Ligand-modified lipid nanoparticle used for delivering nucleic acid to brain cell

Abstract

The purpose of the present invention is to provide a lipid nanoparticle with which a nucleic acid can be efficiently delivered to a brain cell. This lipid nanoparticle is a ligand-modified lipid nanoparticle which contains an ionic lipid, cholesterol, a PEG lipid, a nucleic acid, and a ligand and which is used for delivering a nucleic acid to a brain cell.

Description

Ligand-modified lipid nanoparticles used to deliver nucleic acids to brain cells

[0001] This invention relates to ligand-modified lipid nanoparticles used for delivering nucleic acids to brain cells.

[0002] To put nucleic acid therapy using oligonucleotides such as siRNA and miRNA, or gene therapy using mRNA or pDNA, into practical use, there is a need for effective and safe nucleic acid delivery carriers. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but they have practical problems from a safety standpoint. Therefore, the development of non-viral nucleic acid delivery carriers that can be used more safely is progressing. Among these, lipid nanoparticles, which are carriers using ionic lipids, are currently the most commonly used non-viral nucleic acid delivery carriers.

[0003] Ionic lipids are broadly composed of an amine moiety and a lipid moiety. For example, in ionic lipids, the amine moiety, which is protonated under acidic conditions, interacts electrostatically with a nucleic acid, which is a polyanion, to form lipid nanoparticles, thereby promoting uptake into cells and delivering nucleic acids into the cells.

[0004] A well-known ionic lipid that is widely used in general is, for example, 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP). It is known that by combining such known ionic lipids with phospholipids, cholesterol, and PEG lipids, lipid nanoparticles can be formed to deliver nucleic acids into cells (see, for example, Non-Patent Document 1).

[0005] Patent Document 1 describes an ionic lipid having a structure in which compounds consisting of one or two amine moieties and one lipid moiety are linked by a biodegradable disulfide bond. Patent Document 1 shows that this ionic lipid improves in vivo dynamics such as blood stability and tumor targeting ability. Furthermore, it has been shown that by changing the structure around the amine moiety, the pKa of the lipid membrane structure can be adjusted to a value favorable for endosome escape within the cell, and that by utilizing the cleavage of disulfide bonds within the cell, it has the effect of dissociating nucleic acids from the lipid membrane structure. In fact, compared to the known ionic lipid DODAP, it shows a higher nucleic acid delivery efficiency, making it clear that this ionic lipid can improve intracellular dynamics such as the efficiency of nucleic acid delivery into the cytoplasm.

[0006] Patent Document 2 describes a lipid membrane structure that enhances the ability to fuse with the endosomal membrane and further improves the efficiency of nucleic acid delivery to the cytoplasm by using ionic lipids that have an aromatic ring introduced near the lipid moiety in addition to the tertiary amine moiety and disulfide bond.

[0007] As mentioned above, ionic lipids have been developed that improve intracellular dynamics by enhancing endosomal escape efficiency and membrane fusion ability. On the other hand, for lipid nanoparticles containing ionic lipids to exert more practical effects in vivo as nucleic acid delivery carriers, it is necessary to deliver nucleic acids effectively to target organs and cells. Examples of such target sites include the liver, lungs, spleen, and brain.

[0008] Currently, there are no marketed drug formulations for disease treatment that utilize lipid nanoparticles targeting the brain. Several examples of therapeutic drugs delivered to the brain have been reported. For example, aducanumab, an Alzheimer's disease treatment, is a monoclonal antibody that targets amyloid-beta in the brain and is administered intravenously. Non-patent document 2 evaluates the inhibitory effect of antisense oligonucleotides on target genes in the brain after intraventricular administration.

[0009] WO 2013 / 073480 A1 WO 2019 / 188867 A1

[0010] Mol. Pharmaceutics 2018, 15, 2060-2067

[0011] Among all organs, the brain is one of the most difficult to deliver drugs to. Because of this, many neurological diseases lack effective treatments, and there is a need for methods to efficiently deliver therapeutic nucleic acids to the brain.

[0012] Non-patent document 3 states that nearly 100% of large-molecule drugs and 98% of small-molecule drugs cannot reach the brain. Therefore, the development of more efficient drug delivery methods is desired. Non-patent document 4 shows an example of delivering nucleic acids to brain tissue by directly administering lipid nanoparticles containing nucleic acids into the cerebral ventricles. However, direct administration of drugs into the cerebral ventricles places a significant burden on the patient, so a less invasive method is desirable.

[0013] In view of the above circumstances, the present invention aims to provide lipid nanoparticles that can efficiently deliver nucleic acids to brain cells.

[0014] As a result of diligent research by the inventors, we have found that ligand-modified lipid nanoparticles can efficiently deliver nucleic acids to brain cells. Based on this finding, the present invention is as follows.

[0015] [1] Ligand-modified lipid nanoparticles used to deliver nucleic acids to brain cells, comprising ionic lipids, cholesterol, PEG lipids, nucleic acids, and ligands. [2] The ligand-modified lipid nanoparticles according to [1], further comprising phospholipids.

[0016] [3] Ionic lipids are given by formula (1):

[0017]

[0018] (In formula (1), R １ａ and R １ｂ each independently represent an alkylene group having 1 to 6 carbon atoms, X ａ and X ｂ each independently represent an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R ２ａ and R ２ｂ each independently represent an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms, Y ａ and Y ｂ each independently represent an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, Z ａ and Z ｂ each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom, n ａ and n ｂ each independently represent 0 or 1, and R ３ａ and R ３ｂ each independently are any one of: a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride; a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride; an aliphatic hydrocarbon group having 1 to 40 carbon atoms; an alkyl group having 3 to 40 carbon atoms having a cyclopropane ring; and formula (2): *-R ６ -L １ -R ７ (2) (In formula (2), * represents a bonding position, R ６ represents an alkylene group having 2 to 10 carbon atoms, R ７ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon cyclic group having 3 to 12 carbon atoms, one methylene group in R ７ may be replaced with one ether bond, and L １represents an ester bond or a carbamate bond. ) A group represented by formula (3):

[0019]

[0020] (In formula (3), * indicates the bonding position, and R ８ This represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ８ The group may be substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms.) The group represented by formula (4):

[0021]

[0022] (In equation (4), * indicates the bonding position, R ９ R represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, and R ９ R may be substituted with substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms. １０ and R １１ Each of these independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, R １０ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １１ At least one ethylene group or at least one trimethylene group in the compound may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R １０ and R １１ Each of these may be independently substituted with a substituent selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms, and L ２) represents an oxygen atom, NH, or sulfur atom. ) A group represented by formula (5):

[0023]

[0024] (In formula (5), * represents the bond position, and R １２ is a hydrogen atom, a benzyl group, or *-Si(R １３ ) (Caution １４ ) (Caution １５ ) group (wherein * represents a bond position, and R １３ ~R １５ Each of these independently represents an alkyl group or phenyl group having 1 to 4 carbon atoms. ) Represents a group represented by ) Formula (6):

[0025]

[0026] (In equation (6), * represents the bond position, R １６ R represents an alkylene group having 5 to 10 carbon atoms, and １７ ~R １９ Each independently represents 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, R １７ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １８ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １９ At least one ethylene group or at least one trimethylene group in the compound may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R １７ ~R １９ Each of these may be independently substituted with a substituent selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms.) A group represented by formula (7):

[0027]

[0028] (In equation (7), * represents the bond position, and R ２0 R represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and ２1 and R ２2 Each of these independently represents 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.) represents a group, represents an ionic lipid represented by ). Formula (10):

[0029]

[0030] (In formula (10), R ２６ and R ２７ Each of these independently represents 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, or R ２６ and R ２７ These atoms bond to each other, forming a 5- or 6-membered nitrogen-containing heterocycle with the nitrogen atoms to which they bond, R ２９ and R ２８ Each of these independently represents an aliphatic hydrocarbon group having 5 to 50 carbon atoms, and R ２８ At least one ethylene group in the group may be replaced by an ester bond, and R ２９ At least one ethylene group in the compound may be replaced by an ester bond, and R ３０This represents an alkylene group having 1 to 10 carbon atoms. Ionic lipids represented by ), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, Ligand-modified lipid nanoparticles according to [1] or [2], comprising at least one selected from the group consisting of (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

[0031] [4] Ionic lipids are ionic lipids represented by formula (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, The ligand-modified lipid nanoparticles according to [3], comprising at least one selected from the group consisting of 1,1'-[[2-[4-[2-[[2-[[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

[0032] [5] Ligand-modified lipid nanoparticles according to any one of [2] to [4], wherein the phospholipid comprises at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dimiristoyl-sn-glycero-3-phosphocholine.

[0033] [6] Ligand-modified lipid nanoparticles according to any one of [1] to [5], wherein the amount of ionic lipids is 15 to 75 mol%, the amount of phospholipids is 0 to 45 mol%, the amount of cholesterol is 15 to 65 mol%, and the amount of PEG lipids is 0.5 to 5 mol% of the total amount of ionic lipids, phospholipids, and cholesterol.

[0034] [7] A ligand-modified lipid nanoparticle according to any one of [1] to [6], wherein the ligand is an anti-transferrin receptor antibody. [8] A ligand-modified lipid nanoparticle according to any one of [1] to [7], wherein the amount of ligand is 0.01 to 5 mol% of the total lipids.

[0035] [9] Ligand-modified lipid nanoparticles according to any one of [1] to [8], wherein the brain cells are cerebral vascular endothelial cells.

[10] A pharmaceutical composition used for the treatment of a brain disease, comprising ligand-modified lipid nanoparticles according to any one of [1] to [9].

[0036] The ligand-modified lipid nanoparticles of the present invention can efficiently deliver nucleic acids to brain cells. Therefore, brain diseases can be efficiently treated using the ligand-modified lipid nanoparticles of the present invention.

[0037] Each description herein can be combined with others unless it is clearly stated that they cannot be combined. Embodiments of the present invention are described below, but the present invention is not limited thereto.

[0038] The present invention relates to ligand-modified lipid nanoparticles (hereinafter sometimes abbreviated as "the lipid nanoparticles of the present invention") used to deliver nucleic acids to brain cells, comprising ionic lipids, cholesterol, PEG lipids, nucleic acids, and ligands, and to pharmaceutical compositions used for the treatment of brain diseases, comprising the lipid nanoparticles of the present invention.

[0039] The brain cells are preferably vascular endothelial cells. These vascular endothelial cells are cells that constitute the blood-brain barrier. In addition, the ligand-modified lipid nanoparticles of the present invention may be used to deliver nucleic acids to brain cells other than vascular endothelial cells by crossing the blood-brain barrier.

[0040] Lipid nanoparticles In this specification, "lipid nanoparticles" (sometimes abbreviated as "LNP") means particles having a membrane structure in which the hydrophilic groups of amphiphilic lipids are arranged toward the aqueous phase side of the interface, and having a particle diameter of less than 1 μm.

[0041] In this specification, "ligand-modified lipid nanoparticles" means lipid nanoparticles on which a ligand is presented on the surface of the lipid nanoparticles so as to interact with molecules expressing specific affinity on target cells.

[0042] In this specification, "amphiphilic lipid" means a lipid having both hydrophilic and hydrophobic groups. Examples of amphiphilic lipids include ionic lipids, phospholipids, and PEG lipids. In this specification, "PEG" means polyethylene glycol, and "PEG lipid" means a lipid to which PEG is bound. A lipid having both a phospholipid portion and a PEG lipid portion (for example, DSPE-PEG) is a lipid to which PEG is bound. Therefore, in this specification, lipids having both a phospholipid portion and a PEG lipid portion are included in PEG lipids.

[0043] The lipid nanoparticles of the present invention include ionic lipids, cholesterol, and PEG lipids as constituent materials of the membrane. Optionally, the lipid nanoparticles of the present invention may further contain phospholipids.

[0044] In this specification, "total lipids" refers to all lipids. Examples of lipids include ionic lipids, cholesterol, PEG lipids, phospholipids, and the like.

[0045] The particle size of the lipid nanoparticles of the present invention is preferably 10 nm to 500 nm, and more preferably 30 nm to 200 nm. The particle size can be measured using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles of the present invention can be appropriately adjusted by the manufacturing method. In this specification, "particle size" means the average particle size (zeta mean) measured by dynamic light scattering.

[0046] The surface potential (zeta potential) of the lipid nanoparticles of the present invention is preferably -20 mV to +20 mV, and more preferably -15 mV to 15 mV. The surface potential (zeta potential) can be measured using a zeta potential measuring device such as a Zetasizer Nano (Malvern). The surface potential (zeta potential) of the lipid nanoparticles of the present invention can be adjusted by the composition of its constituent components.

[0047] Ionic Lipids In one embodiment of the present invention, the ionic lipid is preferably an ionic lipid represented by the following formula (1) (which may be abbreviated as "ionic lipid (1)" in this specification), an ionic lipid represented by the following formula (10) (which may be abbreviated as "ionic lipid (10)" in this specification), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione A lipid comprising at least one selected from the group consisting of 1,1'-[[2-[4-[2-[[2-[[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate, more preferably an ionic lipid (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,It comprises at least one selected from the group consisting of 29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

[0048] In one embodiment of the present invention, the ionic lipid preferably comprises at least one selected from the group consisting of ionic lipid (1), ionic lipid (10), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, more preferably comprising ionic lipid (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and even more preferably comprising ionic lipid (1).

[0049] In one embodiment of the present invention, the ionic lipid is preferably ionic lipid (1), ionic lipid (10), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, At least one selected from the group consisting of 1,1'-[[2-[4-[2-[[2-[[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate, more preferably an ionic lipid (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,It is at least one selected from the group consisting of 29-diyl diacetate and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

[0050] In one embodiment of the present invention, the ionic lipid is preferably at least one selected from the group consisting of ionic lipid (1), ionic lipid (10), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and more preferably at least one selected from the group consisting of ionic lipid (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate.

[0051] In one embodiment of the present invention, the ionic lipid is preferably ionic lipid (1), ionic lipid (10), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, or di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate, more preferably ionic lipid (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyldiacetate, or di(pentadecan-8-yl)4,It is 4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

[0052] In one embodiment of the present invention, the ionic lipid is preferably ionic lipid (1), ionic lipid (10), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, more preferably ionic lipid (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and even more preferably ionic lipid (1).

[0053] The following describes ionic lipids represented by the following formula (1). Ionic lipid (1) may be used alone or in combination of two or more types.

[0054]

[0055] (In formula (1), R １ａ and R １ｂ Each of these independently represents an alkylene group with 1 to 6 carbon atoms, X ａ and X ｂ Each of these independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R ２ａ and R ２ｂ Each of these independently represents an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms, Y ａ and Y ｂ Each of these independently represents an ester bond, amide bond, carbamate bond, ether bond, or urea bond, Z ａ and Z ｂEach of these independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, possessing at least one aromatic ring, and possibly having a heteroatom, n ａ and n ｂ Each independently represents either 0 or 1, and R ３ａ and R ３ｂ Each of these independently consists of: a residue derived from the reaction product of a lipid-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride; a residue derived from the reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride; an aliphatic hydrocarbon group having 1 to 40 carbon atoms; an alkyl group having 3 to 40 carbon atoms and a cyclopropane ring; Formula (2): *-R ６ -L １ -R ７ (2) (In equation (2), * represents the bond position, R ６ R represents an alkylene group with 2 to 10 carbon atoms. ７ R represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms. ７ One of the methylene groups may be replaced by one ether bond, and L １ represents an ester bond or a carbamate bond. ) A group represented by formula (3):

[0056]

[0057] (In formula (3), * indicates the bonding position, and R ８ This represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ８ The group may be substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms.) The group represented by formula (4):

[0058]

[0059] (In equation (4), * indicates the bonding position, R ９represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, and R ９ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, and R １０ and R １１ each independently represent an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, and R １０ at least one ethylene group or at least one trimethylene group in may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R １１ at least one ethylene group or at least one trimethylene group in may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and R １０ and R １１ each independently may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, and L ２ represents an oxygen atom, NH, or a sulfur atom. ), a group represented by formula (5):

[0060]

[0061] (in formula (5), * represents a bonding site, and R １２ represents a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ ) group (in said formula, * represents a bonding site, and R １３ to R １５ each independently represent an alkyl group having 1 to 4 carbon atoms or a phenyl group.)), a group represented by formula (6):

[0062]

[0063] (in formula (6), * represents a bonding site, R １６R represents an alkylene group having 5 to 10 carbon atoms, and １７ ~R １９ Each independently represents 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, R １７ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １８ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １９ At least one ethylene group or at least one trimethylene group in the compound may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R １７ ~R １９ Each of these may be independently substituted with a substituent selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms.) A group represented by formula (7):

[0064]

[0065] (In equation (7), * represents the bond position, and R ２0 R represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and ２1 and R ２2 Each of these independently represents 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.

[0066] The definitions of each symbol in equation (1) will be explained in order below. R １ａ and R １ｂEach of these independently represents an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 4, more preferably 1 to 2. Specific examples of alkylene groups having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, propylene, tetramethylene, isobutylene, pentamethylene, neopentylene, and the like. １ａ and R １ｂ Preferably, each of these groups is independently a methylene group, an ethylene group, a trimethylene group, a propylene group, or a tetramethylene group, and more preferably each is an ethylene group.

[0067] R １ａ is R １ｂ It may be the same as or different from, but preferably R １ａ is R １ｂ It is the same base as [another base].

[0068] X ａ and X ｂ Each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, preferably each independently represents a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0069] The C1-C6 alkyl group in an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group may be linear or branched. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of C1-C6 alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl groups, with methyl, ethyl, propyl, or isopropyl groups being preferred, and methyl groups being more preferred.

[0070] A preferred specific structure of an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is X １ This is shown by X. １ The two ends of the following equation indicate bond positions, not carbon atoms.

[0071]

[0072] X １ R ５ represents an alkyl group having 1 to 6 carbon atoms, which may be linear or branched. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of alkyl groups having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, 1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, and the like. Preferably, it is a methyl group, ethyl group, propyl group, or isopropyl group, and more preferably a methyl group.

[0073] In a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, the number of carbon atoms is preferably 4 to 5. Specifically, the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is an aziridinediyl group, an azetidinediyl group, a pyrrolidinediyl group, a piperidinediyl group, an imidazolidinediyl group, or a piperazinediyl group, preferably a pyrrolidinediyl group, a piperidinediyl group, or a piperazinediyl group, and more preferably a piperidinediyl group. In this specification, "compound name + diyl group (e.g., aziridinediyl group)" means a divalent group having a structure obtained by removing two hydrogen atoms from the compound (e.g., aziridine).

[0074] A preferred specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group is X ２ This is shown by X. ２ The two ends of the following equation indicate bond positions, not carbon atoms.

[0075]

[0076] X ２ p is either 1 or 2. When p is 1, X ２ This is a pyrrolidinediyl group, and when p is 2, X ２ is a piperidinediyl group. Preferably, p is 2.

[0077] A preferred specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is X ３ This is shown by X. ３ The two ends of the following equation indicate bond positions, not carbon atoms.

[0078]

[0079] X ３ w is either 1 or 2. When w is 1, X ３ is an imidazolidinediyl group, and when w is 2, X ３ This is a piperazinediyl group.

[0080] X ａ is Xｂ It may be the same as or different from X ａ is X ｂ It is the same base as [another base].

[0081] R ２ａ and R ２ｂ Each of these independently represents an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms, preferably each independently representing an alkylene group having 1 to 8 carbon atoms.

[0082] The alkylene group having 1 to 8 carbon atoms may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 6, more preferably 1 to 4. Specific examples of the alkylene group having 1 to 8 carbon atoms include methylene, ethylene, trimethylene, propylene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, and octamethylene groups, with methylene, ethylene, trimethylene, and tetramethylene groups being preferred, and ethylene groups being more preferred.

[0083] In this specification, "oxydialkylene group having 2 to 8 carbon atoms" means an alkylene group (alkylene-O-alkylene, in other words, "alkylene oxyalkylene group") via an ether bond, where the sum of the carbon atoms of the two alkylene groups is 2 to 8. Here, the two alkylenes may be the same or different, but are preferably the same. Specific examples of oxydialkylene groups having 2 to 8 carbon atoms include oxydimethylene group, oxydiethylene group, oxydi(trimethylene) group (i.e., trimethyleneoxytrimethylene group), oxydi(tetramethylene) group (i.e., tetramethyleneoxytetramethylene group), etc. Preferably, it is oxydimethylene group, oxydiethylene group, or oxydi(trimethylene) group, and more preferably, oxydiethylene group.

[0084] R ２ａ is R ２ｂ It may be the same as or different from, but preferably R ２ａ is R ２ｂ It is the same base as [another base].

[0085] Y ａ and Y ｂ Each of these is independently an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, preferably independently an ester bond, an amide bond, or a carbamate bond, more preferably independently an ester bond or an amide bond, and more preferably independently an ester bond.

[0086] In this specification, "ester bond" means -CO-O- or -O-CO- (the "-" in the formula represents a single bond). In this specification, "amide bond" means -CO-NH- or -NH-CO- (the "-" in the formula represents a single bond).

[0087] In this specification, "carbamate bond" means -O-CO-NH- or -NH-CO-O- (where "-" represents a single bond). In this specification, "ether bond" means -O- (i.e., an oxy group) (where "-" represents a single bond).

[0088] In this specification, "urea bond" means -NH-CO-NH- (where "-" in the formula represents a single bond). In this specification, "carbonate bond" means -O-CO-O- (where "-" in the formula represents a single bond).

[0089] Y ａ and Y ｂ The orientation of the bond is not restricted, but Y ａ and Y ｂ When the bond is an ester bond, preferably -Z ａ -CO-O-R ２ａ - and -Z ｂ -CO-O-R ２ｂ It exhibits a - structure (the "-" in the above formula represents a single bond). Y ａ and Y ｂ When the bond is an amide bond, preferably -Z ａ -CO-NH-R ２ａ - and -Z ｂ -CO-NH-R ２ｂ It exhibits a - structure (the "-" in the above formula represents a single bond).

[0090] Y ａ is Y ｂ It may be the same as or different from Y, but preferably Y ａ is Y ｂ It is the same base as [another base].

[0091] Z ａ and Z ｂ Each of these independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly a heteroatom. The aromatic compound preferably has 6 to 12 carbon atoms, more preferably 6 or 7. The aromatic compound also preferably has one aromatic ring.

[0092] Examples of aromatic rings included in aromatic compounds having 3 to 16 carbon atoms include, for aromatic hydrocarbon rings, benzene rings, naphthalene rings, and anthracene rings; and for aromatic hetero rings, imidazole rings, pyrazole rings, oxazole rings, isoxazole rings, thiazole rings, isothiazole rings, triazine rings, pyrrole rings, furan rings, thiophene rings, pyrimidine rings, pyridazine rings, pyrazine rings, pyridine rings, purine rings, pteridine rings, benzimidazole rings, indole rings, benzofuran rings, quinazoline rings, phthalazine rings, quinoline rings, isoquinoline rings, coumarin rings, chromone rings, benzodiazepine rings, phenoxazine rings, phenothiazine rings, acridine rings, etc. Preferably, the rings are benzene rings, naphthalene rings, and anthracene rings, and more preferably, benzene rings.

[0093] The aromatic ring may have substituents, and these substituents may include a C2-C4 acyl group, a C2-C4 alkoxycarbonyl group, a C2-C4 alkylcarbamoyl group, a C2-C4 acyloxy group, a C2-C4 acylamino group, a C2-C4 alkoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a C1-C4 alkylsulfanyl group, a C1-C4 alkylsulfonyl group, a C6-C10 arylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a C1-C4 alkyl group, a ureido group, a C1-C4 alkoxy group, or a C6-C10 atom. Examples include aryl groups, aryloxy groups having 6 to 10 carbon atoms, and preferred examples include acetyl groups, methoxycarbonyl groups, methylcarbamoyl groups, acetoxy groups, acetamide groups, methoxycarbonylamino groups, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methylsulfanyl groups, phenylsulfonyl groups, nitro groups, trifluoromethyl groups, cyano groups, methyl groups, ethyl groups, propyl groups, isopropyl groups, tert-butyl groups, ureido groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups, phenyl groups, and phenoxy groups.

[0094] Z ａ and Z ｂ A preferred specific structure is Z １ These are some examples. Furthermore, Z １ The two ends of the following equation indicate bond positions, not carbon atoms.

[0095]

[0096] In the formula, s represents an integer from 0 to 3, t represents an integer from 0 to 3, u represents an integer from 0 to 4, and u R ４ Each of these independently represents a substituent. In this specification, "s is 0" means Z １ (CH) ２ ) ｓ This means that it does not exist, and that "t is 0" is Z １ (CH) ２ ) ｔ This means that it does not exist, and "u is 0" means Z １ (R) ４ )ｕ This means that it does not exist. Z １ In the above equation, the right side (i.e., (CH ２ ) ｔ -) Y in equation (1) ａ or Y ｂ It combines with the left side (i.e., -(CH ２ ) ｓ It is preferable that it bonds with O in formula (1) (the "-" in the above formula represents a single bond).

[0097] Z １ The value s in the middle is preferably an integer between 0 and 1, and more preferably 0. １ The value of t in the expression is preferably an integer between 0 and 2, and more preferably 1. １ The integer u is preferably an integer between 0 and 2, and more preferably an integer between 0 and 1.

[0098] Z １ R inside ４The substituents are aromatic ring substituents contained in aromatic compounds having 3 to 16 carbon atoms that do not inhibit the reaction in the synthesis process of the ionic lipid. The substituents include acyl groups having 2 to 4 carbon atoms, alkoxycarbonyl groups having 2 to 4 carbon atoms, alkylcarbamoyl groups having 2 to 4 carbon atoms, acyloxy groups having 2 to 4 carbon atoms, acylamino groups having 2 to 4 carbon atoms, alkoxycarbonylamino groups having 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylsulfanyl groups having 1 to 4 carbon atoms, alkylsulfonyl groups having 1 to 4 carbon atoms, arylsulfonyl groups having 6 to 10 carbon atoms, nitro groups, trifluoromethyl groups, cyano groups, alkyl groups having 1 to 4 carbon atoms, ureido groups, alkoxy groups having 1 to 4 carbon atoms, aryl groups having 6 to 10 carbon atoms, and carbon Examples of aryloxy groups with prime numbers 6 to 10 include acetyl groups, methoxycarbonyl groups, methylcarbamoyl groups, acetoxy groups, acetamide groups, methoxycarbonylamino groups, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methylsulfanyl groups, phenylsulfonyl groups, nitro groups, trifluoromethyl groups, cyano groups, methyl groups, ethyl groups, propyl groups, isopropyl groups, tert-butyl groups, ureido groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups, phenyl groups, and phenoxy groups. ４ If there are multiple instances of R, each R ４ They may be the same or different.

[0099] Z ａ is Z ｂ It may be the same as or different from Z, but preferably Z ａ is Z ｂ It is the same base as [another base].

[0100] n ａ and n ｂ Each of these is independently either 0 or 1. In this specification, "n ａ "is 0" means that (Z in equation (1) ａ -Y ａ ) ｎａ This means that it does not exist, "n ｂ "is 0" means that (Z in equation (1) ｂ -Y ｂ ) ｎｂThis means that n does not exist. ａ han ｂ It may be the same as or different from, but preferably n ａ han ｂ It is identical to [the other one].

[0101] R ３ａ and R ３ｂ Each of these is independently a residue derived from a reaction product of a lipid-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and a cyclopropane ring, a group represented by formula (2), a group represented by formula (3), a group represented by formula (4), a group represented by formula (5), a group represented by formula (6), or a group represented by formula (7).

[0102] In one embodiment of the present invention, R ３ａ and R ３ｂ Preferably, each of these is independently a residue derived from a reaction product of a lipid-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by formula (2), a group represented by formula (3), a group represented by formula (4), a group represented by formula (5), or a group represented by formula (6).

[0103] In one embodiment of the present invention, R ３ａ and R ３ｂ More preferably, each of these is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by formula (2), a group represented by formula (3), a group represented by formula (4), a group represented by formula (5), or a group represented by formula (6).

[0104] In one embodiment of the present invention, R ３ａ and R ３ｂ More preferably, each is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by formula (2), or a group represented by formula (3).

[0105] "Residues derived from the reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride" refers to a fat-soluble vitamin having a hydroxyl group where the hydroxyl group is *-O-CO-CH ２ -CH ２ - or * - O - CO - CH ２ -CH ２ -CH ２ This represents a group with a structure that has been replaced by a hyphen. * indicates the binding position with fat-soluble vitamins.

[0106] Examples of fat-soluble vitamins containing a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachisterol, tocopherol, and tocotrienol. Tocopherol is preferred as the fat-soluble vitamin containing a hydroxyl group.

[0107] "Residues derived from the reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride" refers to residues where the hydroxyl group of the sterol derivative having a hydroxyl group is *-O-CO-CH ２ -CH ２ - or * - O - CO - CH ２ -CH ２ -CH ２ This represents a group with a structure that has been replaced by a hyphen. * indicates the bond position with the sterol derivative.

[0108] Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, with cholesterol or cholestanol being preferred.

[0109] Aliphatic hydrocarbon groups having 1 to 40 carbon atoms may be linear or branched. These aliphatic hydrocarbon groups may be saturated or unsaturated. In the case of unsaturated aliphatic hydrocarbon groups, the number of unsaturated bonds in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, but carbon-carbon double bonds are preferred. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and even more preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., but alkyl groups or alkenyl groups are preferred. Aliphatic hydrocarbon groups having 1 to 40 carbon atoms include, specifically, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, tetracontyl, dodecenyl, tridecenyl, tetracosyl, tetracosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, tetracontyl, dodecenyl, tridecenyl, tetracosyl Radecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icocenyl group, henicocenyl group, dococenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadedecadienyl group, hexadedecadienyl group, heptadedecadienyl group, octadedecadienyl group, nonadedecadienyl group, ico Examples include sadienyl group, henicosadienyl group, docosadienyl group, octadecatrienyl group, eicosatrienyl group, eicosatetraenyl group, eicosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group, and 1-decylundecyl group.The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably an octyl group, tridecyl group, pentadecyl group, heptadecyl group, nonadecyl group, heptadecenyl group, heptadecadienyl group, or 1-hexylnonyl group, and is particularly preferably a tridecyl group, heptadecyl group, heptadecenyl group, or heptadecadienyl group.

[0110] In one embodiment of the present invention, R ３ａ and R ３ｂ The aliphatic hydrocarbon group having 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms), represented by , is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in the -CO-O- in formula (1). Specific examples of aliphatic hydrocarbon groups include the heptadecadienyl group when linoleic acid is used as the fatty acid, and the heptadecenyl group when oleic acid is used as the fatty acid.

[0111] R ３ａ and R ３ｂ In this context, "a C3-C40 alkyl group having a cyclopropane ring" means an alkyl group having 3 to 40 carbon atoms that has at least one cyclopropane ring in its alkyl chain. The number of carbon atoms in the alkyl group (C3-C40) does not include the carbon atoms of the cyclopropane ring. The number of cyclopropane rings in the alkyl group is preferably one. ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the above is preferably of formula (8):

[0112]

[0113] The group is represented by (8) (wherein * represents a bond position, b and c each represent an integer independently, and the sum of b and c is an integer between 2 and 39). Preferably, b is an integer between 1 and 20, and c is an integer between 1 and 19. More preferably, b is an integer between 2 and 18, even more preferably between 3 and 17, and even more preferably between 4 and 12. More preferably, c is an integer between 3 and 15, even more preferably between 3 and 11, and even more preferably between 3 and 9. An example of the group represented by (8) is the 7-(2-octylcyclopropyl)heptyl group.

[0114] In equation (2), as explained above, * represents a bond position, not a carbon atom. Therefore, "*-" in equation (2) represents a single bond. The same meaning applies to * in other equations.

[0115] In equation (2), R ６ This is an alkylene group having 2 to 10 carbon atoms. In this specification, the alkylene group may be linear or branched. Examples of alkylene groups include a methylene group, an ethylene group, and a trimethylene group (-(CH4). ２ ) ３ -), propylene group (-CH(CH ３ )CH ２ -ien-CH ２ CH (CH ３ )-), tetramethylene group (-(CH ２ ) ４ -), butylene group (-CH(C ２ H ５ )CH ２ -ien-CH ２ CH(C) ２ H ５ )-), pentamethylene group (-(CH ２ ) ５ -), hexamethylene group (-(CH ２ ) ６ -), heptamethylene group (-(CH ２ ) ７ -), octamethylene group (-(CH ２ ) ８ -), nonamethylene group (-(CH ２ ) ９ -), decamethylene group (-(CH ２ ) １０ (-) is one example (the "-" in the above formula represents a single bond).

[0116] In equation (2), R ６ Preferably, it is an alkylene group having 3 to 9 carbon atoms, more preferably an alkylene group having 3 to 7 carbon atoms, and even more preferably a trimethylene group or a heptamethylene group.

[0117] In equation (2), R ７R is an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms. ７ One of the methylene groups may be replaced by an ether bond.

[0118] In this specification, "R ７ "One of the methylene groups may be replaced by one ether bond" means R ７ This means that one methylene group in the alkyl group, one methylene group in the alkenyl group, or one methylene group in the alkynyl group may each be independently replaced by one ether bond.

[0119] In this specification, alkyl groups may be linear or branched. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, 1-octylnonyl, octadecyl, nonadecyl, and Examples include cosyl group, henicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, hentriacontyl group, dotriacontyl group, tritriacontyl group, tetratriacontyl group, pentatriacontyl group, hexatriacontyl group, tetracontyl group, hentetracontyl group, dotetracontyl group, tritetracontyl group, and tetratetracontyl group.

[0120] In this specification, the alkenyl group may be linear or branched. Also, in this specification, the number of olefinic carbon-carbon double bonds in the alkenyl group may be only one or two or more. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, 1-ethenylhexyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icocenyl, henicocenyl, dococenyl, tricocenyl, tetracocenyl, pentacocenyl, hexacocenyl, heptacocenyl, octacocenyl, nonacocenyl, triacontenyl, hentriacontenyl, and dotriacontenyl groups.

[0121] In this specification, the alkynyl group may be linear or branched. Also, in this specification, the number of carbon-carbon triple bonds in the alkynyl group may be one or two or more. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octinyl, noninyl, desinyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadesinyl, heptadecynyl, octadecynyl, nonadesinyl, icosinyl, henicosinyl, docosinyl, tricosinyl, tetracosinyl, pentacosinyl, hexacosinyl, heptacosinyl, octacosinyl, nonacosinyl, triacontinyl, hentriacontinyl, and dotriacontinyl.

[0122] R ７ is preferably an alkyl group having 1 to 20 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and R ７ One of the methylene groups may be replaced by an ether bond. ７ The group is more preferably a 1-octyrnonyl group, a 1-ethenylhexyl group, or a 2-(2-methylpropoxy)ethyl group.

[0123] In equation (2), L１ This represents an ester bond (-CO-O- or -O-CO-) or a carbamate bond (-O-CO-NH- or -NH-CO-O-) (the "-" in the above formula represents a single bond). L １ If the bond is an ester bond, L １ The carbonyl group (-CO-) inside is R in formula (2) ６ It combines with L １ The oxy group (-O-) inside is R in formula (2) ７ It is preferable that it is bonded with L. １ If it is a carbamate bond, L １ The imino group (-NH-) inside is R in formula (2) ６ It combines with L １ The oxy group (-O-) inside is R in formula (2) ７ It is preferable that it is bonded with.

[0124] In formula (3), * represents a bond position, not a carbon atom, as described above. In formula (3), R ８ This represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ８ It may be substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms.

[0125] In this specification, "R ８ The phrase "may be substituted with substituents selected from the group consisting of 3-14 member heterocyclic groups and 3-12 carbon-12 hydrocarbon ring groups" means that R ８ This means that the alkyl group, alkenyl group, alkynyl group, or hydrocarbon ring group may each be independently substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms. ８ Other expressions similar to "may be substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms" include "R ８This has the same meaning as "may be substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms." In this specification, unless otherwise specified regarding substituents, "alkyl group," "alkenyl group," "alkynyl group," "alkylene group," "alkendiyl group," "alkindiyl group," etc., refer to unsubstituted groups.

[0126] In this specification, "carbon 3-12 hydrocarbon ring group" means a cyclic group in which the ring is composed of 3 to 12 carbon atoms. Examples of carbon 3-12 hydrocarbon ring groups include carbon 3-8 cycloalkyl groups, phenyl groups, naphthyl groups, and adamantyl groups. Examples of carbon 3-8 cycloalkyl groups include cyclopropyl groups, cyclobutyl groups, cyclopentyl groups, cyclohexyl groups, cycloheptyl groups, and cyclooctyl groups. The carbon 3-12 hydrocarbon ring group is preferably a carbon 3-12 non-aromatic hydrocarbon ring group, more preferably a carbon 3-8 cycloalkyl group or adamantyl group, and even more preferably a cyclohexyl group or adamantyl group.

[0127] In this specification, "3- to 14-membered heterocyclic group" means a heterocyclic group having 3 to 14 ring-forming atoms. Examples of 3- to 14-membered heterocyclic groups include 5- to 14-membered aromatic heterocyclic groups and 3- to 14-membered non-aromatic heterocyclic groups. Preferably, the 3- to 14-membered heterocyclic group is a 3- to 14-membered non-aromatic heterocyclic group.

[0128] Examples of 5- to 14-membered aromatic heterocyclic groups as used herein include: (i) 5- to 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, and triazinyl groups; (ii) Benzothiophenyl group, benzofuranyl group, benzimidazolyl group, benzoxazolyl group, benzoisoxazolyl group, benzothiazolyl group, benzoisothiazolyl group, benzotriazolyl group, imidazopyridinyl group, thienopyridinyl group, phlopyridinyl group, pyrrolopyridinyl group, pyrazolopyridinyl group, oxazolopyridinyl group, thiazolopyridinyl group, imidazopyridinyl group, imidazopyridinyl group, thienopyridinyl group, phlopyridinyl group, pyrrolopyridinyl group, pyrazolopyridinyl group, ox 8- to 14-membered condensed polycyclic aromatic heterocyclic groups such as zolopyrimidinyl group, thiazolopyrimidinyl group, pyrazolotriazinyl group, naphtho[2,3-b]thienyl group, phenoxathiinyl group, indolyl group, isoindolyl group, 1H-indazolyl group, prinyl group, isoquinolyl group, quinolyl group, phthalazinyl group, naphthylidinyl group, quinoxalinyl group, quinazolinyl group, synnolinyl group, carbazolyl group, β-carbolinyl group, phenantridinyl group, acridinyl group, phenazinyl group, phenothiazinyl group, and phenoxazinyl group.

[0129] In this specification, examples of 3- to 14-membered non-aromatic heterocyclic groups include: (i) azilidinyl group, oxyranyl group, thyranyl group, azetidinyl group, oxetanyl group, thietanyl group, tetrahydrothienyl group, tetrahydrofuranyl group, pyrrolinyl group, pyrrolidinyl group, imidazolinyl group, imidazolidinyl group, oxazolinyl group, oxazolidinyl group, pyrazolinyl group, pyrazolidinyl group, thiazolinyl group, thiazolidinyl group, tetrahydroisothiazolyl group, dithiolanyl group (e.g., 1,2-dithiolan-3-yl group), tetrahydrooxazolyl group , monocyclic non-aromatic heterocyclic groups of 3 to 8 members such as tetrahydroisoxazolyl group, piperidinyl group, piperazinyl group, tetrahydropyridinyl group, dihydropyridinyl group, dihydrothiopyranyl group, tetrahydropyrimidinyl group, tetrahydropyridazinyl group, dihydropyranyl group, tetrahydropyranyl group, tetrahydrothiopyranyl group, morpholinyl group, thiomorpholinyl group, azepanyl group, diazepanyl group, azepinyl group, oxepanyl group, azokanyl group, and diazokanyl group; (ii) Dihydrobenzofuranyl group, dihydrobenzimidazolyl group, dihydrobenzoxazolyl group, dihydrobenzothiazolyl group, dihydrobenzoisothiazolyl group, dihydronaphtho[2,3-b]thienyl group, tetrahydroisoquinolyl group, tetrahydroquinolyl group, 4H-quinolidinyl group, indolinyl group, isoindolinyl group, tetrahydrothieno[2,3-c]pyridinyl group, tetrahydrobenzoazepinyl group, tetrahydroquinoxalinyl group, tetrahydro 9- to 14-membered condensed polycyclic non-aromatic heterocyclic groups such as lophenanthidinyl group, hexahydrophenothiazinyl group, hexahydrophenoxazinyl group, tetrahydrophthalazinyl group, tetrahydronaphthilidinyl group, tetrahydroquinazolinyl group, tetrahydrosinnolinyl group, tetrahydrocarbazolyl group, tetrahydro-β-carbolinyl group, tetrahydroacridinyl group, tetrahydrophenazinyl group, tetrahydrothioxanthenyl group, and octahydroisoquinolyl group.

[0130] In one embodiment of the present invention, R ８Preferably, the alkyl group is a C1-C20 alkyl group, a C2-C20 alkenyl group, a C2-C20 alkynyl group, or a C3-C12 hydrocarbon ring group, and the alkyl group may be substituted with a C3-C12 hydrocarbon ring group or a 3-C14 non-aromatic heterocyclic group.

[0131] In one embodiment of the present invention, R ８ The more preferably, the C1-C20 alkyl group, the C2-C20 alkenyl group, the C2-C20 alkynyl group, or the cyclohexyl group, and the alkyl group may be substituted with a cyclohexyl group, an adamantyl group, or a dithiolanyl group.

[0132] In one embodiment of the present invention, R ８ The C1-C10 alkyl group, the C2-C10 alkenyl group, the C2-C10 alkynyl group, or the cyclohexyl group, and the alkyl group may be substituted with a cyclohexyl group, an adamantyl group, or a dithiolanyl group.

[0133] In formula (4), * represents a bond position, not a carbon atom, as described above. In formula (4), R ９ R represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, and R ９ It may be substituted with substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms.

[0134] In this specification, "alkendiyl group" means a divalent group having a structure obtained by removing two hydrogen atoms from an alkene. In this specification, the alkenediyl group may be linear or branched. In this specification, the number of olefinic carbon-carbon double bonds in the alkene or alkenediyl group may be only one or two or more. Examples of alkenediyl groups include ethendiyl, propendiyl, butendiyl, pentendiyl, hexendiyl, heptendiyl, octendiyl, nonendiyl, and decendiyl groups.

[0135] In this specification, "alkynediyl group" means a divalent group having a structure obtained by removing two hydrogen atoms from an alkyne. In this specification, the alkynediyl group may be linear or branched. In this specification, the number of carbon-carbon triple bonds in the alkyne or alkynediyl group may be only one or two or more. Examples of alkynediyl groups include ethindiyl group, propindiyl group, butindiyl group, pentindiyl group, hexindiyl group, heptindiyl group, octindiyl group, nonindiyl group, and decinediyl group.

[0136] In equation (4), R ９ Preferably, it is an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, more preferably an alkylene group having 2 to 9 carbon atoms, even more preferably an alkylene group having 2 or 3 carbon atoms, and particularly preferably a trimethylene group.

[0137] In equation (4), R １０ and R １１ Each of these independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, R １０ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １１ At least one ethylene group or at least one trimethylene group in the compound may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R １０ and R １１ Each of these may be independently substituted with a substituent selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms.

[0138] R １０ and R １１Preferably, each is independently a C1-C17 alkyl group, a C2-C17 alkenyl group, or a C2-C17 alkynyl group; more preferably, each is independently a C1-C17 alkyl group; even more preferably, a C1-C10 alkyl group; and particularly preferably, both are propyl groups.

[0139] In equation (4), L ２ L represents an oxygen atom, NH, or sulfur atom. ２ is preferably an oxygen atom or NH, and more preferably an oxygen atom.

[0140] In formula (5), * represents a bond position, not a carbon atom, as described above. In formula (5), R １２ is a hydrogen atom, a benzyl group, or *-Si(R １３ ) (Caution １４ ) (Caution １５ ) group (wherein * represents a bond position, and R １３ ~R １５ Each of these independently represents an alkyl group or phenyl group having 1 to 4 carbon atoms.

[0141] R １２ Preferably a hydrogen atom, a benzyl group, or *-Si(R １３ ) (Caution １４ ) (Caution １５ ) group (In the above formula, * represents the bond position, R １３ represents a tert-butyl group, and R １４ and R １５ (both represent a methyl group or a phenyl group.) and more preferably a hydrogen atom.

[0142] In formula (6), * represents a bond position, not a carbon atom, as described above. In formula (6), R １６ R represents an alkylene group with 5 to 10 carbon atoms. １６ The group is preferably a heptamethylene group.

[0143] In equation (6), R １７ ~R １９each independently represents 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, R １７ wherein at least one ethylene group or at least one trimethylene group in R １８ may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and at least one ethylene group or at least one trimethylene group in R １９ may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, and at least one ethylene group or at least one trimethylene group in R １７ to R １９ may each independently be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0144] R １７ to R １９ are preferably each independently 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 each independently an alkyl group having 1 to 10 carbon atoms.

[0145] In formula (7), * represents a bonding position, not a carbon atom, as described above. In formula (7), R ２０ represents an alkylene group having 1 to 10 carbon atoms, an alkanediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms. R ２０ is preferably an alkylene group having 1 to 10 carbon atoms, and more preferably an alkylene group having 2 to 5 carbon atoms.

[0146] In formula (7), R ２１ and R ２２Each of these independently represents 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. ２１ and R ２２ Preferably, each is independently a C2-C10 alkyl group or a C2-C10 alkenyl group, and more preferably, each is independently a C2-C10 alkenyl group.

[0147] R ３ａ is R ３ｂ It may be the same as or different from, but preferably R ３ａ is R ３ｂ It is the same base as [another base].

[0148] In one embodiment of the present invention, R １ａ is R １ｂ It is identical to X ａ is X ｂ It is identical to R ２ａ is R ２ｂ It is identical to Y ａ is Y ｂ It is identical to Z ａ is Z ｂ It is identical to R ３ａ is R ３ｂ It is identical to the following. Preferred examples of ionic lipids (1) include the following ionic lipids.

[0149] [Ionic lipids (1-1)] R １ａ and R １ｂ However, each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene group, ethylene group); X ａ and X ｂ However, each is independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., -N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ However, each is independently an alkylene group having 1 to 8 carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Y ａ and Y ｂHowever, each is independently either an ester bond or an amide bond; Z ａ and Z ｂ However, each independently, a divalent group (e.g., -C) is derived from an aromatic compound having 3 to 16 carbon atoms, possessing at least one aromatic ring, and possibly having a heteroatom. ６ H ４ -CH ２ -ien-CH ２ -C ６ H ４ -CH ２ -) and; n ａ and n ｂ However, each is independently either 0 or 1; and R ３ａ and R ３ｂ However, each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), and a group represented by formula (2) (wherein formula (2), R ６ R represents an alkylene group with 3 to 9 carbon atoms. ７ R represents an alkyl group having 1 to 20 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and ７ One of the methylene groups may be replaced by one ether bond, and L １ represents an ester bond or a carbamate bond. ), the group represented by formula (3) (in formula (3), R ８ R represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and the alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms or a non-aromatic heterocyclic group having 3 to 14 members. ), the group represented by formula (4) (in formula (4), R ９ R represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms. １０ and R １１ Each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, and L２ represents an oxygen atom or NH.), the group represented by formula (5) (in formula (5), R １２ is a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ ) group (in the above formula, * represents a bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ each represent a methyl group or a phenyl group).), or a group represented by the above formula (6) (in formula (6), R １６ represents an alkylene group having 5 to 10 carbon atoms, and R １７ to R １９ each independently represent 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.); which is an ionic lipid (1). In this ionic lipid (1), R ３ａ and R ３ｂ are preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by the above formula (2), or a group represented by the above formula (3).

[0150] [Ionic Lipid (1-2)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group); X ａ and X ｂ are each independently X １ :

[0151]

[0152] (wherein R ５ represents an alkyl group having 1 to 3 carbon atoms (e.g., methyl group)), or X ２ :

[0153]

[0154] (wherein p represents 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Yａ and Y ｂ However, each is independently either an ester bond or an amide bond; Z ａ and Z ｂ However, each is independent of Z １ :

[0155]

[0156] (wherein s represents an integer between 0 and 1, t represents an integer between 0 and 2, u represents an integer between 0 and 2 (preferably 0), and u R ４ Each of these independently represents a substituent. ) and; n ａ and n ｂ However, each is independently either 0 or 1; and R ３ａ and R ３ｂ However, each is independently an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), and a group represented by formula (2) (wherein formula (2), R ６ R represents an alkylene group with 3 to 7 carbon atoms. ７ R represents an alkyl group having 1 to 20 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and ７ One of the methylene groups may be replaced by one ether bond, and L １ represents an ester bond or a carbamate bond. ), the group represented by formula (3) (in formula (3), R ８ R represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a cyclohexyl group, and the alkyl group may be substituted with a cyclohexyl group, an adamantyl group, or a dithiolanyl group. ), the group represented by formula (4) (in formula (4), R ９ R represents an alkylene group with 2 to 9 carbon atoms. １０ and R １１ Each of these independently represents an alkyl group having 1 to 17 carbon atoms, and L ２ represents an oxygen atom. ), the group represented by formula (5) (in formula (5), R １２ is a hydrogen atom, a benzyl group, or *-Si(R １３ ) (Caution １４) (Caution １５ ) group (In the above formula, * represents the bond position, R １３ represents a tert-butyl group, and R １４ and R １５ Both represent a methyl group or a phenyl group. ), or a group represented by formula (6) (in formula (6), R １６ represents an alkylene group having 5 to 10 carbon atoms, and R １７ ~R １９ Each of these independently represents an alkyl group having 1 to 10 carbon atoms. ) is; ionic lipid (1). In this ionic lipid (1), R ３ａ and R ３ｂ Preferably, each is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by formula (2), or a group represented by formula (3).

[0157] [Ionic lipids (1-3)] R １ａ and R １ｂ However, each is independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); X ａ and X ｂ However, each is independent of X ２ :

[0158]

[0159] (In the formula, p represents 1 or 2.) and; R ２ａ and R ２ｂ However, each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Y ａ and Y ｂ However, each is independently either an ester bond or an amide bond; Z ａ and Z ｂ However, each is independent of Z １ :

[0160]

[0161] (In the formula, s represents an integer between 0 and 1, t represents an integer between 0 and 2, and u represents 0.) and n ａ and n ｂHowever, each is independently either 0 or 1; and R ３ａ and R ３ｂ However, each is independently an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), and a group represented by formula (2) (wherein formula (2), R ６ R represents an alkylene group with 3 to 7 carbon atoms. ７ R represents an alkyl group having 1 to 20 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and ７ One of the methylene groups may be replaced by one ether bond, and L １ represents an ester bond or a carbamate bond. ), the group represented by formula (3) (in formula (3), R ８ R represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, an alkynyl group having 2 to 10 carbon atoms, or a cyclohexyl group, and the alkyl group may be substituted with a cyclohexyl group, an adamantyl group, or a dithiolanyl group. ), the group represented by formula (4) (in formula (4), R ９ R represents an alkylene group with 2 or 3 carbon atoms. １０ and R １１ Each of these independently represents an alkyl group having 1 to 10 carbon atoms, and L ２ represents an oxygen atom. ), the group represented by formula (5) (in formula (5), R １２ R represents a hydrogen atom. ), or a group represented by formula (6) (wherein R represents a hydrogen atom. １６ represents an alkylene group having 5 to 10 carbon atoms, and R １７ ~R １９ Each of these independently represents an alkyl group having 1 to 10 carbon atoms. ) is; ionic lipid (1). In this ionic lipid (1), R ３ａ and R ３ｂ Preferably, each is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by formula (2), or a group represented by formula (3).

[0162] [Ionic lipids (1-4)] R １ａ and R １ｂHowever, each is independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); X ａ and X ｂ However, each is independent of X ２ :

[0163]

[0164] (In the formula, p represents 1 or 2.) and; R ２ａ and R ２ｂ However, each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Y ａ and Y ｂ However, each is independently either an ester bond or an amide bond; Z ａ and Z ｂ However, each is independent of Z １ :

[0165]

[0166] (In the formula, s represents an integer between 0 and 1, t represents an integer between 0 and 2, and u represents 0.) and n ａ and n ｂ However, each is independently either 0 or 1; and R ３ａ and R ３ｂ However, each is independently an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group), and a group represented by formula (2) (wherein formula (2), R ６ R represents an alkylene group with 3 to 7 carbon atoms. ７ L represents a 1-octyrnonyl group, a 1-ethenylhexyl group, or a 2-(2-methylpropoxy)ethyl group, and L １ represents an ester bond or a carbamate bond. ), the group represented by formula (3) (in formula (3), R ８ R represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, an alkynyl group having 2 to 10 carbon atoms, or a cyclohexyl group, and the alkyl group may be substituted with a cyclohexyl group, an adamantyl group, or a dithiolanyl group. ), the group represented by formula (4) (in formula (4), R９ represents a trimethylene group, and R １０ and R １１ both represent propyl groups, and L ２ represents an oxygen atom. ), a group represented by the above formula (5) (in formula (5), R １２ represents a hydrogen atom), or a group represented by the above formula (6) (in formula (6), R １６ represents a heptamethylene group, and R １７ to R １９ each independently represent an alkyl group having 1 to 10 carbon atoms. ); which is an ionic lipid (1). In this ionic lipid (1), R ３ａ and R ３ｂ are preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, a group represented by the above formula (2), or a group represented by the above formula (3).

[0167] As specific examples of the ionic lipid (1), the compounds described in the following Tables 1-1 to 1-10 may be mentioned. In addition, "TBS" in the structural formula of Compound 35 means a tert-butyldimethylsilyl group.

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] In one embodiment of the present invention, the ionic lipid (1) is preferably at least one selected from the group consisting of the compounds listed in Tables 1-1 to 1-10, more preferably at least one selected from the group consisting of SS-OP, SS-OB, and compounds 1 to 41, and even more preferably at least one selected from the group consisting of SS-OP, compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 19, compound 20, compound 22, compound 23, compound 24, compound 25, compound 27, compound 29, compound 31, compound 34, compound 36, compound 37, compound 38, compound 39, and compound 41. More preferably, it is at least one selected from the group consisting of compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 19, compound 20, compound 22, compound 23, compound 24, compound 25, compound 27, compound 29, compound 31, compound 34, compound 36, compound 37, compound 38, compound 39, and compound 41; particularly preferably, it is at least one selected from the group consisting of compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 20, compound 22, compound 34, and compound 39; and most preferably, it is compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 20, compound 22, compound 34, or compound 39.

[0179] In one embodiment of the present invention, the ionic lipid (1) is preferably at least one selected from the group consisting of the compounds listed in Tables 1-1 to 1-6, more preferably at least one selected from the group consisting of SS-OP, SS-EC, SS-OB, and compounds 1 to 17, even more preferably at least one selected from the group consisting of SS-OP and compounds 1 to 17, even more preferably at least one selected from the group consisting of SS-OP, compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, and compound 16, even more preferably at least one selected from the group consisting of compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, and compound 16, and particularly preferably compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, or compound 16.

[0180] The following describes ionic lipids represented by the following formula (10). Ionic lipid (10) may be used alone or in combination of two or more types.

[0181]

[0182] (In formula (10), R ２６ and R ２７ Each of these independently represents 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, or R ２６ and R ２７ These atoms bond to each other, forming a 5- or 6-membered nitrogen-containing heterocycle with the nitrogen atoms to which they bond, R ２９ and R ２８ Each of these independently represents an aliphatic hydrocarbon group having 5 to 50 carbon atoms, and R ２８ At least one ethylene group in the group may be replaced by an ester bond, and R ２９ At least one ethylene group in the compound may be replaced by an ester bond, and R ３０ (This represents an alkylene group with 1 to 10 carbon atoms.)

[0183] The definitions of each symbol in equation (10) will be explained in order below. R２６ and R ２７ Each of these independently represents 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 may be linear or branched, but is preferably linear. ２６ and R ２７ The number of carbon atoms in the alkyl group, alkenyl group, and alkynyl group is preferably 1 to 4, and more preferably 2, independently of each other.

[0184] R ２６ and R ２７ These may bond with each other, together with the nitrogen atom to which they bond, to form a 5- or 6-membered nitrogen-containing heterocycle. The 5- or 6-membered nitrogen-containing heterocycle may contain only one nitrogen atom (the nitrogen atom in formula (10)) or two or more nitrogen atoms. ２６ and R ２７ Examples of five- or six-membered nitrogen-containing heterocycles include pyrrolidinyl, piperidyl, pyrazolidinyl, imidazolidinyl, and piperazinyl.

[0185] R ２６ and R ２７ Preferably, each is independently a C1-C10 alkyl group, a C2-C10 alkenyl group, or a C2-C10 alkynyl group; more preferably, each is independently a C1-C10 alkyl group; even more preferably, each is independently a C1-C4 alkyl group; and particularly preferably, both are ethyl groups.

[0186] R ２９ and R ２８ Each of these independently represents an aliphatic hydrocarbon group having 5 to 50 carbon atoms, and R ２８ At least one ethylene group in the group may be replaced by an ester bond, and R ２９ At least one ethylene group in the compound may be replaced by an ester bond. ２９ and R ２８ The description of the aliphatic hydrocarbon group is the same as the description of the "aliphatic hydrocarbon group with 1 to 40 carbon atoms" in formula (1) above, except for the number of carbon atoms.２９ and R ２８ The number of carbon atoms in the aliphatic hydrocarbon group is preferably 10 to 40, more preferably 10 to 30, and even more preferably 10 to 25.

[0187] R ２９ and R ２８ Preferably, each is independently a C10-C40 alkyl group, a C10-C40 alkenyl group, a C10-C40 alkynyl group, or formula (11): *-R ３１ -CO-O-R ３２ (11) (In formula (11), * represents the bond position, R ３１ R represents an alkylene group with 1 to 10 carbon atoms. ３２ This represents an alkyl group having 10 to 30 carbon atoms, an alkenyl group having 10 to 30 carbon atoms, or an alkynyl group having 10 to 30 carbon atoms.

[0188] R ２９ and R ２８ Preferably, each is independently a C10-C30 alkyl group, a C10-C30 alkenyl group, a C10-C30 alkynyl group, or formula (11): *-R ３１ -CO-O-R ３２ (11) (In formula (11), * represents the bond position, R ３１ R represents an alkylene group with 1 to 5 carbon atoms. ３２ This represents an alkyl group having 10 to 30 carbon atoms, an alkenyl group having 10 to 30 carbon atoms, or an alkynyl group having 10 to 30 carbon atoms.

[0189] R ２９ and R ２８ More preferably, each independently comprises a C10-C20 alkyl group, a C10-C20 alkenyl group, or formula (11): *-R ３１ -CO-O-R ３２ (11) (In formula (11), * represents the bond position, R ３１ R represents an alkylene group with 1 to 5 carbon atoms. ３２This represents an alkyl group or alkenyl group having 10 to 20 carbon atoms.

[0190] R ３０ This represents an alkylene group having 1 to 10 carbon atoms, preferably an alkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 4 carbon atoms, and even more preferably an ethylene group. The following are examples of suitable ionic lipids (10).

[0191] [Ionic lipids (10-1)] R ２６ and R ２７ However, each is independently 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; R ２９ and R ２８ However, each can independently be an alkyl group having 10 to 40 carbon atoms, an alkenyl group having 10 to 40 carbon atoms, an alkynyl group having 10 to 40 carbon atoms, or formula (11): *-R ３１ -CO-O-R ３２ (11) (In formula (11), * represents the bond position, R ３１ R represents an alkylene group with 1 to 10 carbon atoms. ３２ (represents an alkyl group having 10 to 30 carbon atoms, an alkenyl group having 10 to 30 carbon atoms, or an alkynyl group having 10 to 30 carbon atoms.) and also R ３０ However, it is an alkylene group having 1 to 6 carbon atoms; ionic lipid (10).

[0192] [Ionic lipids (10-2)] R ２６ and R ２７ However, each is independently an alkyl group having 1 to 10 carbon atoms; R ２９ and R ２８ However, each can independently be an alkyl group having 10 to 30 carbon atoms, an alkenyl group having 10 to 30 carbon atoms, an alkynyl group having 10 to 30 carbon atoms, or formula (11): *-R ３１ -CO-O-R ３２ (11) (In formula (11), * represents the bond position, R ３１ R represents an alkylene group with 1 to 5 carbon atoms. ３２R represents an alkyl group having 10 to 30 carbon atoms, an alkenyl group having 10 to 30 carbon atoms, or an alkynyl group having 10 to 30 carbon atoms. ３０ However, it is an alkylene group having 1 to 4 carbon atoms; ionic lipid (10).

[0193] [Ionic lipids (10-3)] R ２６ and R ２７ However, each is independently an alkyl group having 1 to 4 carbon atoms (e.g., ethyl group); R ２９ and R ２８ However, more preferably, each independently, is an alkyl group having 10 to 20 carbon atoms, an alkenyl group having 10 to 20 carbon atoms, or formula (11): *-R ３１ -CO-O-R ３２ (11) (In formula (11), * represents the bond position, R ３１ R represents an alkylene group with 1 to 5 carbon atoms. ３２ ) represents an alkyl group having 10 to 20 carbon atoms or an alkenyl group having 10 to 20 carbon atoms. ) and R ３０ However, it is preferably an ethylene group; an ionic lipid (10).

[0194] Specific examples of ionic lipids (10) include the compounds listed in Table 2 below.

[0195]

[0196] In one embodiment of the present invention, the ionic lipid is preferably SS-OP, SS-EC, SS-OB, Compound 1 to Compound 44, [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl, 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl At least one selected from the group consisting of 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate, More preferably SS-OP, SS-OB, Compound 1 to Compound 44, [(4-hydroxybutyl)azandiyl]bis(hexane-6,At least one selected from the group consisting of 1-diyl)bis(2-hexyldecanoate) and heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, more preferably at least one selected from the group consisting of SS-OP, compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 19, compound 20, compound 22, compound 23, compound 24, compound 25, compound 27, compound 29, compound 31, compound 34, compound 36, compound 37, compound 38, compound 39, and compound 41. More preferably, it is at least one selected from the group consisting of compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 19, compound 20, compound 22, compound 23, compound 24, compound 25, compound 27, compound 29, compound 31, compound 34, compound 36, compound 37, compound 38, compound 39, and compound 41; particularly preferably, it is at least one selected from the group consisting of compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 20, compound 22, compound 34, and compound 39; and most preferably, it is compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, compound 16, compound 18, compound 20, compound 22, compound 34, or compound 39.

[0197] In one embodiment of the present invention, the ionic lipid is preferably SS-OP, SS-EC, SS-OB, Compound 1 to Compound 17, [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl, 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl At least one selected from the group consisting of 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate, More preferably SS-OP, Compounds 1 to 17, [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,At least one selected from the group consisting of 4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate, more preferably at least one selected from the group consisting of SS-OP and compounds 1 to 17, even more preferably at least one selected from the group consisting of SS-OP, compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, and compound 16, particularly preferably at least one selected from the group consisting of compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, and compound 16, and most preferably compound 1, compound 2, compound 4, compound 8, compound 13, compound 14, or compound 16.

[0198] The amount of ionic lipids in the lipid nanoparticles of the present invention is preferably 15 to 75 mol%, more preferably 20 to 72.5 mol%, and even more preferably 30 to 70 mol%, relative to the total amount of ionic lipids, phospholipids, and cholesterol, from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency in brain cells, and stability of lipid nanoparticles. In this specification, "amount of B relative to A (mol%)" means "100 × amount of B (mol) / amount of A (mol)". Therefore, "amount of ionic lipid relative to the total amount of ionic lipids, phospholipids, and cholesterol" means "100 × amount of ionic lipid (1) (mol) / total amount of ionic lipids, phospholipids, and cholesterol (mol)".

[0199] Ionic lipids include commercially available products (e.g., "ALC-0315" from Jenkem Technology USA; "DLin-MC3-DMA" (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) and "SM-102" from Cayman Chemical; Echelon Biosciences "cKK-E12" (i.e., 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione) and "C12-200" (i.e., 1,1'-[[2-[4-[2-[[2-[[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol) manufactured by Inc. can be used. In addition, the ionic lipid (1) can be produced by known methods (e.g., the methods described in WO 2019 / 188867 A1, US 9708628 B2, WO 2021 / 195529 A2, WO 2024 / 203577 A1). Furthermore, the ionic lipid (10) can be produced by known methods (for example, the methods described in WO 2023 / 112939 A1 and WO 2026 / 029040 A1).

[0200] The lipid nanoparticles of the present invention contain phospholipids. One type of phospholipid may be used, or two or more types may be used in combination.

[0201] Examples of phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), and their lyso derivatives. In this specification, phospholipids may be represented by abbreviations. For example, 1,2-diacyl-sn-glycero-3-phosphocholine may be represented as PC, and 1,2-didecanoyl-sn-glycero-3-phosphocholine may be represented as DDPC.

[0202] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) include the following: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), and 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC). 1,2-Diecocenoyl-sn-glycero-3-phosphocholine (DEiPC), 1,2-Dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-Diecocyl-sn-glycero-3-phosphocholine (DEPC), 1-Myristoyl-2-Palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-Myristoyl-2-Stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-Palmitoyl-2-Myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-Palmitoyl-2-Stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-Palmitoyl-2-Oleoyl-sn-glycero-3-phosphocholine (POPC), 1-Stearoyl-2-Oleoyl-sn-Glycerol-3-Phosphocholine (SOPC).

[0203] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) include the following: 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (DDPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1,2-dielcoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine (MPPE), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (MSPE), 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphoethanolamine (PMPE), 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (PSPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

[0204] The phospholipid preferably comprises at least one selected from the group consisting of DOPE, POPE, DOPC, DSPC, DPPC, and DMPC, more preferably at least one selected from the group consisting of DOPE, POPE, DOPC, DSPC, DPPC, and DMPC, even more preferably DOPE, POPE, DOPC, DSPC, DPPC, or DMPC, and particularly preferably DPPC. These phospholipids are thought to produce similar effects because they have similar acyl chain structures.

[0205] The amount of phospholipids in the lipid nanoparticles of the present invention is preferably 0 to 45 mol%, more preferably 0 to 40 mol%, and even more preferably 3 to 35 mol%, relative to the total of ionic lipids, phospholipids, and cholesterol, from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency within brain cells, and stability of lipid nanoparticles.

[0206] The lipid nanoparticles of the present invention contain cholesterol. The amount of cholesterol in the lipid nanoparticles of the present invention is preferably 15 to 65 mol%, more preferably 20 to 60 mol%, and even more preferably 25 to 55 mol%, relative to the total of ionic lipids, phospholipids, and cholesterol, from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency in brain cells, and stability of lipid nanoparticles.

[0207] The lipid nanoparticles of the present invention contain PEG lipids. Only one type of PEG lipid may be used, or two or more types may be used in combination.

[0208] In one embodiment of the present invention, the PEG lipid is preferably of formula (9): CH ２ (OR ２３ )-CH(OR ２４ ) - CH ２ (OR ２５ ) (9) (In formula (9), R ２３ , R ２４ and R ２５ Two of these represent myristoyl groups, and the remaining one represents a C1-C6 alkyl group linked via a polyethylene glycol (PEG) chain with a number average molecular weight of 1,000-3,000. This is dimyristoylglycerol PEG represented by ).

[0209] The number-average molecular weight of the PEG chain in formula (9) is 1,000 to 3,000, preferably 1,500 to 2,500. The number-average molecular weight of the PEG used to form this PEG chain can be measured by gel permeation chromatography (GPC).

[0210] The alkyl group having 1 to 6 carbon atoms may be linear or branched. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of alkyl groups having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, 1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, and 2,3-dimethylbutyl group. The methyl group is preferred.

[0211] In one embodiment of the present invention, the PEG lipid is at least one selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG), and N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), more preferably at least one selected from the group consisting of DMG-PEG and DSPE-PEG. The number-average molecular weight of the PEG chain of these PEG lipids is 1,000 to 6,000, preferably 1,500 to 5,500. The number-average molecular weight of the PEG used to form this PEG chain can be measured by gel permeation chromatography (GPC).

[0212] PEG lipids may have reactive groups that can bind to ligands. Examples of reactive groups that can bind to ligands include azide groups (-N ３Examples of reactive groups include maleimide groups, NHS ester groups (N-succinimidyloxycarbonyl groups), sulfanyl groups, DBCO groups (11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl groups), among which azide groups are preferred. In the following, PEG lipids having reactive groups that can bind to ligands will be referred to as activated PEG lipids, and PEG lipids not having reactive groups that can bind to ligands will be referred to as inactivated PEG lipids.

[0213] The amount of PEG lipid in the lipid nanoparticles of the present invention is preferably 0.5 to 5 mol%, more preferably 0.6 to 4 mol%, and even more preferably 0.7 to 3 mol%, relative to the total amount of ionic lipids, phospholipids, and cholesterol, from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency within brain cells, and stability of lipid nanoparticles. When activated and inactivated PEG lipids are used in combination, the amount of PEG lipid is the sum of the amount of activated and inactivated PEG lipids.

[0214] Other Lipids The lipid nanoparticles of the present invention may also contain lipids other than ionic lipids, cholesterol, PEG lipids, and phospholipids (hereinafter sometimes referred to as "other lipids"). Examples of other lipids include sterols other than cholesterol. Only one type of other lipid may be used, or two or more types may be used in combination. However, it is preferable not to use other lipids in the present invention. In other words, the lipids constituting the lipid nanoparticles of the present invention preferably consist of ionic lipids, cholesterol, and PEG lipids, and optionally phospholipids.

[0215] Lipid Composition In the lipid nanoparticles of the present invention, the molar ratio of ionic lipids:phospholipids:cholesterol:PEG lipids is preferably 15-75:0-45:15-65:0.5-5, more preferably 20-72.5:0-40:20-60:0.6-4, and even more preferably 30-70:3-35:25-55:0.7-3.

[0216] Nucleic acids used in the present invention include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, nucleic acids can be 1- to 3-stranded, but are preferably 1-stranded or 2-stranded. Nucleic acids may be, for example, nucleotides having N-glycosides of purine or pyrimidine bases, oligomers having a non-nucleotide backbone (e.g., commercially available peptide nucleic acids (PNA), etc.), or oligomers having special bonds (however, the oligomer contains nucleotides having a configuration that allows for base pairing and base attachment, as found in DNA and RNA).

[0217] Furthermore, nucleic acids may include, for example, nucleic acids with known modifications, nucleic acids with labels known in the field, capped nucleic acids, methylated nucleic acids, nucleic acids in which one or more natural nucleotides are replaced with related products, nucleic acids with modified intramolecular nucleotides, nucleic acids having uncharged bonds (e.g., methyl sulfonates, phosphotriesters, phosphoramidates, carbamates, etc.), nucleic acids having charged bonds or sulfur-containing bonds (e.g., phosphorothioates, phosphorodithioates, etc.), nucleic acids having side chain groups such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (e.g., monosaccharides, etc.), nucleic acids containing intercurrent compounds (e.g., acridine, psoralen, etc.), nucleic acids containing chelate compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), nucleic acids containing alkylating agents, nucleic acids having modified bonds (e.g., α-anomeric nucleic acids, etc.).

[0218] The type of DNA used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples of DNA include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc. Plasmid DNA, cDNA, and antisense DNA are preferred, and plasmid DNA is more preferred. Circular DNA such as plasmid DNA can be digested with restriction enzymes as appropriate and used as linear DNA.

[0219] The type of RNA used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples of RNA include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. Preferably, gRNA, mRNA, siRNA, miRNA, shRNA, antisense RNA, and RNA replicon.

[0220] In the present invention, the nucleic acids used are preferably purified by methods commonly used by those skilled in the art.

[0221] The amount of nucleic acid in the lipid nanoparticles of the present invention is determined by the N / P ratio (= amount of amino groups of ionic lipids (nmol) / amount of phosphate groups of nucleic acid (nmol)). From the viewpoint of nucleic acid encapsulation efficiency and toxicity reduction, the N / P ratio is preferably 5 to 200 nmol / nmol, more preferably 10 to 150 nmol / nmol, and even more preferably 15 to 100 nmol / nmol.

[0222] Other Components: The lipid nanoparticles of the present invention may contain other components other than nucleic acids and lipids, provided that they do not inhibit the effects of the present invention. These other components may be used individually or in combination of two or more.

[0223] The ligand is preferably an antibody that has specific affinity for a target molecule on the surface of brain cells.

[0224] Examples of target molecules include transferrin receptors, glucose transporters, MDR1, LAT1, BCRP, SMVT1, Claudin 5, CD147, and LDL receptors.

[0225] Antibodies that have specific affinity for target molecules on the surface of brain cells include, but are not limited to, anti-transferrin receptor antibodies, anti-glucose transporter antibodies, anti-insulin receptor antibodies, anti-LAT1 antibodies, anti-MDR1 antibodies, anti-BCRP antibodies, anti-SMVT1 antibodies, anti-Clauudin 5 antibodies, anti-CD147 antibodies, and anti-LDL receptor antibodies. Alternatively, antibodies used as existing antibody drugs or antibodies that recognize the same antigens can be used as ligands to target target cells expressing the antigen.

[0226] The antibodies used in this invention may be complete antibody molecules, or single-chain antibodies (scFv), Fab, F(ab'). ２ This may include any other antibody fragment such as Fab', Fv, reductive antibody (rIgG), dsFv, sFv, diabody, triabody, or an antibody conjugated with an enzyme, etc. (e.g., Pavinafusp alfa), or one into which a reactive group (e.g., a DBCO group) has been introduced. In this specification, unless otherwise specified, "antibody" is used to include antibody fragments, antibodies conjugated with enzymes, etc., and antibodies into which a reactive group has been introduced.

[0227] The ligand is more preferably an anti-transferrin receptor antibody, an anti-glucose transporter antibody, an anti-insulin receptor antibody, an anti-LAT1 antibody, an anti-MDR1 antibody, an anti-BCRP antibody, an anti-SMVT1 antibody, an anti-Clauudin 5 antibody, an anti-CD147 antibody, an anti-LDL receptor antibody, or Pavinafusp alfa, and even more preferably an anti-transferrin receptor antibody.

[0228] In the lipid nanoparticles of the present invention, it is preferable that the ligand exists bound to the lipid, more preferably that the ligand exists bound to the activated PEG lipid, and even more preferably that the ligand to which a reactive group has been introduced exists bound to the activated PEG lipid.

[0229] From the viewpoint of achieving both selectivity for target cells and desirable particle properties, the amount of ligand in the lipid nanoparticles of the present invention is preferably 0.01 to 5 mol%, more preferably 0.05 to 3 mol%, relative to the total lipids in the lipid nanoparticles of the present invention. It is preferable to mix the suspension of nucleic acid-encapsulated lipid nanoparticles with ligand-binding lipids to obtain such an amount of ligand.

[0230] Method for Producing Lipid Nanoparticles of the Present Invention The lipid nanoparticles of the present invention can be produced by known methods such as those described in WO 2023 / 054243 A1. For example, by mixing an alcohol solution containing lipids and a solution containing nucleic acids in an apparatus including a microfluidic channel (e.g., NanoAssemblr® (Precision NanoSystems)) or a vortex mixer, a suspension containing nucleic acid-encapsulated lipid nanoparticles can be produced. Then, a solution containing a ligand can be added, and the nucleic acid-encapsulated lipid nanoparticles and the ligand can be reacted to produce ligand-modified lipid nanoparticles encapsulating nucleic acids (i.e., lipid nanoparticles of the present invention). The solvent for the alcohol solution containing lipids is preferably ethanol. The solvent for the solution containing nucleic acids is preferably a buffer, more preferably an acidic malate buffer. The solvent for the solution containing the ligand is preferably a buffer, more preferably an acidic malate buffer.

[0231] For example, using an apparatus including a microfluidic channel (e.g., NanoAssemblr® (Precision NanoSystems)), a suspension of nucleic acid-free lipid nanoparticles can be produced by mixing an alcohol solution containing lipids with an acidic buffer (step a), mixing the nucleic acid-free lipid nanoparticles with a nucleic acid solution (step b), and mixing the suspension of nucleic acid-encapsulated lipid nanoparticles with a ligand-binding lipid (step c).

[0232] For example, using an apparatus including a microfluidic channel (e.g., NanoAssemblr® (Precision NanoSystems)), a suspension of nucleic acid-free lipid nanoparticles can be produced by mixing an alcohol solution containing lipids with an acidic buffer (step a); a suspension of nucleic acid-free ligand-modified lipid nanoparticles can be produced by mixing the nucleic acid-free lipid nanoparticles with water and a ligand-binding lipid (step c'); and a suspension of nucleic acid-encapsulated ligand-modified lipid nanoparticles (i.e., the lipid nanoparticles of the present invention) can be produced by mixing the nucleic acid-free ligand-modified lipid nanoparticles with a nucleic acid solution (step b').

[0233] For example, a suspension of ligand-modified lipid nanoparticles containing nucleic acids (i.e., the lipid nanoparticles of the present invention) can be produced by mixing nucleic acid-free lipid nanoparticles, a nucleic acid solution, and ligand-binding lipids in an apparatus including a microfluidic channel (e.g., NanoAssemblr® (Precision NanoSystems)).

[0234] It is preferable to mix an alcohol solution containing lipids or a suspension of lipid nanoparticles without encapsulated nucleic acids with a solution containing nucleic acids so that the N / P ratio in the lipid nanoparticles of the present invention falls within the range described above.

[0235] The suspension produced by the method described above contains lipid nanoparticles of the present invention and a dispersion medium (preferably ethanol and a buffer). However, the dispersion medium (especially ethanol) can be removed, the dispersion medium (especially the buffer) can be replaced, etc., by operations such as ultrafiltration, dialysis, and dilution.

[0236] The present invention also provides a pharmaceutical composition for use in the treatment of brain diseases, comprising the lipid nanoparticles of the present invention (i.e., ligand-modified lipid nanoparticles encapsulating nucleic acids). In addition to the lipid nanoparticles of the present invention, the pharmaceutical composition of the present invention typically comprises a pharmaceutically acceptable carrier. The amount of the lipid nanoparticles of the present invention is preferably 0.001 to 20% by weight, more preferably 0.005 to 10% by weight, relative to the entire pharmaceutical composition of the present invention.

[0237] Pharmaceutically acceptable carriers include those commonly used as pharmaceutical materials. For example, in solid formulations, excipients, lubricants, binders, and disintegrants are used, while in liquid formulations, solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics are used. Furthermore, the pharmaceutical composition of the present invention may optionally contain pharmaceutical additives such as preservatives, antioxidants, colorants, and sweeteners.

[0238] The lipid nanoparticles of the present invention can be used in a method for treating brain diseases. This treatment method involves administering the lipid nanoparticles of the present invention (i.e., ligand-modified lipid nanoparticles encapsulating nucleic acids) to a target.

[0239] By administering the lipid nanoparticles of the present invention to a target, the nucleic acids encapsulated within the lipid nanoparticles are delivered to brain cells. The targets to which the lipid nanoparticles of the present invention can be administered are not particularly limited, and examples include mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.). The target to which the lipid nanoparticles of the present invention can be administered is preferably humans or other mammals.

[0240] The method of administering the lipid nanoparticles of the present invention to a target is not particularly limited as long as the lipid nanoparticles of the present invention can deliver nucleic acids to brain cells, and any known administration method (for example, oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, transpulmonary administration, transnasal administration, intraperitoneal administration, spray, etc.)) can be appropriately selected. The dosage of the lipid nanoparticles of the present invention can be appropriately selected considering the type of target, the administration method, etc.

[0241] The present invention will be described in more detail using the following examples, but the present invention is not limited in any way to the following examples.

[0242] In the following examples, ionic lipids other than ALC-0315 and SM-102 are indicated by the names listed in the table above. The meanings of the abbreviations used in the following examples are as follows. ALC-0315: [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) Azide-PEG-DSPE: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azide(polyethylene glycol)-2000] (manufactured by Avanti Polar Lipids) BSA: Bovine serum albumin DBCO-NHS: Dibenzocyclooctin-N-hydroxysuccinimidyl ester (manufactured by Merck-Millipore) DiD: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate DMG-PEG2000: 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (number average molecular weight of PEG chain: 2000, manufactured by NOF Corporation as "SUNBRIGHT® GM-020EX") DMG-PEG5k: 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (number average molecular weight of PEG chain: 5000, manufactured by NOF Corporation as "SUNBRIGHT® GM-050EX") DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPE-PEG2k: N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (number average molecular weight of PEG chain: 2000, manufactured by NOF Corporation as "SUNBRIGHT®") PM-020CN) DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DPPC: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine DSG-PEG2k: 1,2-Distearoyl-rac-glycero-3-methylpolyoxyethylene (Number average molecular weight of PEG chain: 2000, manufactured by NOF Corporation "SUNBRIGHT® GS-020EX") DSG-PEG2k-Azide: 1,2-Distearoyl-rac-glycero-3-(2'-azidoethyl) polyoxyethylene (PEG chain number average molecular weight: 2000, manufactured by NOF Corporation, "SUNBRIGHT® GS-020AZ") DSG-PEG3.4k-Azide: 1,2-Distearoyl-rac-glycero-3-(2'-azidoethyl) polyoxyethylene (PEG chain number average molecular weight: 3400, manufactured by NOF Corporation, "SUNBRIGHT® GS-034AZ") DSG-PEG5k-Azide: 1,2-Distearoyl-rac-glycero-3-(2'-azidoethyl) polyoxyethylene (PEG chain number average molecular weight: 5000, manufactured by NOF Corporation, "SUNBRIGHT®") GS-050AZ) DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine DSPE-PEG2k: N-(methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Number average molecular weight of PEG chain: 2000, manufactured by NOF Corporation, "SUNBRIGHT® DSPE-020CN") DSPE-PEG2k-Azide: N-[(5-azido-1-oxopentyl)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Number average molecular weight of PEG chain: 2000, manufactured by NOF Corporation, "SUNBRIGHT® DSPE-020AZ2") DSPE-PEG3.4k-Azide:N-[(5-azido-1-oxopentyl)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (number-average molecular weight of PEG chain: 3400, manufactured by NOF Corporation as "SUNBRIGHT® DSPE-034AZ2") DSPE-PEG5k-Azide:N-[(5-azido-1-oxopentyl)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (PEG chain number average molecular weight: 5000, manufactured by NOF Corporation, "SUNBRIGHT® DSPE-050AZ2") FACS buffer: A buffer containing 9.6 g of powdered PBS (manufactured by Shimadzu Diagnostics Corporation), 5.0 g of BSA (manufactured by Nacalai Tesque Corporation), and 1.0 g of sodium azide (manufactured by Fujifilm Wako Pure Chemical Industries Ltd.) per 1 L of water LNP: Lipid nanoparticles PBS: Phosphate-buffered saline PBS-BSA buffer: A mixture of 19 mL of PBS and 1 mL of MACS® BSA Stock Solution (manufactured by Milltenyi Biotec) POPE: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine EDTA: Ethylenediaminetetraacetic acid SM-102: Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate Tris・HCl: Tris(hydroxymethyl)aminomethane hydrochloride Triton X-100: Nonionic surfactant (manufactured by Nacalai Tesque Co., Ltd.)

[0243] [Preparation Example 1] Preparation of aqueous solution containing ligand-bound PEG lipid Using Amicon Ultra-4 (MWCO: 50 kDa), the solvent for the aqueous solution of anti-transferrin receptor antibody (manufactured by BioXCell) is 100 mM NaHCO3 ３ The solvent was replaced with an aqueous solution (pH: 9.0). The antibody aqueous solution with the buffer solvent replaced was mixed with 1 equivalent of DBCO-NHS, and the resulting mixture was incubated at room temperature for 2 hours. The mixture was further incubated at 4°C overnight. Using Amicon Ultra-4 (MWCO: 3 kDa), the solvent in the mixture was replaced with ultrapure water to prepare an aqueous DBCO-anti-transferrin receptor antibody solution.

[0244] As described above, 10.1 μL of 74 μM DBCO-anti-transferrin receptor antibody aqueous solution, 7.47 μL of 0.1 mM Azide-PEG-DSPE aqueous solution, and 32.5 μL of 2-morpholinoethanesulfonic acid buffer (pH: 6.0) were mixed, and the resulting mixture was incubated overnight at 4°C to prepare an aqueous solution containing ligand-bound PEG lipid (ligand: anti-transferrin receptor antibody).

[0245] [Examples 1-1 to 1-5] Preparation of ligand-modified LNPs encapsulating mRNA A lipid solution was prepared by mixing a 20 mM SS-OP ethanol solution, a 20 mM cholesterol ethanol solution, and a 20 mM DOPC ethanol solution in a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with a 2 mM DMG-PEG2000 ethanol solution to prepare a lipid solution (amount of DMG-PEG2000 relative to the total of ionic lipids (SS-OP), cholesterol, and phospholipids (DOPC): 1.5 mol%).

[0246] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM Cholesterol concentration: 3.2 mM DOPC concentration: 0.6 mM DMG-PEG2000 concentration: 0.12 mM Total lipid concentration: 8.12 mM

[0247] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min and a lipid solution at a flow rate of 2 mL / min for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0248] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0249] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0250] A 150 μL LNP suspension containing mRNA and an aqueous solution containing ligand-bound PEG lipid obtained in Preparation Example 1 (19.95 μL, 40.05 μL, 79.5 μL, 160.5 μL, or 199.5 μL) were mixed using a vortex mixer under agitation, and the resulting mixture was incubated at 4°C for 30 minutes. After incubation, phosphate buffer (280.05, 259.95, 220.5, 139.5, 100.5 μL, buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare a ligand-modified LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 6.7 μg / mL).

[0251] (Amount of ligand relative to total lipids (hereinafter sometimes abbreviated as "amount of ligand")) Example 1-1: 0.1 mol% Example 1-2: 0.2 mol% Example 1-3: 0.4 mol% Example 1-4: 0.8 mol% Example 1-5: 1.0 mol%

[0252] [Comparative Example 1] Preparation of LNPs encapsulating mRNA but not modified with ligands A lipid solution was prepared by mixing a 20 mM SS-OP ethanol solution, a 20 mM cholesterol ethanol solution, and a 20 mM DOPC ethanol solution in a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with a 2 mM DMG-PEG2000 ethanol solution to prepare a lipid solution (amount of DMG-PEG2000 relative to the total of ionic lipids (SS-OP), cholesterol, and phospholipids (DOPC): 1.5 mol%).

[0253] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM Cholesterol concentration: 3.2 mM DOPC concentration: 0.6 mM DMG-PEG2000 concentration: 0.12 mM Total lipid concentration: 8.12 mM

[0254] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min and a lipid solution at a flow rate of 2 mL / min for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0255] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0256] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0257] [Test Example 1] Evaluation of LNP particle size, PdI, and mRNA encapsulation rate. The particle size, Polydispersity Index (PdI), and mRNA encapsulation rate of ligand-modified LNPs encapsulating mRNA from Examples 1-1 to 1-5 and LNPs encapsulating mRNA from Comparative Example 1 were analyzed. Particle size and PdI were measured by dynamic light scattering using Zetasizer®. The mRNA encapsulation rate was measured by the Ribogreen® assay. The results are shown in Table 3 below.

[0258]

[0259] [Test Example 2] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension containing mRNA from Examples 1-1 to 1-5, an LNP suspension containing mRNA from Comparative Example 1, or PBS (mRNA amount: 1.0 μg).

[0260] 21 hours after the aforementioned administration, the mice were euthanized and their whole brains were removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brains, and the brains were lysed using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13,000 rpm and 4°C, and 20 μL of the supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate. The luminescence derived from NanoLuc® luciferase was measured using GloMax. The protein concentration of a solution obtained by diluting the supernatant 100-fold with ultrapure water was measured by the BCA method, and the luminescence intensity was standardized by the protein content. The results are shown in Table 4.

[0261]

[0262] As shown in Table 4, the ligand-modified LNPs encapsulating mRNA in Examples 1-1 to 1-5 showed improved luminescence intensity compared to the mRNA-encapsulated LNP in Comparative Example 1, and a significant improvement in gene expression activity was observed in mouse brains.

[0263] [Test Example 3] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension containing mRNA from Example 1-2 (ligand amount: 0.2 mol%), an LNP suspension containing mRNA from Comparative Example 1, or PBS. When using the ligand-modified LNP suspension containing mRNA from Example 1-2, the amount of mRNA administered to the mice was 1.0 μg, 5.0 μg, or 18.0 g. When using the LNP suspension containing mRNA from Comparative Example 1, the amount of mRNA administered to the mice was 1.0 μg.

[0264] 21 hours after the aforementioned administration, the mice were euthanized and their whole brains were removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris-HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brains, and the brains were lysed using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13,000 rpm and 4°C for 10 minutes. 20 μL of this supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate, and the luminescence derived from NanoLuc® luciferase was measured using GloMax. The protein concentration of a solution obtained by diluting the supernatant 100-fold with ultrapure water was measured by the BCA method, and the luminescence intensity was standardized by the protein content.

[0265]

[0266] As shown in Table 5, the ligand-modified LNPs encapsulating mRNA in Examples 1-2 showed improved luminescence intensity compared to the mRNA-encapsulated LNP in Comparative Example 1, and a significant improvement in gene expression activity was observed in the mouse brain. Furthermore, increasing the amount of mRNA administered to the mice resulted in a further improvement in gene expression activity.

[0267] [Examples 2-1 to 2-6] Preparation of ligand-modified LNPs encapsulating mRNA Lipid solutions were prepared under the same conditions as in Example 1-2, except that the following phospholipids were used and a 1 mM DiD ethanol solution was added to the lipid solution (amount of DMG-PEG2000 relative to the total of ionic lipids (SS-OP), cholesterol, and phospholipids: 1.5 mol%, amount of DiD relative to the total of ionic lipids (SS-OP), cholesterol, and phospholipids: 0.5 mol%).

[0268] (Phospholipids used) Example 2-1: DOPE Example 2-2: POPE Example 2-3: DOPC Example 2-4: DSPC Example 2-5: DPPC Example 2-6: DMPC

[0269] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM Cholesterol concentration: 3.2 mM Phospholipid concentration: 0.6 mM DMG-PEG2000 concentration: 0.12 mM DiD concentration: 0.04 mM Total lipid concentration: 8.16 mM

[0270] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min, and each lipid solution at a flow rate of 2 mL / min, for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0271] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0272] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0273] A 150 μL LNP suspension containing mRNA and a 40.05 μL aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. After incubation, 259.95 μL of phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare a ligand-modified LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 6.7 μg / mL, amount of ligand relative to total lipids: 0.2 mol%).

[0274] In Examples 2-1, 2-2, and 2-5, which used DOPE, POPE, or DPPC as the phospholipid, a 500 μL LNP suspension containing mRNA and a 133.5 μL aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. The incubated mixture was added to Amicon Ultra-4 (MWCO: 100 kDa) to which 1 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been pre-added, and the resulting mixture was ultrafiltered. To the obtained concentrate (63.35 μL), phosphate buffer (86.65 μL, buffer concentration: 9.6 mM, pH: 7.4) was added to obtain a ligand-modified LNP suspension containing encapsulated mRNA (total mRNA amount: 10 μg, N / P ratio: 36, mRNA concentration: 66.7 μg / mL, ligand amount relative to total lipids: 0.2 mol%).

[0275] [Test Example 4] Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, Polydispersity Index (PdI), and mRNA encapsulation rate of ligand-modified LNPs encapsulating mRNA from Examples 2-1 to 2-6 were analyzed. Particle size and PdI were measured by dynamic light scattering using Zetasizer®. mRNA encapsulation rate was measured by Ribogreen® assay. The results are shown in Table 6 below.

[0276]

[0277] [Test Example 5] Evaluation of LNP uptake in cerebral vascular endothelial cells in vivo C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension containing mRNA from Examples 2-1 to 2-6 (amount of ligand relative to total lipids: 0.2 mol%) or PBS (amount of mRNA administered in Examples 2-1, 2-2, and 2-5: 1.0 μg or 10.0 μg; amount of mRNA administered in Examples 2-3, 2-4, and 2-6: 1.0 μg).

[0278] 21 hours after the aforementioned administration, the mice were euthanized and their entire brains were removed. The removed brains were then cut into eight sections using a scalpel. Subsequently, the cut brains were added to a gentleMACS C Tube (Miltenyi Biotec) to which enzyme mix 1 (1950 μL) from the Adult Brain Dissociation Kit (Miltenyi Biotec) had been added. Enzyme mix 2 (30 μL) was then added to the resulting mixture. The gentleMACS C Tube was set in the gentleMACS Dissociators and the Heating Unit was attached. Program 37C_ABDK_01 was started to dissociate the cells. After the program was completed, the gentleMACS C Tube was removed from the gentleMACS Dissociators, and the cell suspension was transferred to a 50 mL tube fitted with a SmartStrainer (Miltenyi Biotec) (70 μm). The SmartStrainer was washed with PBS (10 mL). The SmartStrainer was removed from the tube and centrifuged at 4°C and 300 G for 10 minutes. The supernatant was then removed with an aspirator to obtain the cell pellet.

[0279] The obtained cell pellet was suspended in PBS (3100 μL), and the resulting cell suspension was transferred to a 15 mL tube. Debris Removal Solution (Miltenyi Biotec) (900 μL) was added to the cell suspension and mixed thoroughly. PBS (4 mL) was gently added to the resulting mixture, and the mixture was centrifuged at 4°C and 2300 G for 10 minutes. Of the three layers formed, the top two were removed using an aspirator to remove the Debris Removal Solution. Then, PBS was added to the mixture after the removal of the Debris Removal Solution until the volume was 15 mL, and after gentle inversion mixing, the mixture was centrifuged at 4°C and 1000 G for 10 minutes. The upper layer was then removed using an aspirator to obtain the cell pellet.

[0280] The obtained cell pellet was suspended in 1 mL of 1 × Red Blood Cell Lysis Solution (Miltenyi Biotec), and the resulting cell suspension was incubated in a refrigerator for 10 minutes. PBS-BSA buffer (10 mL) was added to the incubated cell suspension, and the mixture was centrifuged at 4°C and 300 G for 10 minutes. The supernatant was then removed to obtain a cell pellet from which red blood cells had been removed. The cell concentration was 1 × 10⁶. ７ PBS-BSA buffer was added to the obtained cell pellet to obtain a cell suspension with a cell-to-cell ratio of 90 μL. CD45 MicroBreads (Miltenyi Biotec) (10 μL) were added to the obtained cell suspension and incubated in a refrigerator for 15 minutes. PBS-BSA buffer (1 mL) was added to the incubated cell suspension, and the resulting mixture was centrifuged at 4°C and 300 G for 5 minutes. The upper layer was then removed using an aspirator to obtain the cell pellet.

[0281] PBS-BSA buffer (500 μL) was added to the obtained cell pellet to obtain a cell suspension. An LD column (Miltenyi Biotec) was set in the magnetic location of a MACS separator, and the column was rinsed with PBS-BSA buffer (2 mL). Then, the cell suspension was applied to the column, and the flow-through containing unlabeled cells was collected in a tube. The column was washed twice with PBS-BSA buffer (1 mL), and the unlabeled cells (CD45-negative cells) that passed through were collected in the same tube. The obtained CD45-negative cells were centrifuged at 4°C and 300 G for 10 minutes, and then the supernatant was removed with an aspirator to obtain a cell pellet.

[0282] Cell concentration is 1 × 10 ７PBS-BSA buffer was added to the obtained cell pellet to obtain a cell suspension with a cell-to-cell ratio of 90 μL. CD31 MicroBeads (Miltenyi Biotec) (10 μL) were added to the obtained cell suspension and incubated in a refrigerator for 15 minutes. PBS-BSA buffer (1 mL) was added to the incubated cell suspension, and the resulting mixture was centrifuged at 4°C and 300 G for 5 minutes. The upper layer was then removed using an aspirator to obtain the cell pellet.

[0283] PBS-BSA buffer (500 μL) was added to the obtained cell pellet to obtain a cell suspension. An MS column (Miltenyi Biotec) was set in the magnetic location of the MACS Separator, and the column was rinsed with PBS-BSA buffer (500 μL). The cell suspension was applied to the column, and the flow-through containing unlabeled cells was collected in a tube. The column was washed three times with PBS-BSA buffer (500 μL). The unlabeled cells that passed through were then collected in the same tube. The column was removed from the separator, and PBS-BSA buffer (1 mL) was added to the column. The plunger was firmly pressed into the column, and CD45 － CD31 ＋ The cells were immediately washed away, and the cell suspension was collected in a new 15 mL tube. CD31 ＋ To increase cell purity, a new MS column was used for CD31 ＋ The cell selection procedure was repeated. The resulting CD31 ＋ The cell suspension containing the cells was centrifuged at 4°C and 300G for 10 minutes, and the supernatant was removed using an aspirator to obtain a cell pellet.

[0284] To the obtained cell pellet, 1 mL of FACS buffer was added, the mixture was centrifuged, and the supernatant was removed to obtain a cell pellet. Again, 1 mL of FACS buffer was added to the obtained cell pellet and transferred to a 1.5 mL tube to obtain a cell suspension concentrated with cerebral vascular endothelial cells.

[0285] The obtained cell suspension (18 μL) was mixed with acridine orange (2 μL), and cell counting was performed using the resulting mixture with LUNA-FL (Logos Biosystems). The cell suspension was centrifuged at 4°C and 500 G for 3 minutes, and the supernatant was removed to obtain a cell pellet. A blocking solution was prepared by mixing anti-CD16 / 32 antibody (0.6 μL) and FACS buffer (29.4 μL) to prevent nonspecific staining.

[0286] The obtained blocking solution (15 μL) was added to the obtained cell pellet and tapped. The resulting cell suspension was allowed to stand at 4°C and protected from light for 10 minutes. BV605-labeled CD45 antibody and PE-labeled CD31 antibody were diluted with the blocking solution to the manufacturer's recommended concentrations to obtain an antibody cocktail for staining. The antibody cocktail for staining (17 μL) was added to the cell suspension (15 μL), and the resulting mixture was allowed to stand at 4°C and protected from light for 30 minutes. Five minutes before observation, 7-AAD Viability Staining Solution (BioLegend) (1.5 μL) was added to the mixture, and dead cell staining was performed. To the cell suspension containing the staining antibody and 7-AAD reagent, 1 mL of FACS buffer was added, and the centrifugation and supernatant removal procedure was repeated twice. The resulting cell pellet was suspended in 250 μL of FACS buffer, and the obtained suspension (sample) was analyzed using Novocyte to calculate the percentage of vascular endothelial cells that had taken up LNPs relative to the total number of vascular endothelial cells, as the percentage of DiD-positive cells (%). In other words, the group of cells stained with PE-labeled anti-CD31 antibody among the living cells in the cell suspension was defined as vascular endothelial cells, and the percentage of cells exhibiting DiD fluorescence within this population of vascular endothelial cells was further calculated. The results are shown in Table 7.

[0287]

[0288] As shown in Table 7, ligand-modified LNPs encapsulating mRNA in Examples 2-1 to 2-6, which used different phospholipids, all demonstrated delivery of LNPs to cerebral vascular endothelial cells. Furthermore, increasing the amount of mRNA administered to mice resulted in a further improvement in gene expression activity.

[0289] [Examples 3-1 to 3-6] Preparation of ligand-modified LNPs encapsulating mRNA A lipid solution was prepared under the same conditions as in Example 1-2, except that the following phospholipids were used (amount of DMG-PEG2000 relative to the total of ionic lipids (SS-OP), cholesterol, and phospholipids: 1.5 mol%).

[0290] (Phospholipids used) Example 3-1: DOPE Example 3-2: POPE Example 3-3: DOPC Example 3-4: DSPC Example 3-5: DPPC Example 3-6: DMPC

[0291] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM Cholesterol concentration: 3.2 mM Concentration of each phospholipid: 0.6 mM DMG-PEG2000 concentration: 0.12 mM Total lipid concentration: 8.12 mM

[0292] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min, and each lipid solution at a flow rate of 2 mL / min, for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0293] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0294] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0295] A 150 μL LNP suspension containing mRNA and a 40.05 μL aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. After incubation, 259.95 μL of phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare a ligand-modified LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 6.7 μg / mL, amount of ligand relative to total lipids: 0.2 mol%).

[0296] In Examples 3-1, 3-2, and 3-5, which used DOPE, POPE, or DPPC as the phospholipid, a 500 μL LNP suspension containing mRNA and a 133.5 μL aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. The incubated mixture was added to Amicon Ultra-4 (MWCO: 100 kDa) to which 1 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been pre-added, and the resulting mixture was ultrafiltered. To the obtained concentrate (63.35 μL), phosphate buffer (86.65 μL, buffer concentration: 9.6 mM, pH: 7.4) was added to obtain a ligand-modified LNP suspension containing encapsulated mRNA (total mRNA amount: 10 μg, N / P ratio: 36, mRNA concentration: 66.7 μg / mL, ligand amount: 0.2 mol%).

[0297] [Test Example 6] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension or PBS containing mRNA from Examples 3-1 to 3-6 (amount of mRNA administered in Examples 3-1, 3-2, and 3-5: 1.0 μg or 10.0 μg; amount of mRNA administered in Examples 3-3, 3-4, and 3-6: 1.0 μg).

[0298] 21 hours after the aforementioned administration, the mice were euthanized and their whole brains were removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brains, and the brains were lysed using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13,000 rpm and 4°C, and 20 μL of the supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate. The luminescence derived from NanoLuc® luciferase was measured using GloMax. The protein concentration of a solution obtained by diluting the supernatant 100-fold with ultrapure water was measured by the BCA method, and the luminescence intensity was standardized by the protein content. The results are shown in Table 8.

[0299]

[0300] As shown in Table 8, ligand-modified LNPs encapsulating mRNA in Examples 3-1 to 3-6, which used different phospholipids, all exhibited luminescence in the mouse brain.

[0301] [Example 4] Preparation of ligand-modified LNPs encapsulating mRNA A lipid solution was prepared under the same conditions as in Examples 1-2, except that DPPC was used as the phospholipid (amount of DMG-PEG2000 relative to the total of ionic lipids (SS-OP), cholesterol, and phospholipids (DPPC): 1.5 mol%).

[0302] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: SS-OP concentration: 4.2 mM Cholesterol concentration: 3.2 mM DPPC concentration: 0.6 mM DMG-PEG2000 concentration: 0.12 mM Total lipid concentration: 8.12 mM

[0303] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min, and each lipid solution at a flow rate of 2 mL / min, for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0304] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0305] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding Cre recombinase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed using a vortex mixer under agitation. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0306] A 500 μL LNP suspension containing mRNA and an aqueous solution containing ligand-bound PEG lipid obtained in Preparation Example 1 (133.5 μL) were mixed under agitation using a vortex mixer, and the resulting mixture was incubated at 4°C for 30 minutes. The incubated mixture was added to Amicon Ultra-4 (MWCO: 100 kDa) to which 1 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been pre-added, and the resulting mixture was ultrafiltered. To the resulting concentrate of 63.35 μL, phosphate buffer (86.65 μL, buffer concentration: 9.6 mM, pH: 7.4) was added to obtain a ligand-modified LNP suspension containing mRNA (total amount of mRNA: 12 μg, N / P ratio: 36, mRNA concentration: 66.7 μg / mL, amount of ligand: 0.2 mol%).

[0307] [Test Example 7] Evaluation of LNP gene expression activity in vivo B6. Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)HHe / J mice were administered a ligand-modified LNP suspension containing mRNA from Example 4 via the tail vein (mRNA amount: 1.0 μg).

[0308] Forty-eight hours after the aforementioned administration, the mice were euthanized and their entire brains were removed. The dissected brains were then added to a gentleMACS C Tube (Miltenyi Biotec) containing enzyme mix 1 (1950 μL) from the Adult Brain Dissociation Kit (Miltenyi Biotec). Enzyme mix 2 (30 μL) was then added to the resulting mixture. The gentleMACS C Tube was placed in a gentleMACS Dissociator and fitted with a Heating Unit. Program 37C_ABDK_01 was started to dissociate the cells. After the program was completed, the gentleMACS C Tube was removed from the gentleMACS Dissociators, and the cell suspension was transferred to a 50 mL tube fitted with a SmartStrainer (Miltenyi Biotec) (70 μm). The SmartStrainer was washed with PBS (10 mL). The SmartStrainer was removed from the tube and centrifuged at 4°C and 300 G for 10 minutes. The supernatant was then removed with an aspirator to obtain the cell pellet.

[0309] PBS (3100 μL) was added to the obtained cell pellet to obtain a cell suspension. The obtained cell suspension was transferred to a 15 mL tube. Debris Removal Solution (Miltenyi Biotec) (900 μL) was added to the cell suspension and mixed thoroughly. PBS (4 mL) was gently added to the resulting mixture, and the mixture was centrifuged at 4°C and 2300 G for 10 minutes. Of the three layers formed, the top two layers were removed using an aspirator to remove the Debris Removal Solution. Then, PBS was added to the mixture after the removal of the Debris Removal Solution to a volume of 15 mL, and after gentle inversion mixing, the mixture was centrifuged at 4°C and 1000 G for 10 minutes. The upper layer was then removed using an aspirator to obtain a cell pellet.

[0310] The obtained cell pellet was suspended in 1 mL of 1 × Red Blood Cell Lysis Solution (Miltenyi Biotec), and the resulting cell suspension was incubated in a refrigerator for 10 minutes. PBS-BSA buffer (10 mL) was added to the incubated cell suspension, and the mixture was centrifuged at 4°C and 300 G for 10 minutes. The supernatant was then removed to obtain a cell pellet from which red blood cells had been removed. The cell concentration was 1 × 10⁶. ７ PBS-BSA buffer was added to the obtained cell pellet to obtain a cell suspension with a cell / 90 μL ratio. CD45 MicroBreads (Miltenyi Biotec) (10 μL) were added to the obtained cell suspension and incubated in a refrigerator for 15 minutes. PBS-BSA buffer (1 mL) was added to the incubated cell suspension, and the resulting mixture was centrifuged at 4°C and 300 G for 5 minutes. The upper layer was then removed using an aspirator to obtain the cell pellet.

[0311] The obtained cell pellet was mixed with PBS-BSA buffer (500 μL) to obtain a cell suspension. An LD column (Miltenyi Biotec) was set in the magnetic location of a MACS separator, and the column was rinsed with PBS-BSA buffer (2 mL). The cell suspension was then applied to the column, and the flow-through containing unlabeled cells was collected in a tube. The column was washed twice with PBS-BSA buffer (1 mL), and the unlabeled cells (CD45-negative cells) that passed through were collected in the same tube. The obtained CD45-negative cells were centrifuged at 4°C and 300 G for 10 minutes, and the supernatant was removed with an aspirator to obtain a cell pellet.

[0312] Cell concentration is 1 × 10 ７PBS-BSA buffer was added to the obtained cell pellet to obtain a cell suspension with a cell-to-cell ratio of 90 μL. CD31 MicroBeads (Miltenyi Biotec) (10 μL) were added to the obtained cell suspension and incubated in a refrigerator for 15 minutes. PBS-BSA buffer (1 mL) was added to the incubated cell suspension, and the resulting mixture was centrifuged at 4°C and 300 G for 5 minutes. The upper layer was then removed using an aspirator to obtain the cell pellet.

[0313] PBS-BSA buffer (500 μL) was added to the obtained cell pellet to obtain a cell suspension. An MS column (Miltenyi Biotec) was set in the magnetic location of the MACS Separator, and the column was rinsed with PBS-BSA buffer (500 μL). The cell suspension was applied to the column, and the flow-through containing unlabeled cells was collected in a tube. The column was washed three times with PBS-BSA buffer (500 μL). The unlabeled cells that passed through were then collected in the same tube. The column was removed from the separator, and PBS-BSA buffer (1 mL) was added to the column. The plunger was firmly pressed into the column, and CD45 － CD31 ＋ The cells were immediately washed away, and the cell suspension was collected in a new 15 mL tube. CD31 ＋ To increase cell purity, a new MS column was used for CD31 ＋ The cell selection procedure was repeated. The resulting CD31 ＋ The cell suspension containing the cells was centrifuged at 4°C and 300G for 10 minutes, and the supernatant was removed using an aspirator to obtain a cell pellet.

[0314] To the obtained cell pellet, 1 mL of FACS buffer was added, the mixture was centrifuged, and the supernatant was removed to obtain a cell pellet. Again, 1 mL of FACS buffer was added to the obtained cell pellet and transferred to a 1.5 mL tube to obtain a cell suspension concentrated with cerebral vascular endothelial cells.

[0315] The obtained cell suspension (18 μL) was mixed with acridine orange (2 μL), and cell counting was performed using the resulting mixture with LUNA-FL (Logos Biosystems). The cell suspension was centrifuged at 4°C and 500 G for 3 minutes, and the supernatant was removed to obtain a cell pellet. A blocking solution was prepared by mixing anti-CD16 / 32 antibody (0.6 μL) and FACS buffer (29.4 μL) to prevent nonspecific staining.

[0316] The obtained blocking solution (15 μL) was added to the obtained cell pellet and tapped. The resulting cell suspension was allowed to stand at 4°C and protected from light for 10 minutes. BV605-labeled CD45 antibody and PE-labeled CD31 antibody were diluted in the blocking solution to the manufacturer's recommended concentrations to obtain an antibody cocktail for staining. The antibody cocktail for staining (17 μL) was added to the cell suspension (15 μL), and the resulting mixture was allowed to stand at 4°C and protected from light for 30 minutes. Five minutes before observation, 7-AAD Viability Staining Solution (BioLegend) (1.5 μL) was added to the mixture, and dead cell staining was performed. To a cell suspension containing this staining antibody and 7-AAD reagent, 1 mL of FACS buffer was added, and the centrifugation and supernatant removal procedure was repeated twice. The resulting cell pellet was suspended in 250 μL of FACS buffer, and the resulting suspension (sample) was analyzed using Novocyte to calculate the percentage of brain vascular endothelial cells expressing ZsGreen relative to the total number of brain vascular endothelial cells, which was expressed as the percentage of ZsGreen-positive cells in brain vascular endothelial cells. In other words, the group of cells stained with PE-labeled anti-CD31 antibody among the living cells in the cell suspension was defined as brain vascular endothelial cells, and the percentage of cells exhibiting ZsGreen fluorescence within this population of brain vascular endothelial cells was further calculated. The results are shown in Table 9.

[0317]

[0318] As shown in Table 9, the ligand-modified LNPs encapsulating mRNA from Example 4 were taken up by mouse brain vascular endothelial cells and gene expression was observed.

[0319] [Examples 5-1 to 5-18] Preparation of ligand-modified LNPs containing mRNA A lipid solution was prepared under the same conditions as in Examples 1-2, except that the following ionic lipids were used and DPPC was used as the phospholipid (amount of DMG-PEG2000 relative to the total of ionic lipids, cholesterol, and phospholipid (DPPC): 1.5 mol%).

[0320] (Ionic lipids used) Example 5-1: SS-OP Example 5-2: Compound 1 Example 5-3: Compound 2 Example 5-4: Compound 3 Example 5-5: Compound 4 Example 5-6: Compound 5 Example 5-7: Compound 6 Example 5-8: Compound 7 Example 5-9: Compound 8 Example 5-10: Compound 9 Example 5-11: Compound 10 Example 5-12: Compound 11 Example 5-13: Compound 12 Example 5-14: Compound 13 Example 5-15: Compound 14 Example 5-16: Compound 15 Example 5-17: Compound 16 Example 5-18: Compound 17

[0321] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: Ionic lipid concentration: 4.2 mM Cholesterol concentration: 3.2 mM Phospholipid concentration: 0.6 mM DMG-PEG2000 concentration: 0.12 mM Total lipid concentration: 8.12 mM

[0322] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min, and each lipid solution at a flow rate of 2 mL / min, for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0323] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0324] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0325] A 150 μL LNP suspension containing mRNA and a 40.05 μL aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. After incubation, 259.95 μL of phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare a ligand-modified LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 6.7 μg / mL, amount of ligand relative to total lipids: 0.2 mol%).

[0326] [Test Example 8] Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, Polydispersity Index (PdI), and mRNA encapsulation rate of ligand-modified LNPs encapsulating mRNA from Examples 5-1 to 5-18 were analyzed. Particle size and PdI were measured by dynamic light scattering using Zetasizer®. mRNA encapsulation rate was measured by Ribogreen® assay. The results are shown in Table 10 below.

[0327]

[0328] [Test Example 9] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein one of the ligand-modified LNP suspensions containing mRNA from Examples 5-1 to 5-18 (mRNA amount: 1.0 μg).

[0329] 21 hours after the aforementioned administration, the mice were euthanized and their whole brains were removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brains, and the brains were lysed using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13,000 rpm and 4°C, and 20 μL of the supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate. The luminescence derived from NanoLuc® luciferase was measured using GloMax. The protein concentration of a solution obtained by diluting the supernatant 100-fold with ultrapure water was measured by the BCA method, and the luminescence intensity was standardized by the protein content. The results are shown in Table 11.

[0330]

[0331] As shown in Table 11, ligand-modified LNPs encapsulating mRNA in Examples 5-1 to 5-18, which used different ionic lipids, all exhibited gene expression activity in the mouse brain.

[0332] [Examples 6-1 to 6-14] Preparation of ligand-modified LNPs encapsulating mRNA. Lipid solutions were prepared under the same conditions as in Examples 1-1 to 1-5, except that DPPC was used as the phospholipid and the amounts of each lipid were adjusted as shown in Table 12 below. The reference amounts of each lipid listed in Table 12 are the sum of ionic lipids, phospholipids, and cholesterol. Table 12 also shows the total lipid concentration in the lipid solution.

[0333]

[0334] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min, and each lipid solution at a flow rate of 2 mL / min, for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0335] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0336] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding NanoLuc® luciferase (2.5 μL, mRNA amount: 3 μg) with 2-morpholinoethanesulfonic acid buffer (132.5 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL).

[0337] A 150 μL LNP suspension containing mRNA and a 40.05 μL aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. After incubation, 259.95 μL of phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) was added to the mixture to prepare a ligand-modified LNP suspension containing mRNA (N / P ratio: 36, mRNA concentration: 6.7 μg / mL, amount of ligand relative to total lipids: 0.2 mol%).

[0338] [Test Example 10] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein one of the ligand-modified LNP suspensions containing mRNA from Examples 6-1 to 6-14 (mRNA amount: 1.0 μg).

[0339] 21 hours after the aforementioned administration, the mice were euthanized and their whole brains were removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brains, and the brains were lysed using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13,000 rpm and 4°C, and 20 μL of the supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate. The luminescence derived from NanoLuc® luciferase was measured using GloMax. The protein concentration of a solution obtained by diluting the supernatant 100-fold with ultrapure water was measured by the BCA method, and the luminescence intensity was standardized by the protein content. The results are shown in Table 13.

[0340]

[0341] As shown in Table 13, ligand-modified LNPs encapsulating mRNA from Examples 6-1 to 6-14, which had different lipid compositions (i.e., amounts of each lipid), all exhibited gene expression activity in the mouse brain.

[0342] [Example 7] Preparation of ligand-modified LNPs containing mRNA A lipid solution was prepared under the same conditions as in Examples 1-2.

[0343] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed at a flow rate of 14 mL / min and a lipid solution at a flow rate of 2 mL / min for 15 seconds at 25°C to obtain 4 mL of nucleic acid-free LNP suspension (hereinafter referred to as "empty LNP suspension").

[0344] To the obtained empty LNP suspension (4 mL), 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under stirring with a vortex mixer. The obtained 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been added beforehand, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 14 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the obtained dilution was ultrafiltered. To 100 μL of the obtained concentrate, 100 μL of 320 mg / mL sucrose solution was added to prepare an empty LNP suspension with the dispersion medium replaced. The LNP suspension was stored at 4°C under an argon atmosphere until use.

[0345] An mRNA solution was prepared by mixing an aqueous solution of mRNA encoding the heavy and light chains of lecanemab (3.0 μL, mRNA amount: 3 μg, heavy chain mRNA: light chain mRNA = 2 μg: 1 μg) with 2-morpholinoethanesulfonic acid buffer (132.0 μL, buffer concentration: 20 mM, pH: 6.0). The entire volume of the obtained mRNA solution and 15 μL of an empty LNP suspension from which the dispersion medium had been replaced were mixed under agitation using a vortex mixer. The resulting mixture was incubated at 37°C for 5 minutes to prepare an LNP suspension containing mRNA (N / P ratio: 34.7, mRNA concentration: 20 μg / mL).

[0346] A 500 μL LNP suspension containing mRNA and a 133.5 μL aqueous solution containing ligand-bound PEG lipid obtained in Preparation Example 1 were mixed using a vortex mixer under agitation, and the resulting mixture was incubated at 4°C for 30 minutes. 633.5 μL of the incubated mixture was added to Amicon Ultra-4 (MWCO: 100 kDa) to which 1 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been pre-added, and ultrafiltration was performed. After concentration by ultrafiltration, the obtained concentrate was diluted with 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) (mRNA concentration: 157.9 μg / mL). To the resulting mixture, phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) was added to prepare a ligand-modified LNP suspension containing encapsulated mRNA (N / P ratio: 34.7, mRNA concentration: 66.7 μg / mL, ligand amount relative to total lipids: 0.2 mol%).

[0347] [Preparation Examples 2-1 to 2-5] Preparation of aqueous solutions containing ligand-bound PEG lipids Using Amicon Ultra-4 (MWCO: 50 kDa), the solvent for the aqueous solution of anti-transferrin receptor antibody (manufactured by BioXCell) is 100 mM NaHCO3 ３The solvent was replaced with an aqueous solution (pH: 9.0). The antibody aqueous solution with the buffer solvent replaced was mixed with 1 equivalent of DBCO-NHS, and the resulting mixture was incubated at room temperature for 2 hours while shaking at 700 rpm. The mixture was further incubated overnight at 4°C. The solvent of the mixture was replaced with ultrapure water using an Amicon Ultra-0.5 Centrifugal Filter (MWCO: 3 kDa) to prepare an aqueous DBCO-anti-transferrin receptor antibody solution.

[0348] As described above, 10.1 μL of 74 μM DBCO-anti-transferrin receptor antibody aqueous solution, 7.47 μL of 0.1 mM aqueous solution of the activated PEG lipid shown in Table 14 below, and 32.5 μL of 2-morpholinoethanesulfonic acid buffer (pH: 6.0) were mixed, and the resulting mixture was incubated overnight at 4°C to prepare an aqueous solution containing ligand-bound PEG lipid (ligand: anti-transferrin receptor antibody).

[0349]

[0350] [Examples 8-1 to 8-41] Preparation of ligand-modified LNPs containing mRNA For Examples 8-1 to 8-38, the lipid solution was prepared under the same conditions as in Example 1-2, except that the types of lipids shown in Tables 15-1 and 15-2 below were used (amount of PEG lipid relative to the total of ionic lipids, cholesterol, and phospholipids: 1.5 mol%).

[0351] For Examples 8-39 to 8-41, using the lipids shown in Table 15-2 below, a 20 mM ethanol solution of ionic lipids, a 20 mM ethanol solution of cholesterol, and a 10 mM ethanol solution of phospholipids were mixed so that their molar ratios were 60 / 10 / 30 for Examples 8-39 and 8-40, and 50 / 15.3 / 34.7 for Example 8-41. The resulting mixture was then mixed with a 2 mM ethanol solution of DMG-PEG2000 to prepare a lipid solution (amount of DMG-PEG2000 relative to the total of ionic lipids, cholesterol, and phospholipids: 1.5 mol%).

[0352]

[0353]

[0354] The concentrations of each lipid and the total lipid in the lipid solution obtained as described above were as follows: (Examples 8-1 to 8-38) Ionic lipid concentration: 4.2 mM Cholesterol concentration: 3.2 mM Phospholipid concentration: 0.6 mM PEG lipid (DMG-PEG2k, etc.) concentration: 0.12 mM PEG lipid having an azide group without a ligand (Azide-PEG-DSPE, etc.) Total lipid concentration: 8.12 mM (Examples 8-39, 8-40) Compound 42 concentration: 4.8 mM Cholesterol concentration: 2.4 mM DSPC concentration: 0.8 mM DMG-PEG2000 concentration: 0.12 mM Total lipid concentration: 8.12 mM

[0355] (Example 8-41) Concentration of compound 44: 4.0 mM Concentration of cholesterol: 2.8 mM Concentration of POPE: 1.2 mM Concentration of DMG-PEG2000: 0.12 mM Concentration of total lipids: 8.12 mM

[0356] 3.0 μL of mRNA encoding the heavy and light chains of lecanemab (3 μg of mRNA, with a ratio of 2 μg to 1 μg of heavy chain mRNA to light chain mRNA) was dissolved in citrate buffer (buffer concentration: 20 mM, pH: 5.0).

[0357] Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by Precision NanoSystems Inc.), aqueous solutions of mRNA encoding the heavy and light chains of lecanemab were mixed at a flow rate of 12 mL / min, and each lipid solution at a flow rate of 4 mL / min, for 15 seconds at 25°C to obtain an LNP suspension containing 4 mL of mRNA.

[0358] To each LNP suspension (4 mL) containing the obtained mRNA, 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was quickly added under vortex mixing. The resulting 8.0 mL mixture was added to Amicon Ultra-15 (MWCO: 100 kDa) to which 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been pre-added, and the resulting mixture was ultrafiltered. After concentration by ultrafiltration, the obtained concentrate was diluted with 4 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting dilution was ultrafiltered to prepare an LNP suspension containing mRNA with the dispersion medium replaced (N / P ratio for Examples 8-1 to 8-38: 34.7, N / P ratio for Examples 8-39 to 8-41: 11, mRNA concentration: 20 μg / mL).

[0359] A 500 μL LNP suspension containing mRNA and a 133.5 μL aqueous solution containing ligand-bound PEG lipid obtained in Preparation Example 1 or Preparation Examples 2-1 to 2-5 were mixed using a vortex mixer under stirring, and the resulting mixture was incubated at 4°C for 30 minutes. 633.5 μL of the incubated mixture was added to Amicon Ultra-4 (MWCO: 100 kDa) to which 1 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) had been pre-added, and ultrafiltration was performed. After concentration by ultrafiltration, the obtained concentrate was diluted with 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) (mRNA concentration: 157.9 μg / mL). To the resulting mixture, phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) was added to prepare a ligand-modified LNP suspension containing mRNA (N / P ratio for Examples 8-1 to 8-38: 34.7, N / P ratio for Examples 8-39 to 8-41: 11, mRNA concentration: 66.7 μg / mL, amount of ligand relative to total lipids: 0.2 mol%).

[0360] [Test Example 11] Evaluation of LNP particle size, PdI, and mRNA encapsulation rate The particle size, Polydispersity Index (PdI), and mRNA encapsulation rate of ligand-modified LNPs encapsulating mRNA from Examples 7, 8-1 to 8-41 were analyzed. Particle size and PdI were measured by dynamic light scattering using Zetasizer®. mRNA encapsulation rate was measured by Ribogreen® assay. The results are shown in Tables 16-1 and 16-2 below.

[0361]

[0362]

[0363] [Test Example 12] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension containing mRNA from Examples 1-2, 7, 8-1 to 8-41, or PBS (mRNA amount: 1.0 μg or 10.0 μg).

[0364] 21 hours after the aforementioned administration, the mice were euthanized and their whole brains were removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brains, and the brains were lysed using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13,000 rpm and 4°C, and 20 μL of the supernatant was mixed with 50 μL of an aqueous solution of luciferase substrate. The luminescence derived from NanoLuc® luciferase was measured using GloMax. The protein concentration of a solution obtained by diluting the supernatant 100-fold with ultrapure water was measured by the BCA method, and the luminescence intensity was standardized by the protein content. The results are shown in Tables 17-1 and 17-2.

[0365]

[0366]

[0367] As shown in Tables 17-1 and 17-2, gene expression activity was observed in mouse brains in all cases: Example 7, in which nucleic acids were encapsulated in empty LNPs, and the ligand-modified LNPs encapsulating mRNA from Examples 8-1 to 8-41, which had different lipid species.

[0368] [Example 9] Preparation of ligand-modified LNPs with embedded mRNA Ligand-modified LNPs with embedded mRNA were prepared under the same conditions as in Example 7, except that an aqueous solution of mRNA encoding the heavy and light chains of lecanemab (3.0 μL, mRNA amount: 3 μg, heavy chain mRNA: light chain mRNA = 2 μg: 1 μg) was used as the mRNA.

[0369] [Examples 10-1 to 10-4] Preparation of ligand-modified LNPs with encapsulated mRNA Ligand-modified LNPs with encapsulated mRNA were prepared under the same conditions as in Example 8, except that an aqueous solution of mRNA encoding the heavy and light chains of lecanemab (3.0 μL, mRNA amount: 3 μg, heavy chain mRNA: light chain mRNA = 2 μg: 1 μg) was used as the mRNA, and the ionic lipids, phospholipids, and ligand-binding PEG lipids shown in Table 18 below were used.

[0370]

[0371] [Comparative Example 2] Preparation of LNPs containing mRNA but not modified with ligands LNPs containing mRNA were prepared under the same conditions as in Comparative Example 1, except that an aqueous solution of mRNA encoding the heavy and light chains of lecanemab (3.0 μL, mRNA amount: 3 μg, heavy chain encoding mRNA: light chain encoding mRNA = 2 μg: 1 μg) was used as the mRNA.

[0372] [Test Example 13] Evaluation of LNP particle size, PdI, and mRNA encapsulation rate. The particle size, Polydispersity Index (PdI), and mRNA encapsulation rate of ligand-modified LNPs encapsulating mRNA from Examples 9, 10-1 to 10-4, and comparative example 2 were analyzed. Particle size and PdI were measured by dynamic light scattering using Zetasizer®. mRNA encapsulation rate was measured by Ribogreen® assay. The results are shown in Table 19 below.

[0373]

[0374] [Test Example 14] Evaluation of LNP gene expression activity in vivo C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension containing mRNA from Example 10-1 or an LNP suspension containing mRNA from Comparative Example 2 (mRNA amount: 10.0 μg).

[0375] Blood was collected from the tail vein of the mouse 21 hours after the aforementioned administration, and plasma was recovered. Subsequently, the mouse was anesthetized by intraperitoneal administration of a triple-component anesthetic at a dose of 10.0 μL / g. The diaphragm was incised, and both ends of the sternum were cut to expose the heart. An incision was made in the right atrium, and after inserting a needle into the left ventricle, the heart was perfused with heparin-containing PBS (-). After confirming that the liver had turned pale and blood had been removed, perfusion was terminated and the brain was removed. 50 mg of the removed brain was mixed with 990.0 μL of T-PER (trademark) Tissue Protein Extraction Reagent and 10.0 μL of Halt (trademark) Protease Inhibitor Cocktail, EDTA-Free (100x) to extract lecanemab from the brain tissue. Subsequently, intracerebral and serum recanemab concentrations were calculated using ELISA with Goat anti-Human IgG-Fc Fragment Antibody as the immobilized antibody and Goat anti-Human Kappa Light Chain Antibody HRP Conjugated as the detection antibody. Table 20 below shows the intracerebral recanemab concentration, serum recanemab concentration, and intracerebral recanemab concentration / serum recanemab concentration.

[0376]

[0377] As shown in Table 20, which compares the brain lecanemab concentration / blood lecanemab concentration of Example 10-1 and Comparative Example 2, Example 10-1 was able to express lecanemab in the brain with 1.6 times the efficiency of Comparative Example 2.

[0378] [Test Example 15] Evaluation of LNP gene expression activity in vivo The procedure was the same as in Test Example 14, except that C57BL / 6 mice were administered via tail vein either a ligand-modified LNP suspension (mRNA amount: 10.0 μg) containing mRNA from Examples 9 and 10-2 to 10-4, or PBS. Brain lecanemab concentration and serum lecanemab concentration were calculated. Table 21 below shows the brain lecanemab concentration, serum lecanemab concentration, and brain lecanemab concentration / serum lecanemab concentration.

[0379]

[0380] As shown in Table 21, the ligand-modified LNPs containing mRNA in Examples 9 and 10-2 to 10-4 all showed evidence of lecanemab transfer into the brain.

[0381] The lipid nanoparticles of the present invention are useful for delivering nucleic acids to brain cells.

[0382] This application is based on Japanese Patent Application No. 2025-040654, which is entirely contained herein.

Claims

1. Ligand-modified lipid nanoparticles, comprising ionic lipids, cholesterol, PEG lipids, nucleic acids, and ligands, used for delivering nucleic acids to brain cells.

2. Ligand-modified lipid nanoparticles according to claim 1, further comprising phospholipids.

3. The ionic lipid is represented by formula (1): (In formula (1), R １ａ and R １ｂ each independently represent an alkylene group having 1 to 6 carbon atoms; X ａ and X ｂ each independently represent an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups; R ２ａ and R ２ｂ each independently represent an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms; Y ａ and Y ｂ each independently represent an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂ each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom; n ａ and n ｂ each independently represent 0 or 1; and R ３ａ and R ３ｂ each independently represent any one selected from the group consisting of: a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride; a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride; an aliphatic hydrocarbon group having 1 to 40 carbon atoms; an alkyl group having 3 to 40 carbon atoms having a cyclopropane ring; and a group represented by formula (2): *-R ６ -L １ -R ７ (2) In formula (2), * represents a bonding position; R ６ represents an alkylene group having 2 to 10 carbon atoms; R ７ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon cyclic group having 3 to 12 carbon atoms, wherein one methylene group in R ７ may be replaced by one ether bond; and L １ represents an ester bond or a carbamate bond. ) A group represented by formula (3): (In formula (3), * indicates the bonding position, and R ８ This represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ８ The group may be substituted with substituents selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms.) The group represented by formula (4): (In equation (4), * indicates the bonding position, R ９ R represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, and R ９ R may be substituted with substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms. １０ and R １１ Each of these independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, R １０ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １１ At least one ethylene group or at least one trimethylene group in the compound may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R １０ and R １１ Each of these may be independently substituted with a substituent selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms, and L ２ ) represents an oxygen atom, NH, or sulfur atom. ) A group represented by formula (5): (In formula (5), * represents the bond position, and R １２ is a hydrogen atom, a benzyl group, or *-Si(R １３ ) (Caution １４ ) (Caution １５ ) group (wherein * represents a bond position, and R １３ ~R １５ Each of these independently represents an alkyl group or phenyl group having 1 to 4 carbon atoms. ) Represents a group represented by ) Formula (6): (In equation (6), * represents the bond position, R １６ R represents an alkylene group having 5 to 10 carbon atoms, and １７ ~R １９ Each independently represents 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, R １７ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １８ At least one ethylene group or at least one trimethylene group in R may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, １９ At least one ethylene group or at least one trimethylene group in the compound may be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R １７ ~R １９ Each of these may be independently substituted with a substituent selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups having 3 to 12 carbon atoms.) A group represented by formula (7): (In equation (7), * represents the bond position, and R ２0 R represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and ２1 and R ２2 Each of these independently represents 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.) represents a group, represents an ionic lipid represented by ). Formula (10): (In formula (10), R ２６ and R ２７ Each of these independently represents 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, or R ２６ and R ２７ These atoms bond to each other, forming a 5- or 6-membered nitrogen-containing heterocycle with the nitrogen atoms to which they bond, R ２９ and R ２８ Each of these independently represents an aliphatic hydrocarbon group having 5 to 50 carbon atoms, and R ２８ At least one ethylene group in the group may be replaced by an ester bond, and R ２９ At least one ethylene group in the compound may be replaced by an ester bond, and R ３０ This represents an alkylene group having 1 to 10 carbon atoms. Ionic lipids represented by ), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, Ligand-modified lipid nanoparticles according to claim 1 or 2, comprising at least one selected from the group consisting of (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

4. Ionic lipids are ionic lipids represented by formula (1), [(4-hydroxybutyl)azandiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, The ligand-modified lipid nanoparticle according to claim 3, comprising at least one selected from the group consisting of 1,1'-[[2-[4-[2-[[2-[[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyldiacetate, and di(pentadecan-8-yl)4,4'-[({[3-(dimethylamino)propyl]thio}carbonyl)azandiyl]dibutyrate.

5. The ligand-modified lipid nanoparticle according to claim 2, wherein the phospholipid comprises at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dimiristoyl-sn-glycero-3-phosphocholine.

6. Ligand-modified lipid nanoparticles according to claim 1 or 2, wherein, relative to the total amount of ionic lipids, phospholipids, and cholesterol, the amount of ionic lipids is 15 to 75 mol%, the amount of phospholipids is 0 to 45 mol%, the amount of cholesterol is 15 to 65 mol%, and the amount of PEG lipids is 0.5 to 5 mol%.

7. The ligand-modified lipid nanoparticle according to claim 1 or 2, wherein the ligand is an anti-transferrin receptor antibody.

8. Ligand-modified lipid nanoparticles according to claim 1 or 2, wherein the amount of ligand is 0.01 to 5 mol% relative to the total lipids.

9. The ligand-modified lipid nanoparticles according to claim 1 or 2, wherein the brain cells are cerebral vascular endothelial cells.

10. A pharmaceutical composition for use in the treatment of a brain disease, comprising ligand-modified lipid nanoparticles according to claim 1 or 2.

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