Lipid nanoparticles for delivering nucleic acid to splenic tissue, and method for delivering nucleic acid to splenic tissue using same

Abstract

The purpose of the present invention is to provide: lipid nanoparticles for delivering a nucleic acid to splenic tissue, which can improve the efficiency of delivery of a nucleic acid to splenic tissue cells; and a method for delivering a nucleic acid to splenic tissue using the lipid nanoparticles. Provided are: lipid nanoparticles that are used for the delivery of a nucleic acid to splenic tissue, each of the lipid nanoparticles comprising (A) an ionic lipid represented by formula (1), (B) an anionic lipid or a compound represented by formula (2), (C) cholesterol, and (D) dimyristoyl glycerol PEG represented by the formula CH2(OR6)-CH(OR7)-CH2(OR8) (In the formulae, the definitions for the symbols are as described in the description.); and a method for delivering a nucleic acid to splenic tissue using the lipid nanoparticles.

Description

Lipid nanoparticles for delivering nucleic acids to spleen tissue and method for delivering nucleic acids to spleen tissue using the same

[0001] The present invention relates to lipid nanoparticles used to deliver nucleic acids to spleen tissue, and methods for using the same to deliver nucleic acids to spleen tissue.

[0002] In order to commercialize nucleic acid therapy using oligonucleotides such as siRNA and gene therapy using mRNA, pDNA, etc., an effective and safe nucleic acid delivery carrier is required. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but they have practical safety issues. Therefore, development of non-viral nucleic acid delivery carriers that can be used more safely is underway. 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, the amine moiety, which protonates under acidic conditions, interacts electrostatically with nucleic acids, which are polyanions, to form lipid nanoparticles, which promote cellular uptake and deliver nucleic acids into cells.

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

[0005] For example, 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 connected by a biodegradable disulfide bond. Patent Document 1 shows that the ionic lipid improves in vivo kinetics such as blood stability and tumor targeting. Furthermore, by changing the structure around the amine moiety, the pKa of the lipid membrane structure can be adjusted to a value favorable for endosomal escape within cells, and it has also been shown that the cleavage of disulfide bonds within cells has the effect of dissociating nucleic acids from the lipid membrane structure. In fact, compared to the known ionic lipid DODAP, it exhibits a higher nucleic acid delivery efficiency, revealing that the ionic lipid can improve intracellular kinetics, such as improving the efficiency of delivery of nucleic acids into the cytoplasm.

[0006] For example, Patent Document 2 discloses a lipid membrane structure that uses an ionic lipid having an aromatic ring introduced near the lipid moiety in addition to a tertiary amine moiety and a disulfide bond, thereby enhancing the ability to fuse with an endosomal membrane and further increasing the efficiency of nucleic acid delivery to the cytoplasm.

[0007] As mentioned above, ionic lipids have been developed that improve intracellular dynamics by increasing endosomal escape efficiency and membrane fusion ability. However, in order for lipid nanoparticles made of ionic lipids to be more practical in vivo as nucleic acid delivery carriers, they must be able to target organs and cells.

[0008] Dimyristoylglycerol PEG (DMG-PEG) is one of the PEG lipids widely used in lipid nanoparticles. When lipid nanoparticles using this lipid nanoparticle are administered into the bloodstream, the PEG lipid gradually dissociates from the lipid nanoparticles in the blood, and apolipoprotein E (ApoE) present in the blood adheres to the lipid nanoparticles, thereby increasing their accumulation in the liver, which expresses the ApoE receptor (see, for example, Non-Patent Document 2).

[0009] An example of imparting targeting to organs other than the liver is the use of distearoylglycerol PEG (DSG-PEG), which has a hydrophobic group derived from stearic acid, as the PEG lipid, rather than DMG-PEG, which has a hydrophobic group derived from myristic acid, thereby enhancing tumor accumulation (Non-Patent Document 3). Compared to DMG-PEG, DSG-PEG is less likely to dissociate from lipid nanoparticles in the blood, which prevents adhesion of ApoE in the blood, inhibits liver accumulation, and exhibits high blood retention, resulting in enhanced tumor accumulation.

[0010] Another example of imparting targeting to organs other than the liver is the case where the efficiency of nucleic acid transfer to the spleen was increased by controlling the particle size of lipid nanoparticles to 140 to 230 nm (see, for example, Patent Document 3). It has been shown that lipid nanoparticles with a particle size of 200 nm avoid nucleic acid transfer to the liver and increase the efficiency of nucleic acid transfer to the spleen compared to lipid nanoparticles with a particle size of 100 nm.

[0011] As described above, the efficiency of nucleic acid introduction can be increased by using lipid nanoparticles with improved intracellular dynamics, and by modifying the PEG lipids that are components of lipid nanoparticles or adjusting the particle size of the lipid nanoparticles, it is also possible to efficiently deliver nucleic acids to target tissues such as the liver and spleen.

[0012] U.S. Patent No. 9,708,628 International Publication No. 2019 / 188867 U.S. Patent Application Publication No. 2020 / 0345641

[0013] Molecular Therapy, 25(7): 1467-1475 (2017)J. Control. Release, 235: 236-244 (2016)J. Control. Release, 200: 97-105 (2015)Biomater. Sci, 9: 1449-1463 (2021)

[0014] The spleen is an organ that contains many immune cells and is therefore a potential target for nucleic acid vaccine applications. However, despite advances in this field, the efficiency of nucleic acid delivery to the spleen using conventional lipid nanoparticles is insufficient and there is room for improvement.

[0015] Furthermore, although the lipid nanoparticles described in Patent Document 1 have been shown to have a high efficiency of nucleic acid delivery to the liver, they have not been shown to have any effect on nucleic acid delivery to the spleen.

[0016] In this regard, Non-Patent Document 4 and Patent Document 3 each disclose the use of ionic lipids with different structural features for lipid nanoparticles for splenic delivery. However, in these documents, different phospholipids are used as components of lipid nanoparticles, and the optimal combination with the phospholipid varies depending on the structure of the ionic lipid.

[0017] As mentioned above, there are examples where the efficiency of nucleic acid transfer to the spleen has been improved using lipid nanoparticles. However, because the appropriate phospholipid varies depending on the structure of the ionic lipid, this is not fully satisfactory for practical use, and further improvements in nucleic acid delivery efficiency are required.

[0018] In view of the above problems, the present invention aims to provide lipid nanoparticles for delivering nucleic acids to spleen tissue, which can improve the efficiency of nucleic acid delivery to spleen tissue cells, and a method for delivering nucleic acids to spleen tissue using the same.

[0019] As mentioned above, the appropriate combination of phospholipids in lipid nanoparticles varies depending on the structure of the ionic lipid, and efficient nucleic acid delivery requires optimizing the combination of ionic lipids and phospholipids.

[0020] In view of the above problems, the inventors have conducted extensive research and found that lipid nanoparticles produced using an ionic lipid that has a pKa suitable for endosomal escape and that specifically decomposes in a reductive intracellular environment, and an anionic phospholipid or an anionic cholesterol represented by formula (2), can efficiently transfer nucleic acids into spleen tissue. The present invention, based on this finding, is as follows.

[0021] [1] (A) Formula (1):

[0022]

[0023] (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms;ａ and X ｂ each independently represents a non-cyclic 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 one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms; Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom; ａ and n ｂ are each independently 0 or 1; ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (4): R ９ —O—CO—(CH ２ )a- (4) (In formula (4), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10. ) represents a group represented by the formula: (B) an ionic lipid represented by the formula (2):

[0024]

[0025] (in formula (2), T represents a divalent aliphatic hydrocarbon group having 1 to 8 carbon atoms), (C) cholesterol, and (D) a compound represented by formula (3): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８) (3) (In formula (3), R ６ , R ７ and R ８ wherein any two of the groups represent myristoyl groups, and the remaining group represents an alkyl group having 1 to 6 carbon atoms linked via a polyethylene glycol chain having a number average molecular weight of 1,000 to 3,000.

[0026] [2] The lipid nanoparticle according to [1], wherein the anionic phospholipid is 1,2-diacyl-sn-glycero-3-phosphoglycerol or 1,2-diacyl-sn-glycero-3-phosphoserine.

[0027] [3] The anionic phospholipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DSPG ...sn-glycero-3-phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-dioleoyl-sn The lipid nanoparticle according to [1] or [2], which is at least one selected from the group consisting of 1-palmitoyl-sn-glycero-3-phosphoserine (DPPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS), and 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS).

[0028] [4] The lipid nanoparticle according to any one of [1] to [3], wherein the anionic phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), or 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS).

[0029] [5] The lipid nanoparticle according to any one of [1] to [4], wherein T in formula (2) is a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms.

[0030] [6] The ionic lipid represented by formula (1) is represented by the following formula:

[0031]

[0032] or

[0033]

[0034] The lipid nanoparticle according to any one of [1] to [5], wherein the lipid nanoparticle is any ionic lipid represented by the formula:

[0035] [7] The lipid nanoparticle according to any one of [1] to [6], which may contain a neutral phospholipid as a component of the lipid nanoparticle.

[0036] [8] The lipid nanoparticle according to [7], wherein the neutral phospholipid is 1,2-diacyl-sn-glycero-3-phosphocholine or 1,2-diacyl-sn-glycero-3-phosphoethanolamine.

[0037] [9] The neutral phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). [7] or [8] The lipid nanoparticles are at least one selected from the group consisting of glycero-3-phosphoethanolamine.

[0038]

[10] The ratio of the anionic phospholipid or the compound represented by the formula (2) to the neutral phospholipid is 100:0 to 25:75 mol% [7] to [9]. The lipid nanoparticles according to any one of the above.

[0039]

[11] The ionic lipid is 30 to 70 mol% relative to the total of the ionic lipid, the anionic phospholipid or the compound represented by the formula (2), the neutral phospholipid, and the cholesterol, and the anionic phospholipid or the compound represented by the formula (2) is 2.5 to 15 mol%, the neutral phospholipid is 0 to 15 mol%, the cholesterol is 20 to 60 mol%, and dimyristoylglycerol PEG is 0.5 to 1.5 mol% [7] to

[10] . The lipid nanoparticles according to any one of the above.

[0040]

[12] A method for delivering nucleic acid to spleen tissue, comprising intravenously administering to a living body the lipid nanoparticles according to any one of [1] to

[11] , in which nucleic acid is encapsulated.

[0041]

[13] (A) Formula (1):

[0042]

[0043] (In formula (1), each symbol is as defined in [1]), (B) an anionic phospholipid or a lipid represented by formula (2):

[0044]

[0045] (In formula (2), T is as defined in [1]), (C) cholesterol, and (D) a compound represented by formula (3): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (3) (In formula (3), each symbol is as defined in [1].) Use of lipid nanoparticles containing dimyristoylglycerol PEG represented by the following formula (3) for producing a pharmaceutical used for delivering a nucleic acid to spleen tissue.

[0046]

[14] The use according to

[13] , wherein the anionic phospholipid is 1,2-diacyl-sn-glycero-3-phosphoglycerol or 1,2-diacyl-sn-glycero-3-phosphoserine.

[0047]

[15] The anionic phospholipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DSPG ...sn-glycero-3-phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-sn-glycero The use according to

[13] or

[14] , wherein the glyceroglycoside is at least one selected from the group consisting of palmitoyl-sn-glycero-3-phosphoserine (DPPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS), and 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS).

[0048]

[16] The use according to any one of

[13] to

[15] , wherein the anionic phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), or 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS).

[0049]

[17] The use according to any one of

[13] to

[16] , wherein T in formula (2) is a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms.

[0050]

[18] The ionic lipid represented by formula (1) is an ionic lipid represented by the following formula:

[0051]

[0052] or

[0053]

[0054] The use according to any one of

[13] to

[17] , wherein the ionic lipid is any one of the ionic lipids represented by the formula:

[0055]

[19] The use according to any one of

[13] to

[18] , wherein a neutral phospholipid may be contained as a component of the lipid nanoparticles.

[0056]

[20] The use according to

[19] , wherein the neutral phospholipid is 1,2-diacyl-sn-glycero-3-phosphocholine or 1,2-diacyl-sn-glycero-3-phosphoethanolamine.

[0057]

[21] The use according to

[19] or

[20] , wherein the neutral phospholipid is at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

[0058]

[22] The use according to any one of

[19] to

[21] , wherein the ratio of the anionic phospholipid or the compound represented by formula (2) to the neutral phospholipid is 100:0 to 25:75 mol%.

[0059]

[23] The use according to any one of

[19] to

[22] , wherein the ionic lipid is 30 to 70 mol%, the anionic phospholipid or the compound represented by formula (2), the neutral phospholipid, and the cholesterol are present in an amount of 2.5 to 15 mol%, the neutral phospholipid is 0 to 15 mol%, the cholesterol is 20 to 60 mol%, and the dimyristoylglycerol PEG is 0.5 to 1.5 mol%, based on the total amount of the ionic lipid, the anionic phospholipid or the compound represented by formula (2), the neutral phospholipid, and the cholesterol.

[0060] The lipid nanoparticles of the present invention can deliver nucleic acids to spleen tissue more efficiently than lipid compositions of the prior art by combining an ionic lipid with an anionic phospholipid or anionic cholesterol, which has a pKa suitable for endosomal escape and specifically decomposes in the reductive environment within the cell, and by optimizing the lipid composition.

[0061] 1 is a graph showing gene expression activity in the spleen of lipid nanoparticles (LNPs) of Examples 1 and 2, and LNPs of Comparative Example 1, which are compositions for liver delivery. This graph shows the accumulation of LNPs in the spleen of LNPs of Example 3 and LNPs of Comparative Example 2, which are compositions for liver delivery. This graph shows gene expression activity in the spleen of LNPs of Example 3 and LNPs of Comparative Example 2, which are compositions for liver delivery. This graph shows gene expression activity in the spleen of LNPs of Examples 1 and 4 to 10, and LNPs of Comparative Example 1, which are compositions for liver delivery. This graph shows gene expression activity in the spleen of LNPs of Examples 5 and 11 to 24. This graph shows gene expression activity in the spleen of LNPs of Examples 18 and 25 to 29. This graph shows gene expression activity in the spleen of LNPs of Example 30 and LNPs of Comparative Example 3, which are compositions for liver delivery. This graph shows CTL activity when mice were administered with LNPs of Examples 18 and 24, which encapsulate OVA-mRNA, and LNPs of Comparative Example 1, which are compositions for liver delivery. 1 is a graph showing CTL activity when mice were administered the LNP of Example 30 encapsulating OVA-mRNA, the LNP of Comparative Example 3 which is a composition for liver delivery, and the LNP of Comparative Example 4 which is a composition for spleen delivery. 2 is a graph showing CTL activity when mice were administered the LNP of Example 31 encapsulating OVA-mRNA and the LNP of Comparative Example 4 which is a composition for spleen delivery. 3 is a graph showing the pathology scores of mice administered the LNP of Example 32 encapsulating MOG-mRNA or luc-mRNA or PBS. 4 is a graph showing the weight change (%) (relative to Day 10) of mice administered the LNP of Example 32 encapsulating MOG-mRNA or luc-mRNA or PBS.

[0062] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention relates to lipid nanoparticles containing an ionic lipid represented by formula (1) (i.e., an ionic lipid having a tertiary amino group, a lipid moiety, and a disulfide bond as a biodegradable group), an anionic phospholipid or anionic cholesterol represented by formula (2), cholesterol, and dimyristoylglycerol PEG represented by formula (3), and a method for delivering nucleic acids to spleen cells using the same.

[0063] Lipid Nanoparticles In this specification, "lipid nanoparticles" (sometimes abbreviated as "LNPs" in this specification) refer to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are aligned toward the aqueous phase side of the interface, and having a particle diameter of less than 1 μm, and "amphipathic lipid" refers to a lipid having both a hydrophilic group and a hydrophobic group.

[0064] The particle size of the lipid nanoparticles of the present invention is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Measurement of particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted depending on the method for producing the lipid nanoparticles. In this specification, "particle size" refers to the average particle size (zeta mean) measured by dynamic light scattering.

[0065] Examples of amphipathic lipids include ionic lipids, phospholipids, and PEG lipids. In this specification, "PEG" refers to polyethylene glycol, "PEG lipid" refers to a lipid modified with PEG, and "Y modified with X" (e.g., X:PEG, Y:lipid) refers to Y bound to X. In other words, "PEG lipid" refers to a lipid bound to PEG.

[0066] The lipid nanoparticles of the present invention may contain lipids other than the ionic lipid represented by formula (1), anionic phospholipids or anionic cholesterol represented by formula (2), neutral phospholipids, cholesterol, and dimyristoylglycerol PEG represented by formula (3) (hereinafter referred to as "other lipids"). Examples of other lipids include sterols other than cholesterol and PEG lipids other than dimyristoylglycerol PEG represented by formula (3).

[0067] The amount of other lipids in the lipid nanoparticles of the present invention is preferably 0 to 50 mol%, more preferably 0 to 30 mol%, and even more preferably 0 to 10 mol% relative to the total amount of lipids in the lipid nanoparticles. Here, "total amount of lipids in lipid nanoparticles" means, for example, when the lipid nanoparticles contain an ionic lipid represented by formula (1), a phospholipid, cholesterol, and a dimyristoylglycerol PEG represented by formula (3) and other lipids as constituent components, "the total amount of the ionic lipid represented by formula (1), the phospholipid, cholesterol, and the dimyristoylglycerol PEG represented by formula (3) and other lipids." Also, in this specification, "the amount of B relative to A (mol%)" means "100 × the amount of B (mol) / the amount of A (mol)." For example, "the amount of other lipids relative to the total amount of lipids (mol%)" means "100 × the amount of other lipids (mol) / the total amount of lipids (mol)."

[0068] It is most preferable that no other lipids are used in the present invention, that is, the lipids constituting the lipid nanoparticles of the present invention consist of an ionic lipid represented by formula (1), an anionic phospholipid or anionic cholesterol represented by formula (2), a neutral phospholipid, cholesterol, and dimyristoylglycerol PEG represented by formula (3).

[0069] Ionic Lipid The ionic lipid used in the present invention is an ionic lipid represented by the following formula (1) (sometimes abbreviated as "ionic lipid (1)" in this specification). Only one type of ionic lipid (1) may be used, or two or more types may be used in combination.

[0070]

[0071] (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic 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 one or two tertiary amino groups; R ２ａ and R２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms; Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom; ａ and n ｂ are each independently 0 or 1; ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (4): R ９ —O—CO—(CH ２ )a- (4) (In formula (4), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.

[0072] R １ａ and R １ｂ are each independently 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, and more preferably 1 to 2. Specific examples of alkylene groups having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. R １ａ and R １ｂ are preferably each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each are an ethylene group.

[0073] R１ａ is R １ｂ may be the same as or different from, but preferably, R １ａ is R １ｂ is the same group as

[0074] X ａ and X ｂ each independently represents a non-cyclic 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 one or two tertiary amino groups, and preferably each independently represents a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups.

[0075] The alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0076] A preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is: X １ It is shown as follows.

[0077]

[0078] X １ R ５represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0079] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups is preferably 4 or 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups include an aziridylene group, an azetidylene group, a pyrrolidine group, a piperidylene group, an imidazolidylene group, and a piperaziylene group, preferably a pyrrolidine group, a piperidylene group, and a piperaziylene group, and most preferably a piperidylene group.

[0080] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is X ２ It is shown as follows.

[0081]

[0082] X ２ In the formula, p is 1 or 2. When p is 1, X ２ is a pyrrolidine group, and when p is 2, X ２ is a piperidylene group. Preferably, p is 2.

[0083] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X ３ It is shown as follows.

[0084]

[0085] X３ The w in is 1 or 2. When w is 1, X ３ is an imidazolidylene group, and when w is 2, X ３ is a piperazylene group.

[0086] X ａ is X ｂ may be the same as or different from, but preferably, X ａ is X ｂ is the same group as

[0087] R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, and preferably each independently represents an alkylene group having 8 or less carbon atoms.

[0088] The alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear. The number of carbon atoms contained in the alkylene group is preferably 6 or less, and most preferably 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group. Preferred are a methylene group, an ethylene group, a trimethylene group, and a tetramethylene group, and most preferably an ethylene group.

[0089] In this specification, an "oxydialkylene group having 8 or less carbon atoms" refers to an alkylene group (alkylene-O-alkylene, in other words, an "alkyleneoxyalkylene group") bonded via an ether bond, in which the total number of carbon atoms in the two alkylene groups is 8 or less. Here, the two alkylene groups may be the same or different, but are preferably the same. Specific examples of oxydialkylene groups having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydi(trimethylene) group (i.e., a trimethyleneoxytrimethylene group), and an oxydi(tetramethylene) group (i.e., a tetramethyleneoxytetramethylene group). Preferred are an oxydimethylene group, an oxydiethylene group, and an oxydi(trimethylene) group, and most preferred is an oxydiethylene group.

[0090] R ２ａ is R ２ｂ may be the same as or different from, but preferably, R ２ａ is R ２ｂ is the same group as

[0091] Y ａ and Y ｂ are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or a carbamate bond, more preferably each independently an ester bond or an amide bond, and most preferably each an ester bond. ａ and Y ｂ The bond direction of Y is not limited. ａ and Y ｂ is an ester bond, preferably -Z ａ -CO-O-R ２ａ - and -Z ｂ -CO-O-R ２ｂ - structure.

[0092] Y ａ Is Y ｂ may be the same as or different from, but preferably, Y ａ Is Y ｂ is the same group as

[0093] Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. The aromatic compound preferably contains 6 to 12 carbon atoms, and most preferably 6 to 7 carbon atoms. The aromatic compound preferably contains one aromatic ring.

[0094] Examples of the aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms include, for example, a benzene ring, a naphthalene ring, and an anthracene ring as aromatic hydrocarbon rings, and an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furanthiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring as aromatic hetero rings. Of these, a benzene ring, a naphthalene ring, and an anthracene ring are preferred, and a benzene ring is most preferred.

[0095] The aromatic ring may have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, a methyl group having 1 to 4 ... Examples of the alkyl group include an aryl group having 0 carbon atoms and an aryloxy group having 6 to 10 carbon atoms, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0096] Z ａ and Z ｂ Preferred specific structures of Z １ Examples include:

[0097]

[0098] In the formula, s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R ４ each independently represents a substituent.

[0099] Z １ In the formula, s is preferably an integer of 0 to 1, and more preferably 0. １ t is preferably an integer of 0 to 2, more preferably 1. １ The u in the formula (1) is preferably an integer of 0 to 2, and more preferably an integer of 0 to 1.

[0100] Z １ R ４is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the synthesis process of the ionic lipid. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, Examples of such an aryloxy group include those having 6 to 10 carbon atoms, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group. ４ When there are multiple R ４ may be the same or different.

[0101] Z ａ is Z ｂ may be the same as or different from, but preferably, Z ａ is Z ｂ is the same group as

[0102] n ａ and n ｂ are each independently 0 or 1.

[0103] n ａ is n ｂ may be the same as or different from, but preferably, n ａ is n ｂ is the same as

[0104] R ３ａand R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (4): R ９ —O—CO—(CH ２ )a- (4) (In formula (4), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.) Preferably, each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and most preferably, each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0105] The residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride is a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with a hydroxyl group of *-O-CO-CH ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure in which the hydroxyl group of a sterol derivative is replaced by *-O-CO-CH. * represents the bonding position with a fat-soluble vitamin. The residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride is a residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride. ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure where a - is replaced with *. The symbol * represents the bonding position with the sterol derivative.

[0106] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0107] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and preferably cholesterol or cholestanol.

[0108] The aliphatic hydrocarbon group having 1 to 40 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained 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, with carbon-carbon double bonds being preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., with alkyl or alkenyl groups being preferred. Specific examples of the aliphatic hydrocarbon group having 1 to 40 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a henicosyl group, a docosyl group, a tricosyl group, a tetracosyl group, a pentacosyl group, a hexacosyl group, a heptacosyl group, an octacosyl group, a nonacosyl group, a triacontyl group, a tetracontyl group, a dodecenyl group, a tridecenyl group, a tetracont ... Adecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosenyl group, docosenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosenyl group Examples of such groups include a 1-hexyl group, a 1-hexylnonyl group, a 1-octylundecyl group, a 1-decylundecyl group, and a 1-decylundecyl group.The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0109] 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 the formula (1) is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). Specific examples of the aliphatic hydrocarbon group include a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.

[0110] R ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the formula (I) means an alkyl group having 3 to 40 carbon atoms and having at least one cyclopropane ring in the alkyl chain. The number of carbon atoms in the alkyl group, 3 to 40, does not include the number of carbon atoms in 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 formula (5):

[0111]

[0112] (In formula (5), b and c are each independently an integer, and the sum of b and c is 2 to 39.) Preferably, b is an integer of 1 to 20, and c is an integer of 1 to 19. More preferably, b is an integer of 2 to 18, even more preferably an integer of 3 to 17, and even more preferably an integer of 4 to 12. More preferably, c is an integer of 3 to 15, even more preferably an integer of 3 to 11, and even more preferably an integer of 3 to 9. Examples of the group represented by formula (5) include 7-(2-octylcyclopropyl)heptyl.

[0113] In formula (4), R９The aliphatic hydrocarbon group having 2 to 20 carbon atoms represented by the formula (I) may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained 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, with carbon-carbon double bonds being preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, even more preferably 13 to 19, and most preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., with alkyl groups or alkenyl groups being preferred, and alkyl groups being more preferred. Specific examples of the aliphatic hydrocarbon group having 2 to 20 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, and a heptadecenyl group. Examples of the alkyl group include an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosenyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group, a tetradecadienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-ethylnonyl group, a 1-butylnonyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, and a 3-octylundecyl group.The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0114] In formula (4), a is preferably an integer of 3 to 9, more preferably an integer of 3 to 7, even more preferably an integer of 5 to 7, and most preferably 5 or 7.

[0115] R ３ａ is R ３ｂ may be the same as or different from, but preferably, R ３ａ is R ３ｂ is the same group as

[0116] In one embodiment of the present invention, R １ａ is R １ｂ is the same as X ａ is X ｂ is the same as R ２ａ is R ２ｂ is the same as Y ａ Is Y ｂ is identical to Z ａ is Z ｂ is the same as R ３ａ is R ３ｂ is the same as

[0117] Suitable examples of the ionic lipid (1) include the following ionic lipids: [Ionic lipid (1-1)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., a piperidylene group); R ２ａ and R ２ｂare each independently an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a hetero atom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0118] [Ionic lipid (1-2)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., a piperidylene group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; ａ and Z ｂare each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0119] [Ionic lipid (1-3)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ are each independently, X １ :

[0120]

[0121] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0122]

[0123] (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; ａ and Z ｂ are each independently, Z １ :

[0124]

[0125] (wherein s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R ４ each independently represents a substituent; ａ and n ｂ are each independently 0 or 1; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0126] Specific examples of the ionic lipid (1) include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, and TS-P4C2.

[0127] Specific examples of the ionic lipid (1) include Lipid 1 to Lipid 20 described in WO 2021 / 195529 A2, and in particular, the following Lipid 1, Lipid 5, and Lipid 8.

[0128]

[0129]

[0130]

[0131] Among specific examples of the ionic lipid (1), SS-OP or SS-EC is preferred. That is, the ionic lipid (1) is preferably an ionic lipid represented by the following formula:

[0132]

[0133]

[0134] The amount of ionic lipid (1) in the lipid nanoparticles of the present invention is preferably 30 to 70 mol%, more preferably 40 to 65 mol%, and even more preferably 50 to 60 mol%, based on the total amount of ionic lipid (1), anionic phospholipid or compound represented by formula (2), neutral phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0135] The ionic lipid (1) can be produced by known methods (for example, the methods described in WO 2019 / 188867 A1, US 9708628 B2, and WO 2021 / 195529 A2).

[0136] The lipid nanoparticles of the present invention contain anionic phospholipids as the phospholipids. The phospholipids may be anionic phospholipids alone or may be used in combination with other neutral phospholipids.

[0137] Specific examples of anionic phospholipids include 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), and lyso forms thereof.

[0138] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoserine (PS) include 1,2-didecanoyl-sn-glycero-3-phosphoserine (DDPS), 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS), and 1,2-dierucoyl-sn-glycero-3-phosphoserine (DEPS). Examples of phospholipids include 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphoserine (MPPS), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphoserine (MSPS), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphoserine (PMPS), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphoserine (PSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS). Note that, in the present specification, phospholipids may be referred to by their abbreviations. For example, 1,2-diacyl-sn-glycero-3-phosphoserine is sometimes referred to as PS, and 1,2-didecanoyl-sn-glycero-3-phosphoserine is sometimes referred to as DDPS.

[0139] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG) include 1,2-didecanoyl-sn-glycero-3-phosphoglycerol (DDPG), 1,2-dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol (DLoPG), Examples of the glycerols include 1,2-dierucoyl-sn-glycero-3-phosphoglycerol (DEPG), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphoglycerol (MPPG), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphoglycerol (MSPG), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphoglycerol (PMPG), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphoglycerol (PSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (SOPG).

[0140] Specific examples of 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA) include 1,2-didecanoyl-sn-glycero-3-phosphatidic acid (DDPA), 1,2-dilauroyl-sn-glycero-3-phosphatidic acid (DLPA), 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dilinoleoyl-sn-glycero-3-phosphatidic acid (DLoPA), Examples of phosphatidic acid include 1,2-dierucoyl-sn-glycero-3-phosphatidic acid (DEPA), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphatidic acid (MPPA), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphatidic acid (MSPA), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphatidic acid (PMPA), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphatidic acid (PSPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (SOPA).

[0141] The anionic phospholipid is preferably 1,2-diacyl-sn-glycero-3-phosphoglycerol, more preferably at least one selected from the group consisting of DMPG, DPPG, POPG, DOPG, SOPG, and DSPG, and even more preferably POPG or DOPG. These phospholipids have similar acyl chain structures and are therefore thought to have similar effects.

[0142] Other forms of anionic phospholipids include preferably 1,2-diacyl-sn-glycero-3-phosphoserine, more preferably at least one selected from the group consisting of DPPS, POPS, DOPS, DLoPS, SOPS, and DSPS, and even more preferably DLoPS. These phospholipids have similar acyl chain structures and are therefore thought to have similar effects.

[0143] Other examples of neutral phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), and lyso forms thereof.

[0144] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) include 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-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-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLiPC), 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), 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), Examples include 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

[0145] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) include 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), and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE). Examples of the phosphoethanolamine include 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), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

[0146] The other neutral phospholipids are not particularly limited, but are preferably at least one selected from the group consisting of DPPC, DOPC, DSPC, DEPC, POPC, SOPC, POPE and DOPE, more preferably at least one selected from the group consisting of DSPC, DOPC and DEPC, and most preferably DEPC.

[0147] When an anionic phospholipid and another neutral phospholipid are used in combination, the ratio of the anionic phospholipid to the neutral phospholipid is preferably 100:0 to 25:75 mol%, more preferably 75:25 to 25:75 mol%, and even more preferably 50:50 mol%.

[0148] The total amount of anionic phospholipids in the lipid nanoparticles of the present invention is preferably 1 to 20 mol %, more preferably 2.5 to 15 mol %, and even more preferably 2.5 to 10 mol %, based on the total amount of ionic lipid (1), anionic phospholipid or compound represented by formula (2), neutral phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0149] The total amount of neutral phospholipids in the lipid nanoparticles of the present invention is preferably 0 to 15 mol%, more preferably 2 to 10 mol%, and even more preferably 2.5 to 7.5 mol%, based on the total amount of ionic lipid (1), anionic phospholipid or compound represented by formula (2), neutral phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0150] Cholesterol The lipid nanoparticles of the present invention may contain anionic cholesterol of formula (2) (sometimes abbreviated as "anionic cholesterol (2)" in this specification) instead of anionic phospholipid.

[0151]

[0152] (In formula (2), T represents a divalent aliphatic hydrocarbon group having 1 to 8 carbon atoms.)

[0153] The divalent aliphatic hydrocarbon group having 1 to 8 carbon atoms represented by T may be linear or branched, or may contain unsaturation. T is preferably a linear divalent saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, and more preferably a linear divalent saturated aliphatic hydrocarbon group having 1 to 3 carbon atoms. A specific example of anionic cholesterol (2) is cholesteryl hemisuccinate.

[0154] From the viewpoints of nucleic acid encapsulation efficiency, nucleic acid release efficiency in cells, and lipid nanoparticle stability, the total amount of anionic cholesterol (2) in the lipid nanoparticles of the present invention is preferably 1 to 20 mol %, more preferably 2.5 to 15 mol %, and even more preferably 2.5 to 10 mol %, based on the total amount of ionic lipid (1), anionic phospholipid or anionic cholesterol (2), neutral phospholipid, and cholesterol.

[0155] The lipid nanoparticles of the present invention contain cholesterol. From the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability, the amount of cholesterol in the lipid nanoparticles of the present invention is preferably 20 to 60 mol%, more preferably 25 to 50 mol%, and even more preferably 30 to 40 mol% relative to the total amount of the ionic lipid (1), anionic phospholipid or anionic cholesterol (2), neutral phospholipid, and cholesterol.

[0156] Dimyristoylglycerol PEG The lipid nanoparticles of the present invention are represented by the formula (3): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (3) (In formula (3), R ６ , R ７ and R ８ wherein any two of the groups represent myristoyl groups, and the remaining group represents an alkyl group having 1 to 6 carbon atoms linked via a polyethylene glycol chain (PEG chain) having a number-average molecular weight of 1,000 to 3,000.)

[0157] The number average molecular weight of the PEG chain in formula (3) is 1,000 to 3,000, and preferably 1,500 to 2,500. The number average molecular weight of the PEG used to form the PEG chain can be measured by gel permeation chromatography (GPC).

[0158] The alkyl group having 1 to 6 carbon atoms may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group is preferred.

[0159] The amount of dimyristoylglycerol PEG (3) in the lipid nanoparticles of the present invention is preferably 0.5 to 1.5 mol %, more preferably 1.0 mol %, based on the total amount of the ionic lipid (1), anionic phospholipid or anionic cholesterol (2), neutral phospholipid, and cholesterol, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability.

[0160] Optimal Composition The optimal molar ratio of ionic lipid (1): anionic phospholipid or anionic cholesterol (2): neutral phospholipid: cholesterol: dimyristoylglycerol PEG (3) in the lipid nanoparticles of the present invention is 55:5:5:35:1.0.

[0161] Method for Producing Lipid Nanoparticles The lipid nanoparticles of the present invention can be produced by dispersing a lipid raw material containing an ionic lipid (1), an anionic phospholipid or anionic cholesterol (2), cholesterol, and dimyristoylglycerol PEG (3) in an appropriate dispersion medium (e.g., an aqueous dispersion medium, an alcoholic dispersion medium), and optionally performing an operation to induce organization.

[0162] Examples of the "operation to induce organization" for producing the lipid nanoparticles of the present invention include alcohol (e.g., ethanol, tert-butanol, etc.) dilution using a microchannel or vortex, simple hydration, ultrasonic treatment, heating, vortexing, ether injection, French press method, cholic acid method, Ca ２＋Examples of known methods include fusion, freeze-thaw, and reverse-phase evaporation. The alcohol dilution method using a microchannel or vortex is preferred, and the alcohol dilution method using a microchannel is more preferred. In the alcohol dilution method using a microchannel, for example, NanoAssemblr (registered trademark) (Precision NanoSystems) can be used to produce a dispersion containing lipid nanoparticles by mixing an acidic buffer solution containing nucleic acid with an ethanol solution of lipids. The dispersion produced by this method contains lipid nanoparticles and a dispersion medium (an acidic buffer solution and an alcohol). The dispersion medium (particularly alcohol) can be removed or replaced by ultrafiltration, dialysis, dilution, or other procedures.

[0163] The present invention also provides a method for delivering nucleic acid to spleen cells, which comprises administering to a subject lipid nanoparticles of the present invention encapsulating nucleic acid. In this method, the lipid nanoparticles are preferably administered intravenously to the subject.

[0164] Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, any one of mono-, tri-, and tri-stranded nucleic acids can be used, but single- or double-stranded nucleic acids are preferred. Nucleic acids may be, for example, nucleotides having N-glycosides of purine or pyrimidine bases, oligomers having non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs)), or oligomers having special linkages (provided that the oligomers contain nucleotides having a configuration that allows base pairing or base attachment as found in DNA or RNA).

[0165] Furthermore, the nucleic acid may be, for example, a nucleic acid having a known modification added thereto, a nucleic acid labeled as known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more naturally occurring nucleotides are replaced with an analogue, a nucleic acid having a modified nucleotide, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid containing an intercurrent compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-anomeric nucleic acid, etc.), etc.

[0166] The type of DNA that can be 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., and preferably plasmid DNA, cDNA, and antisense DNA, and more preferably plasmid DNA. Circular DNA such as plasmid DNA can also be digested with appropriate restriction enzymes and used as linear DNA.

[0167] The type of RNA that can be 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., and preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.

[0168] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0169] The nucleic acid used in the present invention is preferably one that has preventive and / or therapeutic activity against a specific disease (prophylactic / therapeutic nucleic acid), such as nucleic acid used in so-called gene therapy.

[0170] The particle size of the lipid nanoparticles encapsulating nucleic acid is not particularly limited, but is preferably 10 nm to 500 nm, and more preferably 30 nm to 300 nm. Measurement of the particle size can be performed using a particle size distribution analyzer such as Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles encapsulating nucleic acid can be adjusted appropriately depending on the manufacturing method.

[0171] The surface potential (zeta potential) of lipid nanoparticles encapsulating nucleic acids for delivery into spleen cells is preferably −30 to +10 mV, more preferably −25 to 0 mV. Previous gene transfer techniques primarily used positively charged particles. This is useful as a method for promoting electrostatic interactions with heparin sulfate on the negatively charged cell surface and facilitating cellular uptake. However, a positive surface potential can (a) inhibit the release of nucleic acids from the carrier due to intracellular interaction with the delivered nucleic acid, and (b) inhibit protein synthesis due to interaction between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface potential (zeta potential) within the above range. Measurement of the surface potential (zeta potential) can be performed using a zeta potential measuring device such as a Zetasizer Nano. The surface potential (zeta potential) of lipid nanoparticles can be adjusted by the composition of the lipid nanoparticle's components.

[0172] By administering the lipid nanoparticles of the present invention encapsulating nucleic acids to a living body, the lipid nanoparticles reach and contact splenic tissue, and the nucleic acid encapsulated in the lipid nanoparticles is delivered to the splenic tissue in the living body. The subjects to which the lipid nanoparticles can be administered are not particularly limited, and examples include cells of mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), and fish (e.g., zebrafish, medaka, etc.). The subjects to which the lipid nanoparticles are introduced are preferably human or other mammalian cells.

[0173] The method of administering lipid nanoparticles encapsulating nucleic acids to a subject is not particularly limited as long as the lipid nanoparticles can deliver nucleic acids to spleen cells, and can be appropriately selected from known administration methods (e.g., oral administration, parenteral administration (e.g., intranasal administration, intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)). Intravenous administration is preferred as the administration method. The dosage of the lipid nanoparticles can be appropriately selected taking into consideration the type of subject, the administration method, etc.

[0174] The lipid nanoparticles of the present invention can be prepared as oral preparations (e.g., tablets, capsules, etc.) or parenteral preparations (e.g., nasal preparations, injections, inhalants, etc.), either as they are or mixed with a pharmaceutically acceptable carrier, preferably as parenteral preparations (more preferably nasal preparations).

[0175] Pharmaceutically acceptable carriers include those commonly used as formulation materials, such as excipients, lubricants, binders, and disintegrants for solid formulations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents for liquid formulations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed. For example, nasal preparations are used in the form of nasal drops or sprays.

[0176] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.

[0177] In the following examples, the ionic lipid (1) is shown by the name shown in the above table. The abbreviations used in the following examples have the following meanings: Chol: cholesterol CHEMS: cholesteryl hemisuccinate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine SOPC: 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine DEPC: 1,2-dierucoyl-sn-glycero-3-phosphocholine POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPG: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol DMPG: 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol DPPG: 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol DSPG: 1,2-distearoyl-sn-glycero-3-phosphoglycerol POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol SOPG: 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoglycerol DLoPS: 1,2-dilinoleoyl-sn-glycero-3-phosphoserine DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (number average molecular weight of PEG (Mn): 2000) MES: 2-morpholinoethanesulfonic acid DiR: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine, iodide PBS: phosphate-buffered saline The mol% of the lipid composition indicates the mol% relative to the total of ionic lipid (1), anionic phospholipid or anionic cholesterol (2), neutral phospholipid, and cholesterol.

[0178] [Manufacturing Example 1] Preparation of mRNA-Encapsulated LNP (1) Preparation of Lipid Alcohol Solution Each lipid was dissolved in ethanol to give 10 mM SS-OP, 5 mM phospholipid, 5 mM CHEMS, 10 mM Chol, and 1 mM DMG-PEG2000. From these solutions, selected lipids were mixed at any ratio so that the total mol amount was 1000 nmol, and a 1 mM DMG-PEG2000 ethanol solution was added to give the desired ratio. Finally, an ethanol solution of lipids was prepared by adding ethanol to a total volume of 450 μL. A similar method was used when using a lipid tert-butanol solution.

[0179] (2) Preparation of Acidic Buffer Solution of Nucleic Acid The acidic buffer solution of nucleic acid was prepared by mixing mRNA encoding the luciferase gene (luc-mRNA) with 20 mM acidic malic acid buffer (pH 3.0) containing 30 mM NaCl to give a concentration of 0.0067 μg / μL.

[0180] (3) Preparation of LNP by alcohol dilution method Using a NanoAssmblr (registered trademark) ultrafast nanomedicine production device (manufactured by Precision NanoSystems), 1200 μL of nucleic acid acidic buffer solution and 300 μL of lipid alcohol solution were mixed at flow rates of 3 mL / min and 1 mL / min, respectively, and 1 mL of LNP solution was recovered. The resulting LNP was diluted with 3 mL of 20 mM MES buffer (pH 6.5) and transferred to an Amicon Ultra 4 (Millipore). The transferred LNP solution was subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, approximately 10 minutes) and concentrated to approximately 500 μL. The resulting concentrate was diluted to 4 mL with PBS and then concentrated to approximately 200 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, approximately 10 minutes). Finally, the concentrate was diluted to a volume of 10 μg / mL with PBS.

[0181] [Test Example 1] Measurement of particle size and surface potential of various mRNA-encapsulated LNPs The particle size and surface potential of the mRNA-encapsulated LNPs of Examples 1 to 10, which had different lipid compositions prepared in Production Example 1, and the mRNA-encapsulated LNPs of Comparative Examples 1 and 2, were measured by dynamic scattering (Zetasizer Nano; Malvern). The results are shown in Table 2. All LNPs had desirable particle sizes and surface charges of -3 mV to -15 mV. The LNPs of Examples 11 to 32 and Comparative Examples 3 and 4 were prepared with the compositions shown in Table 3.

[0182]

[0183]

[0184] [Test Example 2] Evaluation of gene expression activity in the spleen 1 (evaluation of the combination with anionic phospholipids or anionic cholesterol) (1) IV administration to mice The luc-mRNA-encapsulated LNP prepared according to the method described in Production Example 1 was diluted with PBS to an mRNA concentration of 5.0 μg / mL. The diluted mRNA-encapsulated LNP was intravenously administered to 6-week-old female BALB / c mice at a volume of 200 μL per mouse (mRNA dose of 1.0 μg per mouse). (2) Measurement of gene expression activity Six hours after administration, spleens were collected from the mice and stored frozen at -80°C in homogenization tubes containing cell disruption beads. The frozen tube was removed, and 800 μL of lysis buffer (77 mM Tris, 2 mM EDTA 2Na, 0.1% Triton X-100-containing MQ pH 7.8) was added. Homogenization was repeated twice at 4,500 rpm for 30 seconds at -2°C using a Micro Smash (TOMY). 500 μL of the supernatant was transferred to another tube and centrifuged (4°C, 13,000 rpm, 10 minutes), and 300 μL of the supernatant was collected. 50 μL of Luciferase Substrate (Promega) prepared according to the manufacturer's protocol was added to 20 μL of lysis buffer, and luminescence was measured using a Glomax 20 / 20 luminometer (Promega). This was used as the background. Similarly, 50 μL of Luciferase Substrate was added to 20 μL of each sample, and luminescence was measured. 10 μL of the supernatant was diluted 100-fold with MQ to prepare 1 mL of diluted solution, and protein concentration was measured using the BCA method using 25 μL of the diluted solution. Gene expression activity was calculated as relative luminescence units (RLU) using luminescence / (protein concentration x 0.02). The gene expression activity is shown in Figure 1 as a relative value to Comparative Example 1. As a result, it was confirmed that the LNPs of Examples 1 and 2 had higher gene expression activity in the spleen than the LNP of Comparative Example 1, which has a composition for liver delivery. Because gene expression activity depends on the efficiency of nucleic acid delivery into target cells, this indicates that the LNPs were able to efficiently deliver nucleic acids to the spleen.

[0185] [Test Example 3] Evaluation of gene expression activity in the spleen 2 (evaluation in combination with PS) (1) Preparation of fluorescently labeled LNP A lipid alcohol solution containing a fluorescent dye was prepared by adding an alcohol solution of DiR to the lipid alcohol solution described in Production Example 1 at a concentration of 0.2 mol% relative to the total lipid amount. Fluorescently labeled LNP was obtained by preparing LNP using this lipid alcohol solution according to the procedure described in Production Example 1. (2) IV administration to mice The luc-mRNA-encapsulated LNP prepared according to the method described in Production Example 1 was diluted with PBS to an mRNA concentration of 5.0 μg / mL. The diluted mRNA-encapsulated LNP was intravenously administered to 6-week-old female BALB / c mice at a volume of 200 μL per mouse (1.0 μg of mRNA per mouse). (3) Evaluation of LNP Accumulation and Gene Expression Activity Five hours and 55 minutes after administration, luciferin diluted to 15 mg / mL with PBS was intraperitoneally administered to mice at 200 μL per mouse. Five minutes after luciferin administration (6 hours after LNP administration), the mice were euthanized, and the spleen and liver removed to a petri dish were imaged using IVIS (PerkinElmer). Image quantification was used to measure LNP accumulation based on DiR fluorescence intensity, and gene expression based on luciferin luminescence intensity. Figure 2 shows the fluorescence intensity of each LNP, indicating the amount of fluorescently labeled LNP accumulated in the spleen. Figure 3 shows gene expression activity in the spleen, indicating nucleic acid delivery efficiency. It was confirmed that the LNP of Example 3 exhibited higher LNP accumulation and nucleic acid delivery efficiency in the spleen than the LNP of Comparative Example 2, which is an LNP formulated for liver delivery.

[0186] [Test Example 4] Evaluation of Gene Expression Activity in the Spleen 3 (Evaluation of Combination with Neutral Phospholipids) (1) IV Administration to Mice LNPs encapsulating luc-mRNA prepared according to the method described in Production Example 1 were intravenously administered to mice under the same conditions as in Test Example 1 (1.0 μg of mRNA per mouse). (2) Measurement of Gene Expression Activity Measurements were performed using the method described in Test Example 2, and the gene expression activity was calculated as a relative value to that of Comparative Example 1. As a result, it was confirmed that both LNPs using the anionic phospholipid DOPG alone as the phospholipid and those combining DOPG with a neutral phospholipid (1,2-diacyl-sn-glycero-3-phosphocholine or 1,2-diacyl-sn-glycero-3-phosphoethanolamine) showed higher gene expression activity in the spleen than the LNP of Comparative Example 1, which is formulated for liver delivery (FIG. 4).

[0187] [Test Example 5] Evaluation of Gene Expression Activity in the Spleen 4 (Evaluation with Various Lipid Compositions) (1) IV Administration to Mice LNPs encapsulating luc-mRNA prepared according to the method described in Production Example 1 were intravenously administered to mice under the same conditions as in Test Example 1 (1.0 μg of mRNA per mouse). (2) Measurement of Gene Expression Activity Measurements were performed using the method described in Test Example 2, and the gene expression activity was calculated as a relative value to that of Example 5. Gene expression in the spleen was confirmed for LNPs with all lipid compositions. Examples 11, 18, 19, and 21 showed particularly high gene expression activity compared to Example 5 ( FIG. 5 ).

[0188] [Test Example 6] Evaluation of gene expression activity in the spleen 5 (evaluation using various PGs) (1) IV administration to mice LNPs encapsulating luc-mRNA prepared according to the method described in Production Example 1 were intravenously administered to mice under the same conditions as in Test Example 1 (1.0 μg of mRNA per mouse). (2) Measurement of gene expression activity Measurement was performed according to the method described in Test Example 2, and the gene expression activity was calculated as a relative value to that of Example 18. Gene expression activity in the spleen was confirmed for LNPs using any of the PGs. LNPs with lipid compositions containing DOPG or POPG showed particularly high gene expression activity ( FIG. 6 ).

[0189] Test Example 7 Evaluation of Gene Expression Activity in the Spleen 6 (Evaluation Using SS-EC) (1) IV Administration to Mice LNPs encapsulating luc-mRNA prepared according to the method described in Production Example 1 were intravenously administered to mice under the same conditions as in Test Example 1 (1.0 μg of mRNA administered per mouse). (2) Measurement of Gene Expression Activity Measurement was performed using the method described in Test Example 2, and the gene expression activity was calculated as a relative value to that of Comparative Example 3. Compared to Comparative Example 3, which is an LNP formulated for liver delivery, the LNP of Example 30 exhibited high gene expression activity in the spleen ( FIG. 7 ).

[0190] Test Example 8 Evaluation of CTL Activity Using LNP (1) IV Administration to Mice LNP encapsulating mRNA (hereinafter referred to as OVA-mRNA) encoding the ovalbumin gene (hereinafter referred to as OVA) prepared according to the method described in Production Example 1 was diluted with PBS to an mRNA concentration of 5.0 μg / mL. The diluted mRNA-encapsulated LNP was intravenously administered to 6-week-old female C57BL / 6J mice at a volume of 200 μL per mouse (mRNA dose of 1.0 μg per mouse). (2) Evaluation of CTL Activity One week after administration, the following CTL assay was performed to evaluate OVA-specific cytotoxic T cell (CTL) activity. The splenocyte culture medium used was composed of 500 mL of RPMI 1640, 50 mL of FBS, 5 mL of 100 mM sodium pyruvate, 5 mL of 1 M HEPES, 500 μL of 55 mM 2-mercaptoethanol, and 5 mL of penicillin / streptomycin (hereafter referred to as the medium). Untreated mice were euthanized by cervical dislocation, and the spleens were harvested. The spleens were cut in half, and splenocytes were removed from the cut surface using tweezers and suspended in the medium. The cell suspension medium was collected in a 50 mL tube through a 40 μm cell strainer and centrifuged (4°C, 500 g, 5 minutes). The supernatant was discarded, and 1 mL of Red Blood Cell Lysis Buffer (SIGMA) was added per mouse, followed by incubation for 5 minutes. The cells were diluted 5-fold with medium and centrifuged (4°C, 500g, 5 minutes). The cells were then suspended in 10 mL of medium and centrifuged (4°C, 500g, 5 minutes). The cells were then suspended in 30 mL of medium and the cell count was measured. The cells were then passed through a 40 μm cell strainer and divided equally into two 50 mL tubes, followed by centrifugation (4°C, 500g, 5 minutes). 1.0 x 10 ７ The cells were suspended in medium to a concentration of 1.5 × 10 cells / mL. (1) To one of the cell suspensions, 2 mM OVA epitope solution (consisting of the amino acid sequence of positions 257-264 of OVA) was added in an amount of 1 / 400 of the cell suspension, and the mixture was left to stand in a culture incubator for 1 hour. This epitope-pulsed group was centrifuged (4°C, 500 g, 5 minutes). After washing with 10 mL of medium and then with 10 mL of PBS, the cells were counted again and then 3.0 × 10 cells were collected. ７The other cell suspension was incubated without adding the epitope and washed with medium and PBS. ７ The target cells and control cells were washed twice with medium and twice with PBS, and then diluted to 5.0 × 10 ７ The cells were suspended at 100 μL / mL. 100 μL of "target cells" and 100 μL of "control cells" were mixed and administered to immunized mice. The two cells can be distinguished by the intensity of CFSE fluorescence. 20 hours after administration of the cell mixture, spleens were harvested from the mice and analyzed by flow cytometry. The amount of "target cells" was corrected for by the amount of "control cells," and epitope-specific CTL activity was quantified.

[0191] The evaluation results are shown in Figures 8 and 9. The LNPs of Examples 18 and 24 exhibited approximately three times the CTL activity of the LNPs of Comparative Example 1, which are LNPs with a composition for liver delivery. Furthermore, the LNPs of Example 30 exhibited higher CTL activity than the LNPs of Comparative Example 3, which are LNPs with a composition for liver delivery, and the LNPs of Comparative Example 4, which are LNPs with a composition for spleen delivery. CTL activity is an index for evaluating whether adaptive immunity can be obtained, and the higher the CTL activity, the more adaptive immunity can be obtained. Therefore, it was shown that the LNPs of Examples 18, 24, and 30 can enhance adaptive immunity more than the comparative examples.

[0192] Test Example 9: Evaluation of CTL activity using LNP 2 (1) IV administration to mice OVA-mRNA-encapsulated LNP prepared according to the method described in Production Example 1 was diluted with PBS to an mRNA concentration of 1.0 μg / mL. The diluted mRNA-encapsulated LNP was intravenously administered to 6-week-old female C57BL / 6J mice at a volume of 200 μL per mouse (mRNA dose of 0.2 μg per mouse). (2) Evaluation of CTL activity CTL activity was evaluated using the method described in Test Example 8. The results are shown in Figure 10. The LNP with the composition of Example 31 exhibited higher CTL activity than Comparative Example 4.

[0193] Test Example 10: Evaluation of the Therapeutic Effect of Multiple Sclerosis in Mice (1) Preparation of Model Mice Ten-week-old female C57BL / 6J mice were pre-bred for one week to allow them to acclimate to the breeding environment. According to the manufacturer's protocol for a kit product (Hooke Laboratories) for inducing experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model, a mixed emulsion of an autoantigen peptide of myelin oligodendrocyte glycoprotein (MOG) (comprising the amino acid sequence of positions 35-55 of MOG) and complete Freund's adjuvant was subcutaneously administered in 100 μL each to the neck and lumbar regions of mice. The pertussis toxin stock included in the kit product was diluted with PBS to a concentration of 165 ng / 100 μL. The diluted pertussis toxin was administered intraperitoneally twice, at 100 μL per mouse, on the day of administration of the mixed emulsion and the following day. (2) IV Administration to Mice LNPs containing luc-mRNA or LNPs containing mRNA encoding MOG peptide (comprising the amino acid sequence of positions 27-63 of MOG) (hereinafter referred to as MOG-mRNA) prepared according to the method described in Preparation Example 1 were diluted with PBS to an mRNA concentration of 1.0 μg / 200 μL. The diluted mRNA-encapsulated LNPs were intravenously administered to EAE mice three times, at 200 μL per mouse, on days 7, 10, and 13 from the day of EAE induction. (3) Evaluation of Multiple Sclerosis Therapeutic Efficacy From day 10 onwards, the pathology score and body weight of the mice were measured daily. The pathology score was determined according to the following criteria. 0: no symptoms, 1: tail droops when lifted from the base, 2: paralysis of one hind leg / abnormal gait, 3: paralysis of both hind legs, 3.5: score 3 and clear decrease in locomotion, 4: paralysis of forelimbs, 5: moribund / death. To avoid bias in the score measurement, LNP administration and score measurement were performed by a different experimenter, and the scores were measured in a blinded manner.

[0194] The evaluation results are shown in Figures 11 and 12. It was confirmed that the LNP of Example 32 encapsulating MOG-mRNA alleviated EAE symptoms. Furthermore, the LNP of Example 32 encapsulating luc-mRNA also alleviated EAE symptoms, suggesting that the LNP of Example 32 itself contributed to the alleviation of EAE symptoms.

[0195] The lipid nanoparticles of the present invention are useful for delivering nucleic acids to spleen cells.

[0196] This application is based on patent application No. 2022-051918 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. (A) Formula (1): 【Chemistry 1】 (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 or oxydialkylene group having 8 or fewer 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 ｂ each independently represents 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 of these is independently either 0 or 1, 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 having a cyclopropane ring, or formula (4): R ９ -O-CO-(CH ２ )a- (4) (In formula (4), R ９ This represents an aliphatic hydrocarbon group with 2 to 20 carbon atoms. (where 'a' represents an integer between 2 and 10.) (This represents the base represented by [this symbol].) Ionic lipids represented by (B) Anionic phospholipid or formula (2): 【Chemistry 2】 (In formula (2), (T represents a divalent aliphatic hydrocarbon group with 1 to 8 carbon atoms.) Compounds represented by, (C) Cholesterol, and (D) Formula (3): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (3) (In formula (3), R ６ , R ７ and R ８ Two of these represent myristoyl groups, and the remaining one represents a C1-C6 alkyl group linked via a polyethylene glycol chain with a number-average molecular weight of 1,000-3,000. Dimyristoylglycerol PEG Lipid nanoparticles used to deliver nucleic acids containing [specific substances] to spleen tissue.

2. The lipid nanoparticle according to claim 1, wherein the anionic phospholipid is 1,2-diacyl-sn-glycero-3-phosphoglycerol or 1,2-diacyl-sn-glycero-3-phosphoserine.

3. The anionic phospholipids are 1,2-dimiristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (SOPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2- The lipid nanoparticle according to claim 1, which is at least one selected from the group consisting of dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS), and 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS).

4. The lipid nanoparticle according to claim 1, wherein the anionic phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), or 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS).

5. Lipid nanoparticles according to any one of claims 1 to 4, wherein T in formula (2) is a divalent aliphatic hydrocarbon group having 1 to 3 carbon atoms.

6. The ionic lipid represented by formula (1) is given by the following formula: 【Transformation 3】 or 【Chemistry 4】 Lipid nanoparticles according to any one of claims 1 to 4, which are any ionic lipids represented by the formulas.

7. The lipid nanoparticles according to any one of claims 1 to 4, wherein the lipid nanoparticles may contain neutral phospholipids as a component of the lipid nanoparticles.

8. The lipid nanoparticle according to claim 7, wherein the neutral phospholipid is 1,2-diacyl-sn-glycero-3-phosphocholine or 1,2-diacyl-sn-glycero-3-phosphoethanolamine.

9. The lipid nanoparticle according to claim 7, wherein the neutral phospholipid is at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dielcoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

10. Lipid nanoparticles according to claim 7, wherein the ratio of the anionic phospholipid or the compound represented by formula (2) to the neutral phospholipid is 100:0 to 25:75 mol%.

11. Lipid nanoparticles according to claim 7, wherein, with respect to the total of the ionic lipid, the anionic phospholipid or compound represented by formula (2), the neutral phospholipid, and the cholesterol, the ionic lipid is 30 to 70 mol%, the anionic phospholipid or compound represented by formula (2) is 2.5 to 15 mol%, the neutral phospholipid is 0 to 15 mol%, the cholesterol is 20 to 60 mol%, and the dimyristoyl glycerol PEG is 0.5 to 1.5 mol%.

12. A method for delivering nucleic acids to spleen tissue, comprising intravenously administering lipid nanoparticles containing nucleic acids, as described in any one of claims 1 to 4, to a living organism (excluding humans).

13. (A) Formula (1): 【Transformation 5】 (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 or oxydialkylene group having 8 or fewer 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 ｂ Each 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 of these is independently either 0 or 1, 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 having a cyclopropane ring, or formula (4): R ９ -O-CO-(CH ２ )a- (4) (In formula (4), R ９ This represents an aliphatic hydrocarbon group with 2 to 20 carbon atoms. (where 'a' represents an integer between 2 and 10.) (This represents the base represented by [this symbol].) Ionic lipids represented by (B) Anionic phospholipid or formula (2): 【Transformation 6】 (In formula (2), (T represents a divalent aliphatic hydrocarbon group with 1 to 8 carbon atoms.) Compounds represented by, (C) Cholesterol, and (D) Formula (3): CH ２ (OR ６ )-CH(OR ７ )-CH ２ (OR ８ ) (3) (In formula (3), R ６ , R ７ and R ８ Two of these represent myristoyl groups, and the remaining one represents a C1-C6 alkyl group linked via a polyethylene glycol chain with a number-average molecular weight of 1,000-3,000. Dimyristoylglycerol PEG Use of lipid nanoparticles containing [specific substance] for the manufacture of pharmaceuticals used to deliver nucleic acids to spleen tissue.

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