated at 37 °C for 15 minutes. The RIBOGREEN® reagent was diluted 1:200 in TE buffer, and 100 μl of this solution was added to each well. Fluorescence intensity was measured using a plate reader (Tecan 200 Pro, Tecan) at an excitation wavelength of 485 nm and an emission wavelength of 515 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample, and the percentage of free RNA was determined by dividing the fluorescence intensity of the intact sample (without Triton X-100) by the fluorescence value of the disrupted sample (containing Triton X-100).

[0109] The particle size, polydispersity index (PDI), and zeta potential of the nanoparticle composition were measured using a Zetasizer Nano ZS (Malvern Instruments). The particle size was measured in 1×PBS, and the zeta potential was measured in 0.1×PBS.

[0110] <Measurement of pKa value by TNS assay> The apparent pKa values ​​of LNPs were measured using the TNS assay, following the method described by Heyes J. et al., Journal of Controlled Release 107 (2005) 276-287. Briefly, TNS was prepared as a 100 μM stock solution in DMSO. LNPs were diluted to 25 ng / μL of ionizable lipid in 0.1X PBS. This LNP solution was further diluted 10-fold into buffers ranging from approximately pH 2.5 to 9.0. One TNS stock solution was added to the LNP solutions at different pH levels to a final concentration of 6 μM and mixed thoroughly in a black 384-well plate. Fluorescence intensity was monitored using a Tecan Pro200 plate reader at excitation and emission wavelengths of 320 nm and 465 nm. A sigmoidal plot of fluorescence versus buffer pH was generated using the obtained fluorescence values. The logarithm of the inflection point of this curve represents the apparent pKa of the LNP formulation. pKa values ​​were obtained using Prism Software.

[0111] <Animal experiments> All animal studies were approved by the MIT Institutional Animal Care and Use Committee (CAC) and in accordance with local, state, and federal regulations, where applicable. All experimental procedures were performed with ethical compliance and approval under the guidelines of the Department of Comparative Medicine at Massachusetts Institute of Technology. Female C57BL / 6 mice (6 weeks old) were obtained from the Jackson Laboratory, housed in the MIT animal facility, and allowed to acclimate for at least 3 days before the start of the study.

[0112] For intravenous administration, PBS-diluted siRNA-LNPs were injected into the tail vein using a 29 g, 3 / 10 cc insulin syringe (BD Biosciences) after gently warming the animals using a heat lamp.For intramuscular administration, PBS-diluted siRNA-LNPs were injected into the quadriceps muscle using a 29 g, 3 / 10 cc insulin syringe (BD Biosciences). At 48 - 72 hours after injection, organs or tissues including the heart, liver, spleen, lung, kidney, muscle (quadriceps and diaphragm), brain, and tumor were harvested, immersed in RNAlater solution at 4 °C for 12 - 48 hours, and stored at -20 °C after removal of RNAlater.

[0113] <Quantification of tissue mRNA by qPCR> Total RNA was isolated from tissues using TRIzol. Briefly, tissue punches were placed into deep - well 96 - well plates with 4 mm stainless - steel beads and lysed in 350 μL of TRIzol using a GenoGrinder2010. Next, 300 μL of the lysate was transferred to a new deep - well 96 - well plate. The total RNA was further purified using Direct - zol - 96 MagBead (Zymo Research) according to the manufacturer's protocol.

[0114] Gene expression was analyzed by one - step multiplex qPCR using Luna (registered trademark) Universal Probe One - Step RT - qPCR Kit (NEB) and Taqman probes - VE - cadherin (Mm00486938_m1), GusB (Mm01197698_m1), and B2M (Mm00437762_m1). Samples were amplified using a LightCycler 480 qPCR instrument (Roche). VE - cadherin expression was normalized to B2M or GusB.

[0115] Example 1: DC - cholesterol uptake To investigate the potential of synthetic cholesterol analogs with basic functional groups, we replaced cholesterol in standard hepatocyte-targeting LNPs with DC-cholesterol. DC-cholesterol was developed for nucleic acid delivery liposomes and contains an ionizable tertiary amine group with a pKa of 7.8 at the C3 position of cholesterol (Non-Patent Documents 11 and 12). The combination of an ionizable lipid and DC-cholesterol was tested in a subcutaneous mRNA vaccine to enhance mRNA delivery to dendritic cells in lymph nodes, but it showed no advantage over cholesterol and actually reduced delivery efficiency.

[0116] LNP1 is a standard hepatocyte-targeting LNP formulation containing approximately 50 mol% ionizable lipid, approximately 10 mol% DSPC, approximately 38.5 mol% cholesterol, and approximately 1.5 mol% PEG-DMG. This formulation is used in the U.S. Food and Drug Administration-approved drugs Onpattro® and Spikevax®. LNP2 replaces cholesterol with DC-cholesterol at the same lipid ratio as LNP1.

[0117] [Table 1]

[0118] To evaluate gene silencing in endothelial cells of various organs, we used siRNA against VE-cadherin, a cell adhesion molecule whose expression is restricted to the endothelium. A formulation containing VE-cadherin siRNA was administered intravenously to mice at 0.3 mg / kg, and organs were harvested 48 hours after injection. After isolation and purification of total RNA from the organs, VE-cadherin mRNA was quantified relative to the housekeeping gene B2M. A PBS control was also tested. As shown in Figure 1, replacing cholesterol in conventional LNPs with DC-cholesterol enabled endothelial gene silencing in various organs of mice. VE-cadherin mRNA expression was measured based on ΔΔCt calculations compared to PBS on day 3 post-injection (0.3 mg / kg siVEcad).

[0119] Example 2: Generalization to other ionizable lipids To test the generalizability of the DC-cholesterol LNP formulation, various ionizable lipids were tested. Table 2 summarizes the physicochemical properties of the tested formulations. FL-A, FL-B, and FL-C were developed by Fujifilm.

[0120] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0121] [Table 2]

[0122] Mice were intravenously administered 0.5 mg / kg of a formulation containing VE-cadherin siRNA, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, VE-cadherin mRNA was quantified relative to the housekeeping gene GusB. A PBS control was also tested. The results are shown in Figure 2. Figure 2 demonstrates endothelial gene silencing with various ionizable lipids via substitution with DC-cholesterol. VE-cadherin mRNA expression was measured based on ΔΔCt calculations compared to PBS at 3 days post-injection (0.5 mg / kg siVEcad).

[0123] Example 3: Evaluation of other C3-modified cholesterol derivatives To clarify the structural requirements of cholesterol derivatives, various cholesterol derivatives were formulated into LNPs with a fixed lipid composition of FL-A:DSPC:sterol:DMG-mPEG2000 = 50:10:38.5:1.5.

[0124] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0125] [Table 3]

[0126] Mice were intravenously administered 0.5 mg / kg of a formulation containing VE-cadherin siRNA, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, VE-cadherin mRNA was quantified relative to the housekeeping gene GusB. A PBS control was also tested. The results are shown in Figure 3. As shown in Figure 3, various 3'-modified cholesterol analogs demonstrated endothelial gene silencing in various organs of mice. VE-cadherin mRNA expression was measured based on ΔΔCt calculations compared to PBS on day 3 post-injection (0.5 mg / kg siVEcad).

[0127] Example 4: DC-cholesterol ratio (FL-B) To optimize the DC-cholesterol ratio, LNP formulations with different DC-cholesterol ratios were prepared and tested in vivo. In this example, FL-A was used as the ionizable lipid, and the DSPC and DMG-mPEG-2000 ratios were fixed at 10% and 1.5%, respectively. Table 4 provides a summary of formulation contents and properties of lipid components useful in the nanoparticle compositions of the present invention.

[0128] [Table 4]

[0129] Mice were intravenously administered 0.5 mg / kg of a formulation containing VE-cadherin siRNA, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, VE-cadherin mRNA was quantified relative to the housekeeping gene B2M. A PBS control was also tested. The results are shown in Figure 4. As shown in Figure 4, LNPs with different DC-cholesterol ratios demonstrated strong endothelial gene silencing in the lungs and hearts of mice. VE-cadherin mRNA expression was measured based on ΔΔCt calculations compared to PBS on day 3 post-injection (0.5 mg / kg siVEcad).

[0130] Example 5: DC-Cholesterol Ratio 2 (FL-A) To optimize the DC-cholesterol ratio, LNP formulations with different DC-cholesterol ratios were prepared and tested in vivo. In this example, FL-A was used as the ionizable lipid, and the DSPC and DMG-mPEG-2000 ratios were fixed at 10% and 1.5%, respectively. Table 5 provides a summary of formulation contents and properties of lipid components useful in the nanoparticle compositions of the present invention.

[0131] [Table 5]

[0132] Mice were intravenously administered a formulation containing 0.5 mg / kg of siRNA against Cdh5, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene GusB. A PBS control was also tested.

[0133] The results are shown in Figure 5. As shown in Figure 5, LNPs with different DC-cholesterol ratios exhibited strong endothelial gene silencing in the lungs and hearts of mice. Cdh5 mRNA expression was measured based on ΔΔCt calculations compared to PBS on day 3 post-injection (0.5 mg / kg siVEcad).

[0134] Example 6: Generalization to other ionizable lipids To further test the generalizability of the DC-cholesterol LNP formulation, various ionizable lipids were tested. Table 6 summarizes the physicochemical properties of the formulations tested.

[0135] [ka] [ka] [ka] [ka] [ka]

[0136] [Table 6]

[0137] Mice were intravenously administered a formulation containing 0.5 mg / kg of siRNA against Cdh5, and organs were harvested 72 hours after injection. After isolation and purification of total RNA from the organs, Cdh5 mRNA was quantified relative to the housekeeping gene GusB. A PBS control was also tested. DC-cholesterol displacement demonstrates endothelial gene silencing by various ionizable lipids. VE-cadherin mRNA expression is measured based on ΔΔCt calculations compared to PBS at 3 days post-injection (0.5 mg / kg siVEcad).

[0138] Example 7: In vivo endothelial cell RNA delivery with various ionizable lipids To evaluate whether endothelial RNA delivery by ionizable cholesterol (iChol) LNPs can be generalized to various ionizable lipids, we prepared a library of iChol LNPs containing different ionizable lipids (Table 7). For this experiment, we selected siRNA against the Cdh5 gene, which is specifically expressed in endothelial cells. Using siCdh5, we were able to evaluate endothelial delivery efficiency without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Organs were then harvested 48–72 hours after administration, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and Cdh5 gene expression was quantified by real-time PCR. The results are shown in Figure 7. All iChol LNP formulations with different ionizable lipids demonstrated significant gene silencing in the liver, kidney, lung, heart, and skeletal muscle (quadriceps), demonstrating the generalizability of iChol LNP formulations to a variety of ionizable lipids.

[0139] Endothelial cells: siCdh5 5'-ccAAAAGAGAGAcuGGAuudTsdT-3' 5'-AAUCcAGUCUCUCUUUUGGdTsdT-3'

[0140] [Table 7]

[0141] Figure 7 demonstrates the generalizability of the iChol LNP formulation to a variety of ionizable lipids. The graph shows Cdh5 mRNA levels relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD.

[0142] Example 8: In vivo endothelial cell RNA delivery using various ionizable cholesterol ratios (1) HAPC-cholesterol To optimize the ionizable cholesterol ratio, LNP formulations with different DSPC-cholesterol ratios were prepared and tested in vivo. In this example, FL-A was used as the ionizable lipid, and the ratios of FL-A, DSPC, and DMG-mPEG-2000 were fixed at 50%, 10%, and 1.5%, respectively (Table 8). In this experiment, we selected siRNA against the Cdh5 gene, which is specifically expressed in endothelial cells. Using siCdh5, we were able to evaluate endothelial delivery efficiency without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Organs were then harvested 48–72 hours after administration, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and Cdh5 gene expression levels were quantified by real-time PCR. The results are shown in Figure 8. iChol LNPs with different HAPC-cholesterol ratios all showed significant gene silencing in the liver, and iChol LNPs with 20% or more HAPC-cholesterol showed more efficient RNA delivery to extrahepatic organs.

[0143] Endothelial cells: siCdh5 5'-ccAAAAGAGAGAcuGGAuudTsdT-3' 5'-AAUCcAGUCUCUCUUUUGGdTsdT-3'

[0144] [Table 8]

[0145] Figure 8 shows that iChol LNPs deliver RNA to endothelial cells at various HAPC-cholesterol ratios. The graph shows Cdh5 mRNA levels relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD.

[0146] Example 9: In vivo endothelial cell RNA delivery using various ionizable cholesterol ratios (2) DC-cholesterol To optimize the ionizable cholesterol ratio, LNP formulations with different DC-cholesterol ratios were prepared and tested in vivo. In this example, FL-A was used as the ionizable lipid, and the ratios of FL-A, DSPC, and DMG-mPEG-2000 were fixed at 50%, 10%, and 1.5%, respectively (Table 9). In this experiment, we selected siRNA against the Cdh5 gene, which is specifically expressed in endothelial cells. Using siCdh5, we were able to evaluate endothelial delivery efficiency without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Organs were then harvested 48–72 hours after administration, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and Cdh5 gene expression levels were quantified by real-time PCR. The results are shown in Figure 9. All iChol LNPs with different DC-cholesterol ratios showed significant gene silencing in the liver, and iChol LNPs with HAPC-cholesterol of 20% or more showed more efficient RNA delivery to extrahepatic organs.

[0147] [Table 9]

[0148] Figure 9 shows that iChol LNPs deliver RNA to endothelial cells at various DC-cholesterol ratios. The graph shows Cdh5 mRNA levels relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD.

[0149] Example 10: In vivo endothelial cell RNA delivery with various ionizable cholesterol ratios (3) HAPC-Cholesterol To optimize the ionizable cholesterol ratio, LNP formulations with different HAPC-cholesterol ratios were prepared and tested in vivo. In this example, FL-A was used as the ionizable lipid, and the DMG-mPEG-2000 ratio was fixed at 1.5% (Table 10). In this experiment, we selected siRNA against the Cdh5 gene, which is specifically expressed in endothelial cells. Using siCdh5, we were able to evaluate endothelial delivery efficiency without isolating endothelial cells. These LNPs were intravenously administered to mice at 0.5 mg / kg. Organs were then harvested 48–72 hours after administration, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and Cdh5 gene expression levels were quantified by real-time PCR. The results are shown in Figure 10. iChol LNPs with different DC-cholesterol ratios all showed significant gene silencing in the liver, and iChol LNPs with 20% or more HAPC-cholesterol and 20% or more ionizable lipids were more efficient in delivering RNA to extrahepatic organs.

[0150] [Table 10]

[0151] Figure 10 shows that iChol LNPs deliver RNA to endothelial cells at various HAPC-cholesterol ratios. The graph shows the mRNA levels of Cdh5 gene relative to the PBS control group. Each symbol represents an individual animal. Data are shown as mean ± SD.

[0152] Example 11: Serum protein-independent RNA delivery to endothelial cells Apolipoprotein E (ApoE) is a protein present in blood and is involved in the transport of lipids and cholesterol in the body. Recent studies have shown that ApoE plays an important role in the uptake and distribution of LNPs into hepatocytes. Specifically, LNPs are taken up by liver cells through a process mediated by ApoE adsorption to the LNP surface. For LNPs containing constitutive cationic lipids, the cellular uptake mechanism has also been reported to be serum protein-dependent. Among serum proteins, vitronectin has been reported to play an important role in the cellular uptake of cationic LNPs.

[0153] WO2020051220 WO2020051223 Dilliard SA Proc. Nat. Assoc. Sci. 2022. https: / / doi.org / 10.1073 / pnas.2109256118 Dilliard SAJ Cont. Rel. 2023. https: / / doi.org / 10.1016 / j.jconrel.2023.07.058.

[0154] To analyze the serum protein dependency of ionizable cholesterol LNP uptake, in vitro transfection efficiency was evaluated in serum-containing and serum-free media. SiCdh5-encapsulated LNP formulations were prepared as described in Materials and Methods (Table 11), and the resulting LNPs were tested in the bEND.3 mouse brain endothelial cell line. bEND.3 cells were cultured under standard culture conditions and transferred to 96-well plates at a density of 10,000–15,000 cells per well. bEND.3 endothelial cells were transfected with 100 nM siCdh5 per well. After 2 hours of culture, the cell culture medium containing LNPs was removed, and the cells were washed with serum-free medium and then cultured in serum-containing medium. After overnight culture, total RNA was extracted using the QuickExtract RNA Extraction Kit (LGC Biosearch Technologies), and cDNA was synthesized by reverse transcription. Cdh5 expression was quantified by real-time PCR. The results are shown in Figure 11. Hepatocyte LNPs showed more efficient gene silencing in serum-containing medium, whereas ionizable cholesterol LNPs showed similar gene silencing efficiency in serum-free and serum-containing medium, suggesting that the uptake of ionizable cholesterol LNPs is independent of serum proteins.

[0155] [Table 11]

[0156] FIG. 11 shows that endothelial cells take up iChol LNP in a serum-independent manner. Top: Schematic of the in vitro experimental design showing bEND.3 endothelial cells treated with siRNA-LNPs in serum-containing and serum-free medium. Lower panel: The graph shows the Cdh5 mRNA levels relative to the PBS control group. Each symbol represents an individual well. Data are shown as mean ± SD.

[0157] Example 12: In vitro endothelial RNA delivery using various LNP formulations To optimize the DC-cholesterol ratio, LNP formulations encapsulating firefly luciferase mRNA (TriLink) were prepared at various lipid ratios. The resulting LNPs were tested in the bEND.3 mouse brain endothelial cell line. bEND.3 cells were cultured under standard culture conditions and transferred to 96-well plates at a density of 10,000–15,000 cells per well. Prior to transfection, the cell culture medium was replaced with serum-free medium. bEND.3 endothelial cells were transfected at a dose of 1 μg per well. A total of 29 LNP formulations, including hepatocyte LNPs, were transfected into bEND.3 cells. After 2 hours of incubation, luciferase expression was measured using Steady-Glo. TM Quantification was performed using the Luciferase Assay System (Promega). Ionizable lipids 10-80 < 20-70 < 25-50 Phospholipids 0-40 < 5-35 < 10-30 Ionizable sterols 5-60 < 7.5-55 < 10-50 Sterols + Ionizable Sterols 10-60 < 20-50

[0158] [Table 12]

[0159] Example 13: In vitro LNP-mediated endothelial cell injury analysis Endothelial cell damage in vivo potentially leads to internal bleeding (Figure 12). To quantify the cytotoxicity of LNPs to endothelial cells, we used the mouse endothelial cell line bEND. 250 nM siRNA against Cdh5 encapsulated in LNPs was administered to 10,000 cells in a 96-well plate. After incubating these cells for 24 hours after transfection, cell viability was measured using a CCK-8 assay kit (Dojindo) according to the manufacturer's protocol and normalized to the PBS-treated group. The results are shown in Figure 12. These results indicated that LNPs containing ionizable cholesterol were better tolerated than LNPs containing the constitutive cationic lipid, DOTAP.

[0160] [Table 13]

[0161] The ionizable lipid / RNA ratio was fixed at 10.

[0162] FIG. 12 shows that iChol LNPs are less endothelial cell toxic than LNPs containing cationic lipids. Top: Schematic representation of potential hemolysis induced by LNP-mediated endothelial cell injury. Bottom: The graph shows cell viability relative to the PBS control group. Each symbol represents an individual well. Data are presented as mean ± SD.

[0163] Example 14: In vitro hemocompatibility analysis using human primary red blood cells To evaluate the hemocompatibility of the LNPs, a hemolysis assay was performed. For the hemolysis assay, human primary red blood cells (RBCs, Innovative Research) were washed three times with PBS, and 90 μL of a 4% vol / vol RBC suspension in PBS was transferred to a 96-well plate. Then, 10 μL of siRNA LNP solution containing 150 μg / mL total lipids was added to the RBC suspension. After incubation at 37°C for 1 hour, the plate was centrifuged at 1,000 x g for 5 minutes, and 80 μL of the supernatant was transferred to a clear, flat-bottom 96-well plate. Hemoglobin release was quantified by measuring UV-vis absorbance at 490 nm. Triton X-100, a cationic detergent that completely lyses the RBC membrane, was used as a positive control, and the amount of hemoglobin released was normalized to that of the Triton X-100-treated group. The results are shown in Figure 13. 50% DOTAP LNPs exhibited significant hemolysis, whereas LNPs containing ionizable cholesterol, such as DC-cholesterol and HAPC-cholesterol, did not. These data demonstrate the high hemocompatibility of ionizable cholesterol LNPs.

[0164] [Table 14]

[0165] The ionizable lipid / RNA ratio was fixed at 10.

[0166] FIG. 13 shows that iChol LNPs have lower hemolytic activity than LNPs containing cationic lipids. Top: Schematic diagram of hemolysis assay Bottom: Graph shows % hemolysis normalized to the Triton X-100 positive control. Each symbol represents an individual well. Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA and Dunnett's post test. Adjusted P values ​​less than 0.05 were considered statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

[0167] Example 15: In vivo hepatic stellate cell delivery To analyze specific cell types in the liver transfected with ionizable cholesterol LNPs, we performed a thorough evaluation of gene silencing efficiency within individual cell populations. To obtain cell-type-specific information, we employed different siRNAs targeting genes characterized by cell-type-specific expression patterns. LNPs containing the following siRNAs were prepared (Table 15) and intravenously administered to mice. Organs were then excised 48–72 hours after administration, and total RNA was extracted using the Quick-RNA MagBead Kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and the gene expression levels of Cdh5, Reln, and Sirpa were quantified by real-time PCR. For Fvii gene silencing, we used BIOPHEN TM Serum factor VII protein levels were quantified using an FVII assay kit (Aniara). The results are shown in Figure 14. Hepatocyte LNPs demonstrated gene silencing in all cell types evaluated in the liver, while ionizable cholesterol LNPs demonstrated more efficient and specific gene silencing in endothelial cells and hepatic stellate cells.

[0168] Hepatocytes:siFvii 5'-GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT-3' 5'-GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT-3'

[0169] Endothelial cells: siCdh5 5'-ccAAAAGAGAGAcuGGAuudTsdT-3' 5'-AAUCcAGUCUCUCUUUUGGdTsdT-3'

[0170] Hepatic stellate cells:siReln 5'-GGmUmCmUmCAAGmCmCAmCmUmCGmUmUmUdTsdT-3' 5'-AAACGAGUGGCUUGAGACCdTsdT-3'

[0171] Kupffer cell:siSirpa 5'-mCmUAAmCAAmCmCAmCAmCAGAAmUAdTsdT-3' 5'-mUAUUCUGUGUGGUUGUmUAGdTsdT-3'

[0172] [Table 15]

[0173] Figure 14 shows that iChol LNP delivers RNA to hepatic endothelial cells and hepatic stellate cells. The graphs show the mRNA levels of Cdh5 (endothelial cells), Sirpa (Kupffer cells), or Reln (hepatic stellate cells), or serum Factor VII protein levels (hepatocytes), relative to the PBS control group. siRNA-LNPs were intravenously administered to mice at doses of siCdh5 0.3 mg / kg, siFvii 0.3 mg / kg, siSirpa 0.4 mg / kg, and siReln 0.2 mg / kg, respectively. Each symbol represents an individual animal. Data are shown as mean ± SD. Statistical significance was assessed using one-way ANOVA and Dunnett's posttest. Adjusted P values ​​less than 0.05 were considered statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0174] Example 16: In vivo RNA delivery to extrahepatic extracellular matrix-producing cells Because stellate cells are also present in extrahepatic organs such as the pancreas, lungs, kidneys, intestines, spleen, adrenal glands, vas deferens, and vocal cords, we also evaluated RNA delivery to extrahepatic stellate cells using siRNA against Col1a1, which is specifically expressed in extracellular matrix-producing cells such as stellate cells and fibroblasts. LNPs containing siCol1a1 were prepared (Table 16), and these LNPs were intravenously administered to mice at a dose of 0.5 mg / kg siCol1a. 48–72 hours after administration, each organ was harvested, and total RNA was extracted using the Quick-RNA MagBead Kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and Col1a1 expression levels were quantified by real-time PCR. The results are shown in Figures 15 and 16. Compared with hepatocyte LNPs, DC-cholesterol LNPs demonstrated more effective gene silencing in pancreatic cells and colonic stellate cells. Gene silencing was also confirmed in extracellular matrix-producing cells in other organs.

[0175] siCol1a1 5'-GmUmCmUAGAmCAmUGmUmUmCAGmCmUmUdTsdT-3' 5'-AAGCUGAAmCAUGUCmUAGACdTsdT-3'

[0176] [Table 16]

[0177] Figure 15 shows that iChol LNP delivers RNA to extrahepatic stellate cells. Graphs show the mRNA levels of Col1a1 gene in the pancreas (left) and colon (right) relative to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD. Figure 16 shows that iChol LNP delivers RNA to extrahepatic extracellular matrix-producing cells. Graphs show the mRNA levels of Col1a1 gene in the spleen, kidney, lung, and pancreas, respectively, relative to the PBS control group. Each symbol represents an individual animal. Data are shown as mean ± SD.

[0178] Example 17: Intramuscular administration and delivery to muscle cells and endothelial cells Lipid nanoparticles (LNPs) have proven to be highly versatile delivery vehicles beyond their well-known role in vaccine development. When administered intramuscularly, LNPs offer potential advantages in various therapeutic areas by localizing therapeutic effects to affected muscles, reducing the potential for systemic side effects, and improving therapeutic specificity. To evaluate whether the effects of LNPs are localized, iChol LNPs were intramuscularly administered into the quadriceps muscle, as hepatocyte LNPs have been reported to transport large amounts of RNA to the liver upon intramuscular administration. Gene silencing in skeletal muscle cells in the quadriceps muscle (injected side) was also evaluated. To obtain cell type-specific information, we employed different siRNAs targeting genes characterized by cell type-specific expression patterns. LNPs containing the following siRNAs were prepared (Table 17), and these LNPs were intramuscularly administered to mice at a dose of 0.2 mg / kg siMstn. Quadriceps and liver were then harvested 48–72 hours after administration, and total RNA was extracted using the Quick-RNA MagBead kit (Zymo Research). After RNA extraction, cDNA was synthesized by reverse transcription, and gene expression levels of Cdh5 and Mstn were quantified by real-time PCR. The results are shown in Figures 17 and 18. DC-cholesterol LNPs demonstrated stronger gene silencing in muscle endothelial cells but no off-target delivery to hepatic endothelial cells. Regarding muscle cells, hepatocyte LNPs and DC-cholesterol LNPs demonstrated comparable gene silencing. This low off-target delivery during local administration may be advantageous for local tissue regeneration therapy.

[0179] Endothelial cells: siCdh5 5'-ccAAAAGAGAGAcuGGAuudTsdT-3' 5'-AAUCcAGUCUCUCUUUUGGdTsdT-3'

[0180] Muscle cells:siMstn 5'-AmUGGmCAAAGAAmCAAAmUAAmUdTsdT-3' 5'-AUmUAUUUGUUCUUUGCmCAUdTsdT-3'

[0181] [Table 17]

[0182] FIG. 17 shows that intramuscularly administered ionizable cholesterol LNPs deliver RNA to skeletal muscle endothelial cells around the injection site while minimizing off-target delivery to liver endothelial cells. (Left) Liver endothelial cells; (Right) Muscle endothelial cells at the injection site. Graphs show Cdh5 mRNA levels in the liver (left) and quadriceps (right) compared to the PBS control group. Each symbol represents an individual animal. Data are shown as mean ± SD. Statistical significance was assessed using one-way ANOVA with Dunnett's post-test. Adjusted p values ​​less than 0.05 were considered statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Figure 18 shows that intramuscularly administered LNPs deliver RNA to skeletal muscle cells surrounding the injection site. The graph shows Mstn mRNA levels in the quadriceps compared to the PBS control group. Each symbol represents an individual animal. Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA and Dunnett's post-test. Adjusted p values ​​less than 0.05 were considered statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

[0183] Example 18: In vitro cancer cell delivery To test the applicability of ionizable cholesterol LNPs to cancer cells, we tested them in a mouse cancer model. We used a B16F10 mouse melanoma lung metastasis model and administered cancer cells intravenously. LNPs containing siRNA against the CD47 gene, which encodes the CD47 protein, were prepared (Table 18). CD47 is a ubiquitous membrane receptor that interacts with signal-regulatory receptor protein alpha (SIRPα) expressed on macrophages, transmitting signals that suppress the phagocytic activity of macrophages. It has been reported that CD47 expression levels are elevated in various cancer cells, resulting in escape from immune surveillance by the innate immune system and tumor progression. In vitro transfection efficiency was evaluated in serum-free medium. SiCD47-loaded LNP formulations were prepared as described in Materials and Methods, and the resulting LNPs were tested in the B16F10 mouse melanoma cell line. B16F10 cells were cultured under standard culture conditions and transferred to 96-well plates at a density of 10,000–15,000 cells per well. B16F10 cells were transfected with 30 nM siCD47 per well. After 2 hours of culture, the cell culture medium containing LNPs was removed, and the cells were washed with serum-free medium and then cultured in serum-containing medium. After overnight culture, total RNA was extracted using the QuickExtract RNA Extraction Kit (LGC Biosearch Technologies), and cDNA was synthesized by reverse transcription. Cd47 expression was quantified by real-time PCR. The results are shown in Figure 19. Ionizable cholesterol LNPs demonstrated more efficient gene silencing than hepatocyte LNPs.

[0184] siCd47 5'- mCmCGAAGAAAmUGmUmUmUGmUGAAdTsdT -3' 5'- UUmCAmCAAAmCAUUUCUUCGGdTsdT -3'

[0185] [Table 18] < / sirna>

Claims

1. 1. A method for delivering a therapeutic agent to an endothelial cell, a mesenchymal cell, or a cancer cell, comprising administering a lipid composition to a subject, the lipid composition comprises the therapeutic agent and lipid nanoparticles; The lipid nanoparticles comprise an ionizable lipid and a compound represented by formula (1) or a salt thereof. 【Chemistry 1】 In the formula, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L Ｙ represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, X represents a basic functional group.

2. 2. The method according to claim 1, wherein the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5- or 6-membered heterocyclic alkyl group, or a 5- or 6-membered heterocyclic aryl group.

3. The method according to claim 1 or 2, wherein the compound represented by formula (1) is a compound represented by formula (2). 【Chemistry 2】 In the formula, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L Ｙ represents a single bond, an alkylene group having 1 to 14 carbon atoms, a substituted alkylene group having 1 to 14 carbon atoms, a heteroalkylene group having 1 to 14 carbon atoms, or a substituted heteroalkylene group having 1 to 14 carbon atoms, R ２ , R ３ and R ４ are each independently a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or —C(NH ２ ) = NH ２ indicates R ２ , R ３ and R ４ One of them may not be present.

4. The method according to claim 3, wherein the compound represented by formula (2) is a compound represented by formula (3). 【Transformation 3】 In the formula, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L １ represents a single bond or an alkylene group having 1 to 6 carbon atoms, R １ represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms, G ２ represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—, L ２ represents an alkylene group having 1 to 6 carbon atoms which may have an amino group, R ２ , R ３ and R ４ are each independently a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or —C(NH ２ ) = NH ２ indicates, R ２ , R ３ and R ４ One of them may not be present.

5. The method according to claim 4, wherein the compound represented by formula (3) is a compound represented by formula (3A). 【Chemistry 4】 In the formula, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L １ represents a single bond or an alkylene group having 1 to 6 carbon atoms, R １ represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms, G ２ represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—, L ２ represents an alkylene group having 1 to 6 carbon atoms which may have an amino group, R ２ , R ３ are each independently a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or —C(NH ２ ) = NH ２ Shows.

6. G １ The method according to any one of claims 1 to 5, wherein represents -C(O)- or -C(O)O-.

7. R １ The method according to claim 5, wherein represents a hydrogen atom or an aminoalkyl group having 1 to 4 carbon atoms.

8. R １ The method of claim 5 , wherein represents a hydrogen atom.

9. G ２ The method according to claim 5, wherein represents a single bond or —C(O).

10. G ２ The method of claim 5 , wherein represents a single bond.

11. L ２ represents an alkylene group having 1 to 3 carbon atoms, and R ２ and R ３ and each independently represent a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms which may be substituted with a hydroxyl group.

12. The method according to claim 1 or 2, wherein the compound represented by formula (1) or a salt thereof is any one of the following: 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】

13. The method according to any one of claims 1 to 12, wherein the content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol% based on the total lipids.

14. 14. The method of any one of claims 1 to 13, wherein the therapeutic agent is a nucleic acid.

15. 15. The method of any one of claims 1 to 14, wherein the therapeutic agent is DNA or RNA.

16. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent is mRNA or siRNA.

17. 17. The method according to any one of claims 1 to 16, wherein the ionizable lipid has at least one ionizable amino group and at least one biodegradable group, and the biodegradable group is represented by -O(CO)O-, -O(CO)-, or -(CO)O-.

18. 18. The method of any one of claims 1 to 17, wherein the ionizable lipid is a compound represented by formula (4): 【Chemistry 13】 In the formula, X is —NR １ - or -O-, R １ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or 【Chemistry 14】 indicates R ２２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or 【Chemistry 15】 indicates R ３２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R ４ and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, -NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c, and d each independently represent an integer of 0 to 3, provided that a+b is 1 or more, and c+d is 1 or more.

19. 18. The method of any one of claims 1 to 17, wherein the ionizable lipid is a compound represented by formula (1): 【Chemistry 16】 During the ceremony, R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , and O-R ５６ and optionally substituted with one or more substituents selected from R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R ８ -L １ -R ９ where R ５ and R ６ are both hydrocarbon groups having 1 to 8 carbon atoms, R ７ is -R １０ -L ２ -R １１ -L ３ -R １２ indicates, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R ５３ , R ５４ , R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ may be substituted with The aryl group having 6 to 20 carbon atoms is —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ may be substituted with R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, R ５７ is -OH, COOH, -NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , or -O-R ６６ Shows. R ６１ and R ６２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R ６３ , R ６４ , R ６５ , and R ６６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R ６３ , R ６４ , R ６５ , and R ６６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ６８ may be substituted with The aryl group having 6 to 20 carbon atoms is —OH, —COOH, —NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ or -(C1-C12 hydrocarbon group)-R ６７ may be substituted with R ６８ represents a hydrocarbon group having 1 to 12 carbon atoms, L １ , L ２ , and L ３ each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—. R ８ represents a hydrocarbon group having 1 to 12 carbon atoms, R ９ represents a hydrocarbon group having 1 to 24 carbon atoms, R １０ represents a hydrocarbon group having 1 to 8 carbon atoms, R １１ represents a hydrocarbon group having 1 to 24 carbon atoms, R １２ represents a hydrocarbon group having 1 to 24 carbon atoms, R ９ , and R １２ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ is defined as above, R １１ The hydrocarbon group represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ or —OC(O)—R ５５ and R ５３ , R ５４ , and R ５５ The definition of is as above.

20. 18. The method according to any one of claims 1 to 17, wherein the ionizable lipid is a compound represented by the following formula (5): 【Chemistry 17】 In the formula, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The substituent A is a hydroxyl group, -G ２０ -CH(R ５５ ) (R ５６ ), -N(R ５８ ) (R ５９ ), or -G ２０ -R ６０ represents a group represented by G ２０ represents —O(CO)— or —(CO)O—, R ５５ and R ５６ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R ５８ and R ５９ each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, Substituent B is —N(R ６１ ) (R ６２ ) and R ６１ and R ６２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R ６０ represents a hydrocarbon group having 1 to 18 carbon atoms, L １０ represents a hydrocarbon group having 1 to 18 carbon atoms, G ３０ is -S-(CO)-NR ６４ - indicates R ６４ Ha,-L ３０ -G ２０ -CH(R ５５ ) (R ５６ ) represents a group represented by a represents 0 or 1; L ３０ represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G １０ is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R ６３ ) - indicates R ６３ represents a hydrocarbon group having 1 to 18 carbon atoms, L ２０ represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1; R ５３ , R ５４ and R ５７ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is —(CO)O —R ６５ , or -O(CO) -R ６５ represents a group represented by R ６５ represents a hydrocarbon group having 1 to 18 carbon atoms or -L ４０ -CH(R ６６ ) (R ６７ ) represents a group represented by L ４０ represents a hydrocarbon group having 1 to 6 carbon atoms, R ６６ and R ６７ represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms.

21. 21. The method of claim 1, wherein the lipid nanoparticles further comprise a lipid having a non-ionic hydrophilic polymer.

22. 22. The method of any one of claims 1 to 21, wherein the lipid nanoparticles further comprise a phospholipid.

23. 23. The method of any one of claims 1 to 22, wherein the lipid composition is administered to the subject by intravenous or intramuscular injection.

24. 24. The method of any one of claims 1 to 23, wherein the mesenchymal cells are muscle cells.

25. 24. The method of any one of claims 1 to 23, wherein the mesenchymal cells are extracellular matrix-producing cells.

26. 24. The method of any one of claims 1 to 23, wherein the extracellular matrix cells are astrocytes or fibroblasts.

27. 24. The method of any one of claims 1 to 23, wherein the stellate cells are hepatic stellate cells, pancreatic stellate cells, or colonic stellate cells.

28. 1. A lipid composition comprising a therapeutic agent and lipid nanoparticles, The lipid nanoparticles comprise a compound represented by formula (1) or a salt thereof and an ionizable lipid having a biodegradable group. Lipid composition. [Chemistry 18] In the formula, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L Y represents a single bond or an alkylene group having 1 to 14 carbon atoms, a substituted alkylene, a heteroalkylene, or a substituted heteroalkylene; X represents a basic functional group.

29. The lipid composition according to claim 28, wherein the basic functional group represented by X is an amino group, a substituted amino group, a guanidino group, a 5- to 6-membered heterocyclic alkyl group, or a 5- to 6-membered heterocyclic aryl group.

30. The lipid composition according to claim 28 or 29, wherein the compound represented by formula (1) is a compound represented by formula (2). 【Chemistry 19】 During the ceremony, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L Y represents a single bond or an alkylene group, substituted alkylene group, heteroalkylene group or substituted heteroalkylene group having 1 to 14 carbon atoms, R ２ , R ３ and R ４ are each independently a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or —C(NH ２ ) = NH ２ indicates R ２ , R ３ and R ４ One of them may not be present.

31. The lipid composition according to claim 30, wherein the compound represented by formula (2) is a compound represented by formula (3). 【Chemistry 20】 During the ceremony, G １ represents —C(O)—, —OC(O)—, —O(CO)O— or —C(O)O—, L １ represents a single bond or an alkylene group having 1 to 6 carbon atoms, R １ represents a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms, or an aminoalkyl group having 1 to 4 carbon atoms, G ２ represents a single bond, —C(O)—, —OC(O)—, —O(CO)O—, or —C(O)O—, L ２ represents an alkylene group having 1 to 6 carbon atoms which may have an amino group, R ２ , R ３ and R ４ are each independently a hydrogen atom, a hydrocarbon group having 1 to 4 carbon atoms which may be substituted with a hydroxyl group, or —C(NH ２ ) = NH ２ indicates, R ２ , R ３ and R ４ One of them may not be present.

32. 32. The lipid composition according to any one of claims 28 to 31, wherein the ionizable lipid having a biodegradable group is a compound represented by formula (4). 【Chemistry 21】 In the formula, X is —NR １ - or -O-, R １ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or 【Chemistry 22】 indicates R ２２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or 【Chemistry 23】 indicates R ３２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms, R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, R ４ and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ any one or more pairs of may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom, The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, The substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, -NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, a, b, c, and d each independently represent an integer of 0 to 3, provided that a+b is 1 or more, and c+d is 1 or more.

33. 32. The lipid composition of any one of claims 28 to 31, wherein the ionizable lipid is a compound represented by formula (1). 【Chemistry 24】 During the ceremony, R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , and O-R ５６ and optionally substituted with one or more substituents selected from R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R ８ -L １ -R ９ where R ５ and R ６ are both hydrocarbon groups having 1 to 8 carbon atoms, R ７ is -R １０ -L ２ -R １１ -L ３ -R １２ indicates, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R ５３ , R ５４ , R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ may be substituted with The aryl group having 6 to 20 carbon atoms is —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ may be substituted with R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, R ５７ is -OH, COOH, -NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , or -O-R ６６ Shows. R ６１ and R ６２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms, R ６３ , R ６４ , R ６５ , and R ６６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms, R ６３ , R ６４ , R ６５ , and R ６６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ６８ may be substituted with The aryl group having 6 to 20 carbon atoms is —OH, —COOH, —NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ or -(C1-C12 hydrocarbon group)-R ６７ may be substituted with R ６８ represents a hydrocarbon group having 1 to 12 carbon atoms, L １ , L ２ , and L ３ each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—. R ８ represents a hydrocarbon group having 1 to 12 carbon atoms, R ９ represents a hydrocarbon group having 1 to 24 carbon atoms, R １０ represents a hydrocarbon group having 1 to 8 carbon atoms, R １１ represents a hydrocarbon group having 1 to 24 carbon atoms, R １２ represents a hydrocarbon group having 1 to 24 carbon atoms, R ９ , and R １２ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ is defined as above, R １１ The hydrocarbon group represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ or —OC(O)—R ５５ and R ５３ , R ５４ , and R ５５ The definition of is as above.

34. 32. The lipid composition according to any one of claims 28 to 31, wherein the ionizable lipid having a biodegradable group is a compound represented by formula (5). 【Chemistry 25】 In the formula, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, The substituent A is a hydroxyl group, -G ２０ -CH(R ５５ ) (R ５６ ), -N(R ５８ ) (R ５９ ), or -G ２０ -R ６０ represents a group represented by G ２０ represents —O(CO)— or —(CO)O—, R ５５ and R ５６ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R ５８ and R ５９ each independently represents a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, Substituent B is —N(R ６１ ) (R ６２ ) and R ６１ and R ６２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R ６０ represents a hydrocarbon group having 1 to 18 carbon atoms, L １０ represents a hydrocarbon group having 1 to 18 carbon atoms, G ３０ is -S-(CO)-NR ６４ - indicates R ６４ Ha,-L ３０ -G ２０ -CH(R ５５ ) (R ５６ ) represents a group represented by a represents 0 or 1; L ３０ represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G １０ is -O(CO)-, -(CO)O-, -O(CO)O- or -N(C(O)R ６３ ) - indicates R ６３ represents a hydrocarbon group having 1 to 18 carbon atoms, L ２０ represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1; R ５３ , R ５４ and R ５７ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, The substituent C is —(CO)O —R ６５ , or -O(CO) -R ６５ represents a group represented by R ６５ represents a hydrocarbon group having 1 to 18 carbon atoms or -L ４０ -CH(R ６６ ) (R ６７ ) represents a group represented by L ４０ represents a hydrocarbon group having 1 to 6 carbon atoms, R ６６ and R ６７ represents a hydrocarbon group or alkoxy group having 1 to 10 carbon atoms.

35. The lipid composition according to any one of claims 28 to 34, wherein the content of the compound represented by formula (1) or a salt thereof is 5 to 80 mol% based on the total lipids.

36. 36. The lipid composition of any one of claims 28 to 35, wherein the therapeutic agent is a nucleic acid.

37. 37. The lipid composition of any one of claims 28 to 36, wherein the therapeutic agent is DNA or RNA.

38. 38. The lipid composition of any one of claims 28 to 37, wherein the therapeutic agent is mRNA or siRNA.

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