Nucleic acid-ionized lipid complex

Abstract

The present invention relates to: a nucleic acid-ionized lipid complex which contains a nucleic acid that can be introduced into a cell by endocytosis and an ionized lipid; the nucleic acid-ionized lipid complex, in which the nucleic acid is modified with a molecule that binds to a receptor present on the surface of a cell; any of the nucleic acid-ionized lipid complexes, in which the ionized lipid has a tertiary amino group in a polar head portion; any of the nucleic acid-ionized lipid complexes, in which the nucleic acid is a nucleic acid for suppressing gene expression in a cell; and a pharmaceutical composition which contains any of the nucleic acid-ionized lipid complexes as an active ingredient.

Description

Nucleic acid-ionized lipid complex

[0001] This invention relates to nucleic acids, such as nucleic acid drugs, that are taken up into cells and function within them, and to pharmaceutical compositions containing such nucleic acids as active ingredients. This application claims priority based on Japanese Patent Application No. 2025-007482, filed in Japan on January 20, 2025, the contents of which are incorporated herein by reference.

[0002] In recent years, research into nucleic acid drugs using oligonucleotides has been progressing. Nucleic acid drugs have advantages such as high specificity for target molecules and fewer side effects. However, nucleic acid drugs have low cell membrane permeability, making it difficult to deliver them to target molecules present inside cells. In particular, because siRNA is double-stranded, both its molecular weight and negative charge are larger than those of antisense RNA, resulting in lower cell membrane permeability and the need for drug delivery via a carrier. Drug delivery agents that use lipid nanoparticles (Patent Document 1) or cationic polymer nanoparticles (Patent Document 2) are known. However, there is much room for improvement in terms of cell membrane permeability efficiency and toxicity concerns.

[0003] Compounds with polyfluoro structures are known to be stable and low-toxicity in vivo, and to have excellent uptake into cells and exit from endosomes (Non-Patent Document 1). Taking advantage of these properties, studies are underway to introduce polyfluoro structures into oligonucleotides and peptide nucleic acids as a cell membrane-permeable portion (Patent Documents 3 and 4, Non-Patent Documents 2-5).

[0004] Nucleic acid drugs face the challenge of stability when administered to the body, as phosphodiester bonds in nucleic acids are susceptible to degradation by nucleases. Low stability can lead to degradation within the body before reaching the target tissue, preventing the desired therapeutic effect from being achieved. To improve the stability of nucleic acids, chimeric nucleic acids are used, which combine natural nucleic acids with artificial nucleic acids that exhibit superior nuclease resistance. Examples of such artificial nucleic acids include acyclic glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), acyclic threoninol nucleic acids (aTNAs), and serinol nucleic acids (SNAs) (Non-Patent Literature 6). It has also been reported that cell membrane permeability can be further improved by introducing an alkyl group, which may be substituted with a fluorine atom, into the side chain of acyclic threoninol-type nucleic acids (aTNA-type nucleic acids) (Patent Literature 5).

[0005] International Publication No. 2011 / 036557, International Publication No. 2017 / 212006, International Publication No. 2012 / 130941, Japanese Patent Publication No. 2006-321797, International Publication No. 2022 / 186350

[0006] Zhang et al., MRS Communications, 2018, vol.8, p.303-313.Godeau et al., Medicinal Chemistry Communications, 2010, vol.1, p.76-78.Ellipilli et al., Chemical Communications, 2016, vol.52, p.521-524.Rochambeaua et al., Polymer Chemistry, 2016, vol.7, p. 4998-5003.Metelev et al., Theranostics, 2017, vol.7, p.3354-3368.Murayama et al., Chemistry A European Journal, 2013, vol.19, p.14151-14158.

[0007] Many nucleic acid drugs are used to induce the expression of foreign genes or to control the expression of specific genes after being taken up into target cells. Therefore, it is desirable for nucleic acid drugs to have not only high uptake efficiency into target cells but also excellent ability to escape from endosomes within the cell. However, some nucleic acids whose cell membrane permeability has been improved to increase uptake efficiency into cells have difficulty escaping from endosomes, resulting in insufficient therapeutic effect.

[0008] The present invention aims to provide nucleic acids with excellent ability to escape from endosomes, and pharmaceutical compositions containing such nucleic acids.

[0009] The inventors of the present invention have discovered that the ability of nucleic acids, which are taken into cells by endocytosis, to escape from endosomes can be improved by complexing them with ionized lipids, and have completed the present invention.

[0010] In other words, the present invention includes the following embodiments: [1] A nucleic acid-ionized lipid complex comprising a nucleic acid that can be introduced into a cell by endocytosis and an ionized lipid. [2] The nucleic acid-ionized lipid complex of [1], wherein the nucleic acid is introduced into the cell via a receptor present on the cell surface. [3] The nucleic acid-ionized lipid complex of [2], wherein the receptor is a scavenger receptor, an asialoglycoprotein receptor, a glucagon-like peptide-1 receptor, or an integrin. [4] The following general formula (A1) or (A2)

[0011]

[0012] [In the formula, R ０[1] A nucleic acid-ionized lipid complex comprising a nucleic acid having a structure represented by [1], n11 and n12 are each independently integers of 1 or more; B is a nucleic acid base; black circles indicate bonding hands] and an ionized lipid. [5] Any nucleic acid-ionized lipid complex of [1] to [4], wherein the ionized lipid has a tertiary amino group in its polar head portion. [6] Any nucleic acid-ionized lipid complex of [1] to [5], wherein the hydrophobic tail portion of the ionized lipid has two or more saturated hydrocarbon chains. [7] The nucleic acid-ionized lipid complex of [6] wherein an ester bond is inserted between carbon atoms in at least one saturated hydrocarbon chain of the two or more saturated hydrocarbon chains. [8] The nucleic acid-ionized lipid complex of any of [1] to [6] wherein the ionized lipid is selected from the group consisting of ALC-315, Lipid 5, SM-102, C12-200, and derivatives thereof. [9] The R ０ However, the nucleic acid-ionized lipid complex of any of the above [4] to [8] is an alkyl group having 1 to 30 carbon atoms substituted with at least two fluorine atoms.

[10] The R ０ However, the nucleic acid-ionized lipid complex of any of [4] to [8] is a perfluoroalkyl group having 1 to 10 carbon atoms, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a perfluoroalkyl group having 1 to 10 carbon atoms.

[11] The R ０

[12] A nucleic acid-ionized lipid complex according to any of [4] to [8], wherein the nucleic acid is an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom.

[13] A nucleic acid-ionized lipid complex according to any of [1] to

[12] , wherein the nucleic acid is a nucleic acid for suppressing gene expression in a cell.

[14] The nucleic acid-ionized lipid complex according to

[13] , wherein the nucleic acid for suppressing gene expression in a cell is siRNA.

[15] A nucleic acid-ionized lipid complex according to any of [1] to

[14] , which does not contain any lipids other than the ionized lipid.

[16] A pharmaceutical composition comprising any nucleic acid-ionized lipid complex according to [1] to

[15] as an active ingredient.

[17] A method for producing a nucleic acid-ionized lipid complex, comprising mixing a solution containing a nucleic acid that can be introduced into cells by endocytosis with a solution containing an ionized lipid to complex the nucleic acid and the ionized lipid, thereby producing any of the nucleic acid-ionized lipid complexes described in [1] to

[15] above.

[0013] The present invention provides a nucleic acid-ionized lipid complex that exhibits excellent escape ability from endosomes after being taken into cells by endocytosis, and a pharmaceutical composition containing the nucleic acid-ionized lipid complex.

[0014] In Example 1, (F17)4-siRNA FAM (F17)4-siRNA from HeLa-Luc cells introduced with IL FAM image( ＦＡＭ R Ｆ The images are -siRNA / IL), LysoTracker Red stained image (Lysosome), and a merged image of both.

[0015] In the present invention and the specification of the present application, "nucleic acid" means a molecule in which nucleotides are linked by phosphodiester bonds. The nucleotides include not only natural nucleotides such as DNA and RNA (nucleotides that exist in nature), but also artificial nucleotides obtained by modifying natural nucleotides and capable of forming phosphodiester bonds with natural nucleotides. Artificial nucleotides include those in which the side chains etc. of natural nucleotides are modified with functional groups such as amino groups, those in which the hydroxyl group at the 2'-position of the ribose skeleton is substituted with a methoxy group, a fluoro group, a methoxyethyl group, etc., phosphorothioate-type nucleotides (in which the oxygen atom of the phosphate group is substituted with a sulfur atom), morpholino-type nucleotides (in which ribose and deoxyribose are substituted with a morpholine ring), BNA (Bridged nucleic acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threose nucleic acid), GNA (Glycerol nucleic acid), CeNA (Cyclohexenyl nucleic acid), etc. "Nucleic acid" includes a molecule in which only one or more natural nucleotides are linked by phosphodiester bonds, a molecule in which one or more natural nucleotides and one or more artificial nucleotides are linked by phosphodiester bonds, and a molecule in which only one or more artificial nucleotides are linked by phosphodiester bonds.

[0016] In the present invention and the specification of the present application, "C" ｐ１－ｐ２ "(where p1 and p2 are positive integers satisfying p1 < p2) means a group having a carbon number of p1 to p2.

[0017] In the present invention and the specification of the present application, "C" １－３０ alkyl group" is an alkyl group having 1 to 30 carbon atoms, which may be linear or branched. "C" ２－３０ alkyl group" is an alkyl group having 2 to 30 carbon atoms, which may be linear or branched. C １－３０Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, and triacontyl groups.

[0018] In the present invention and this specification, "C １－２０ "Alkyl group" is an alkyl group having 1 to 20 carbon atoms, and may be a straight chain or a branched chain. ２－２０ An alkyl group is an alkyl group having 2 to 20 carbon atoms, and may be a straight chain or a branched chain. １－２０ Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl groups.

[0019] In the present invention and this specification, "C １－１０ "Alkyl alkyl group" is an alkyl group having 1 to 10 carbon atoms, and may be a straight chain or a branched chain. ２－１０ An alkyl group is an alkyl group having 2 to 10 carbon atoms, and may be a straight chain or a branched chain. １－１０ Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, and decyl groups.

[0020] In the present invention and this specification, "C １０－３０ An alkyl group is an alkyl group having 10 to 30 carbon atoms, and may be a straight chain or a branched chain. １０－３０ Examples of alkyl groups include undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, and triacontyl groups.

[0021] In the present invention and this specification, "C １－６ An alkyl group is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. １－６ Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl groups.

[0022] In the present invention and this specification, "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be a linear or branched chain. Examples of alkylene groups include methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, methylmethylene group, ethylmethylene group, methylethylene group, methylpropylene group, ethylethylene group, dimethylmethylene group, 1,2-dimethylethylene group, 1,1-dimethylethylene group, 1-ethylpropylene group, 2-ethylpropylene group, 1,2-dimethylpropylene Examples include the 1-propylpropylene group, 2,2-dimethylpropylene group, 1-propylpropylene group, 2-propylpropylene group, 1-methyl-1-ethylpropylene group, 1-methyl-2-ethylpropylene group, 1-ethyl-2-methylpropylene group, 2-methyl-2-ethylpropylene group, 1-methylbutylene group, 2-methylbutylene group, 3-methylbutylene group, 2-ethylbutylene group, 1-methylpentylene group, 2-ethylpentylene group, and 1-methylhexylene group.

[0023] In the present invention and this specification, "C １－２０ A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group having 1 to 20 carbon atoms are replaced with fluorine atoms. １－１０ Examples of perfluoroalkyl groups include perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluoroisopropyl, perfluorobutyl, perfluoroisobutyl, perfluorosec-butyl, perfluorotert-butyl, perfluoropentyl, perfluoroisopentyl, perfluoroneopentyl, perfluorotert-pentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, perfluorodecyl, perfluoroundecyl, perfluorododecyl, perfluorotridecyl, perfluorotetradecyl, perfluoropentadecyl, perfluorohexadecyl, perfluoroheptadecyl, perfluorooctadecyl, perfluorononadecyl, and perfluoroeicosyl groups.

[0024] In the present invention and this specification, a "perfluoroalkylene group" is a group in which all hydrogen atoms of an alkylene group are replaced with fluorine atoms. An example of a perfluoroalkylene group is the group in which all hydrogen atoms of the alkylene group mentioned above are replaced with fluorine atoms.

[0025] In the present invention and this specification, "ether-bonded oxygen atom" refers to an oxygen atom that links carbon atoms, and does not include oxygen atoms linked in series. The maximum number of ether-bonded oxygen atoms that an alkyl group with Nc (where Nc is an integer of 2 or more) may have is Nc-1. ２－１０ "Alkyl alkyl group" refers to C ２－１０ This group has at least one ether-bonded oxygen atom between the carbon atoms of the alkyl group. Hereinafter, alkyl groups having an ether-bonded oxygen atom may be referred to as "ether-bonded alkyl groups."

[0026] In the present invention and this specification, "having an ether-bonded oxygen atom between carbon atoms, C ２－１０ "Perfluoroalkyl group" is C ２－１０ Ether bond-containing C having at least one ether-bonded oxygen atom between the carbon atoms of the alkyl group ２－１０ This is a group in which all hydrogen atoms of the alkyl group are replaced with fluorine atoms. Hereinafter, perfluoroalkyl groups having ether-bonded oxygen atoms may be referred to as "ether-bonded perfluoroalkyl groups."

[0027] In the present invention and this specification, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. "Halogen atom other than a fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. Examples of "halogen atoms other than a fluorine atom" are preferably chlorine atoms or bromine atoms, and chlorine atoms are particularly preferred.

[0028] Hereafter, "compound (X)" refers to the compound represented by formula (X).

[0029] <Nucleic Acid-Ionized Lipid Complex> The nucleic acid-ionized lipid complex according to this embodiment contains a nucleic acid that can be introduced into cells by endocytosis and an ionized lipid. Because the nucleic acid-ionized lipid complex according to this embodiment is complexed with an ionized lipid, it has a superior ability to escape from endosomes compared to when the nucleic acid exists alone. Note that the nucleic acid-ionized lipid complex according to this embodiment is a complex in which the nucleic acid and the ionized lipid are directly complexed, and is different from nanoparticles in which the nucleic acid is encapsulated within lipid nanoparticles that simply contain the ionized lipid as a constituent lipid. It is preferable that the nucleic acid-ionized lipid complex according to this embodiment does not contain lipids other than the ionized lipid. By having only the ionized lipid as the lipid contained in the nucleic acid-ionized lipid complex according to this embodiment, the effect obtained by the direct complexation of the ionized lipid and the nucleic acid is more fully exhibited. It is particularly preferable that the nucleic acid-ionized lipid complex according to this embodiment is a complex consisting only of nucleic acid and an ionized lipid.

[0030] (Nucleic Acids) The nucleic acids constituting the nucleic acid-ionized lipid complex according to this embodiment are not particularly limited as long as they are nucleic acids that can be introduced into cells by endocytosis. The nucleic acids may be, for example, unmodified nucleic acids (molecules consisting only of structures in which native nucleotides and / or artificial nucleotides are linked by phosphate diester bonds), or nucleic acids modified with molecules other than nucleic acids. The nucleic acids may be single-stranded nucleic acids or double-stranded nucleic acids.

[0031] The nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment may consist entirely of natural nucleotides. The nucleic acid preferably contains one or more artificial nucleotides for greater stability in vivo, and may consist entirely of artificial nucleotides. Examples of such artificial nucleotides include aTNA, GNA, PNA, and SNA. If the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment contains artificial nucleotides, there may be one type of artificial nucleotide or two or more types.

[0032] The nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is preferably a nucleic acid having a functional nucleic acid portion that exhibits some kind of physiological activity when taken up into a target cell in a living organism. For example, the nucleic acid to be introduced into the cell to exert a function (introduced nucleic acid) can be a nucleic acid modified by another molecule directly or via an appropriate linking group, and this can constitute the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment. The other molecule that modifies the introduced nucleic acid may be a nucleic acid or a molecule other than a nucleic acid.

[0033] The nucleic acid to be introduced is not particularly limited; it may be a nucleic acid in which all of the nucleotides it contains are of the natural type, or it may be a nucleic acid in which some or all of the nucleotides are artificial. The nucleic acid to be introduced may be a single-stranded nucleic acid or a double-stranded nucleic acid. The nucleic acid to be introduced may be DNA, RNA, or a chimeric nucleic acid of DNA and RNA.

[0034] The nucleic acids to be introduced are not particularly limited, as long as they are nucleic acids that are introduced into cells to exert some kind of physiological function. Specifically, examples of such nucleic acids to be introduced include nucleic acids for expressing proteins in cells (protein expression nucleic acids), nucleic acids for suppressing gene expression in cells (gene expression suppression nucleic acids), nucleic acids for performing gene modification, nucleic acid aptamers that specifically bind to target biomolecules, and nucleic acids that affect the physiological function of cells. Examples of nucleic acids for protein expression include cDNA and mRNA that code for proteins, and may also be expression plasmid vectors into which the region coding for the target protein is incorporated. Examples of nucleic acids for gene expression suppression include nucleic acids used for RNA interference such as siRNA, miRNA, shRNA, and antisense oligonucleotides, and may also be RNAi vectors. Examples of nucleic acids for performing gene modification include fragments of genomic DNA. Examples of nucleic acids that affect the physiological function of cells include microRNA, decoy nucleic acids, and CpG (cytosine-phosphate-guanine) oligonucleotides.

[0035] The nucleic acid-ionized lipid complex according to this embodiment exhibits excellent ability to escape from endosomes. Therefore, by applying the nucleic acid-ionized lipid complex according to this embodiment to a functional nucleic acid that exhibits some kind of physiological activity in cells, that is, by making the target nucleic acid in the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment a functional nucleic acid, the function of the functional nucleic acid can be exerted more efficiently in cells. For example, if the nucleic acid-ionized lipid complex according to this embodiment has siRNA as the target nucleic acid, the nucleic acid-ionized lipid complex according to this embodiment can exert a higher gene expression suppression effect than the siRNA alone.

[0036] The linking group is not particularly limited as long as it is a group capable of linking the nucleic acid to be introduced with another molecule to be linked, and any divalent or trivalent organic group can be used. Examples of such linking groups include alkylene groups, alkenylene groups, carbonyl groups, amino groups, ether bonds, thioether bonds, ester bonds, amide bonds, polyethylene glycol groups (PEG:-(C) ２ H ４ Examples of linking groups include O)n-), siloxane bonds, silyl ether bonds, sugars, peptides, and nucleotide chains. Groups obtained by removing two or three hydrogen atoms from rings such as pyrrole rings, pyrazole rings, imidazole rings, triazole rings, pyridine rings, pyrimidine rings, pyrazine rings, oxazole rings, thiazole rings, furan rings, thiophene rings, and benzene rings can also be used as linking groups. Furthermore, groups obtained by appropriately combining these can also be used as linking groups.

[0037] As the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment, nucleic acids that are introduced into cells via membrane molecules such as receptors present on the cell surface are preferred due to their high efficiency in introducing them into cells. Examples of such receptors include scavenger receptors, asialoglycoprotein receptors, glucagon-like peptide-1 (GLP-1) receptors, integrins, transferrin receptors, epidermal growth factor receptors, insulin receptors, mannose 6-phosphate receptors, and GM1 gangliosides.

[0038] The nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is preferably a nucleic acid in which the target nucleic acid is directly or via an appropriate linking group to a site derived from a molecule that binds to a receptor present on the cell surface. Nucleic acids having a site that binds to a receptor are efficiently taken up into cells by endocytosis via the receptor. The molecule that binds to the receptor may be a nucleic acid or a molecule other than a nucleic acid.

[0039] Examples of molecules that bind to the asialoglycoprotein receptor include galactose (Gal) and N-acetylgalactosamine (GalNAc). Gal and GalNAc may be present as single molecules per nucleic acid molecule, or as oligosaccharides in which two or more molecules, for example, 2 to 5 molecules, are linked in tandem. An example of a molecule that binds to the GLP-1 receptor is GLP-1. An example of a molecule that binds to integrin is a peptide having an RGD sequence (arginine-glycine-aspartic acid). Examples of molecules that bind to the transferrin receptor, epidermal growth factor receptor, insulin receptor, mannose 6-phosphate receptor, and sialylated ganglioside receptor include transferrin, epidermal growth factor, insulin, mannose 6-phosphate, and sialylated ganglioside, respectively. The nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is preferably a nucleic acid in which the target nucleic acid is directly or via an appropriate linking group bound to a molecule that binds to these receptors.

[0040] As the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment, a nucleic acid modified with a group containing a fluorine atom is preferred from the viewpoint of enhancing cell membrane permeability. When the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is a nucleic acid bound to a molecule that binds to a receptor, the group containing a fluorine atom may be introduced into the nucleic acid portion, into the molecule that binds to the receptor, or into other portions. As the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment, a nucleic acid containing an artificial nucleotide modified with a group containing a fluorine atom is more preferred because it has good in vivo stability and cell membrane permeability, and a nucleic acid having aTNA modified with a group containing a fluorine atom is even more preferred.

[0041] In this embodiment, nucleic acids having a structure represented by the following general formula (A1) or (A2) are particularly preferred as the nucleic acids constituting the nucleic acid-ionized lipid complex. Hereafter, "the structure represented by general formula (A1)" may be referred to as "structure (A1)," and "the structure represented by general formula (A2)" may be referred to as "structure (A2)." Nucleic acids having these structures are nucleic acids with excellent cell membrane permeability that are taken into cells by endocytosis via scavenger receptors. That is, structures (A1) and (A2) correspond to sites that bind to scavenger receptors.

[0042]

[0043] In general formulas (A1) and (A2), R ０ C is a carbon atom substituted with at least one fluorine atom. １－３０ Alkyl(C) １－３０ (Fluoroalkyl groups), or C atoms not substituted with a fluorine atom １０－３０ It is an alkyl group. If the alkyl group has two or more carbon atoms, it may have one to five ether-bonded oxygen atoms between carbon atoms. In the present invention and this specification, "C １－３０ Fluoroalkyl groups (if the alkyl group has two or more carbon atoms, the alkyl group may have one to five ether-bonded oxygen atoms between carbon atoms) are defined as "C １－３０ Fluoroalkyl groups, or C ２－３０ This refers to a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a fluoroalkyl group.

[0044] R ０ C １－３０ In the case of fluoroalkyl groups, one or more hydrogen atoms bonded to the carbon atom may be further substituted with halogen atoms other than fluorine atoms. Structure (A1) or structure (A2) is R ０ C is a carbon atom substituted with at least two fluorine atoms. １－３０ It is preferable that it be a fluoroalkyl group. In particular, R ０ C １－２０ Fluoroalkyl groups are preferred, C １－１５Fluoroalkyl groups are more preferred, C ２－１５ A fluoroalkyl group is more preferably C ６－10 Fluoroalkyl groups are even more preferred. ０ C １－３０ In the case of fluoroalkyl groups, the number of hydrogen atoms substituted for fluorine atoms is not particularly limited as long as there is one or more; for example, three or more are preferred, six or more are more preferred, and seven or more are even more preferred.

[0045] R ０ C １－３０ In the case of a fluoroalkyl group, R ０ Specifically, a group represented by the following general formula (f-1) or (f-2) is preferred. Here, Rf Ｐ is a fully halogenated C １－２０ Alkyl(C) １－２０ A group in which all of the hydrogen atoms of an alkyl group are replaced by halogen atoms, and which has one or more fluorine atoms. Rf Ｐ If the carbon number is 2 or more, that is, completely halogenated C ２－２０ In the case of alkyl groups, there may be 1 to 5 ether-bonded oxygen atoms between the carbon atoms. In the present invention and this specification, "a fully halogenated C which may have 1 to 5 ether-bonded oxygen atoms between the carbon atoms" ２－２０ "Alkyl alkyl group" refers to "completely halogenated C ２－２０ Alkyl alkyl groups, or fully halogenated C ２－２０ This refers to a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group.

[0046] In the general formula (f-2), two Rf Ｐ These groups may be of the same kind or different kinds. Rf Ｐ C １－２０ Perfluoroalkyl groups (C １－２０ It is preferable that the alkyl group is a group in which all of the hydrogen atoms are replaced by fluorine atoms.

[0047] In the following general formulas (f-1) or (f-2), n1 is an integer from 0 to 10, and n2 is an integer from 0 to 9. When both n1 and n2 are 0, both represent a simple combination. That is, when n1 is 0, the base represented by general formula (f-1) is -Rf Ｐ And when n2 is 0, the group represented by the general formula (f-2) is -CH(Rf Ｐ ) ２ That is the case.

[0048]

[0049] R ０ If the group is represented by the general formula (f-1), then R ０ Rf Ｐ The group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 4. Ｐ However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 2. Ｐ However, the group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n1 is an integer from 0 to 2 (however, when n1 is 1, Rf Ｐ More preferably, Rf (excluding groups where is a trifluoromethyl group) Ｐ However, a group that is a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, where n1 is 0, is even more preferred.

[0050] R ０ If the group is represented by the general formula (f-2), then R０ Rf Ｐ The group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 4. Ｐ However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 2. Ｐ The group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is an integer from 0 to 2 (however, n2 is 0 or 1, and Rf Ｐ More preferably, Rf (excluding groups where is a trifluoromethyl group) Ｐ However, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is 0, is even more preferred.

[0051] R ０ C １－３０ In the case of a fluoroalkyl group, R ０Examples include a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, a perfluorodecyl group, a difluoromethyl group, a 1,1-difluoroethyl group, a 2,2-difluoroethyl group, a 1,1,2,2-tetrafluoroethyl group, a 1,1,2,2,3,3-hexafluoropropyl group, a 1,1,2,3,3,3-hexafluoropropyl group, a 1,1,2,2,3,3-hexafluorohexyl group, a 1,1,2,2,3,3-hexafluorooctyl group, a 1,1,2,2,3,3-hexafluorodecyl group, a 1,1,2,2,3,3-hexafluorooctadecyl group, a 1,1,2,2,3,3-hexafluorodocosyl group, and the like. As the nucleic acid constituting the nucleic acid-lipid complex according to the present embodiment, in the general formula (A1) or (A2), R ０ is C １－３０ preferably a nucleic acid having a structure in which it is a perfluoroalkyl group, and in the general formula (A1) or (A2), R ０ is C １－２０ more preferably a nucleic acid having a structure in which it is a perfluoroalkyl group, and in the general formula (A1) or (A2), R ０ is C １－１０ even more preferably a nucleic acid having a structure in which it is a perfluoroalkyl group.

[0052] When R ０ is C １０－３０ an alkyl group, as the structure (A1) or structure (A2), R ０ is preferably C １０－２５ an alkyl group, more preferably C １５－２５ an alkyl group, and even more preferably C １５－２３ an alkyl group. An alkyl group of sufficient length has high hydrophobicity similar to a fluoroalkyl group and contributes to the cell membrane permeability of the nucleic acid having the structure (A1) or structure (A2).

[0053] In general formula (A2), B is a nucleic acid base. Preferred nucleic acid bases include those found in natural nucleic acids, nucleic acid bases structurally similar to natural nucleic acid bases, and modified bases that have undergone various modifications. Modifications of the base include alkylation, hydroxylation, alkoxylation, acylation, dihydrolation, amination, formylation, halogenation, etc. Nucleic acid bases structurally similar to natural nucleic acid bases include triazole, imidazole, azapyrimidine, azapurine, etc. Specifically, the nucleic acid base of B is adenine, guanine, cytosine, thymine, uracil, 1-methyladenine, N6-methyladenine, 7-methylguanine, 5-methylcytosine, 1-methylthymine, 5-methyluracil, 5-hydroxymethylcytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, dihydrouracil, dihydrothymine, dihydrocytosine, 2,6-diaminoadenine, 2,6-diamino Examples include guanine, 6-thioguanine, 2-thioadenine, 2-thiocytosine, 4-thiouracil, 5-fluorouracil, 5-iodouracil, 5-halogenocytosine, 5-fluorocytosine, 5-trihalogenomethyluracil, 5-trifluoromethyluracil, 5-azathymine, 5-azacytosine, 6-azauracil, 8-azaadenine, 7-deazaadenine, 7-deazaguanine, and 3-deazauracil. The nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is preferably a nucleic acid having a structure in which B in general formula (A2) is adenine, guanine, cytosine, thymine, or uracil.

[0054] In general formulas (A1) and (A2), n11 and n12 are each independent integers of 1 or more. n11 and n12 are the number of repeats per molecule of each structure, and the larger the number, the higher the hydrophobicity. This improves the cell membrane permeability of the nucleic acid. In the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment, it is preferable that n11 and n12 are 2 or more, and more preferably 3 or more. Since it is easier to obtain structures similar to natural double-stranded nucleic acids and single-stranded nucleic acids, it is preferable that n11 and n12 are 10 or less, more preferably 8 or less, and even more preferably 6 or less. In the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment, n11 is preferably 2 or more and 10 or less, more preferably 2 or more and 8 or less, even more preferably 2 or more and 6 or less, even more preferably 3 or more and 5 or less, and particularly preferably 4. When n11 and n12 are 2 or more, the multiple structures (A1) or structures (A2) may be the same as each other or may be different structures.

[0055] In general formulas (A1) and (A2), black circles indicate binding sites. The nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment may be any nucleic acid having structure (A1) or structure (A2), and the position in which structure (A1) or structure (A2) is introduced is not particularly limited, and it may be introduced at any site as long as it does not impair the function of the nucleic acid. For example, structure (A1) or structure (A2) may be directly or indirectly bound to the 5' or 3' end of the nucleic acid, or introduced between two nucleotides.

[0056] In the case where the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is a nucleic acid having structure (A1) or structure (A2) at its 5' end, it is preferable that the binding hand extending from the carbon atom at the end of structure (A1) or structure (A2) is bound to a hydroxyl group, and the binding hand extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) is bound to the sugar at the 5' end of the nucleic acid. In the case where the nucleic acid constituting the nucleic acid-ionized lipid complex according to this embodiment is a nucleic acid having structure (A1) or structure (A2) at its 3' end, it is preferable that the binding hand extending from the carbon atom at the end of structure (A1) or structure (A2) is bound to the phosphate group at the 3' end of the nucleic acid, and the binding hand extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) is bound to a hydrogen atom.

[0057] In this embodiment, if the nucleic acid constituting the nucleic acid-ionized lipid complex is a nucleic acid in which structure (A1) or structure (A2) is introduced between two nucleotides, it is preferable that the structure is more closely approximated to that of a natural nucleic acid, so that the binding hands extending from the carbon atom at the end of structure (A1) or structure (A2) are bound to the phosphate group of the other nucleotide, and the binding hands extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) are bound to the sugar of the other nucleotide.

[0058] Structure (A1) or structure (A2) can be introduced into the target nucleic acid by various coupling reactions. For example, by performing the phosphoramidite method using a phosphoramidite containing structure (A1) or structure (A2) as a raw material, structure (A1) or structure (A2) can be easily introduced into nucleic acid. Commonly used automated nucleic acid synthesizers utilize the phosphoramidite method. Therefore, by using a phosphoramidite containing structure (A1) or structure (A2) as a raw material, nucleic acids with structure (A1) or structure (A2) introduced at the desired position can be easily synthesized in an automated synthesizer for nucleic acids with various base sequences.

[0059] Examples of phosphoramidites containing structure (A1) or structure (A2) include compounds in which a sugar and a phosphate group are linked via structure (A1) among phosphoramidites generally used for nucleic acid synthesis, and compounds in which a nucleoside moiety is substituted with an organic group containing structure (A2).

[0060] The synthesized target nucleic acid can be isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse-phase chromatography, normal-phase chromatography, and the like.

[0061] R ０ groups have high hydrophobicity and high affinity for cell membranes. When a nucleic acid having structure (A1) or structure (A2) forms a DNA double helix structure, the R ０ groups are exposed outside the helix structure, and not only can a more stable double helix structure be formed, but the cell membrane permeability can be improved by the R ０ groups exposed on the surface. Therefore, by using a nucleic acid having structure (A1) or structure (A2) as the nucleic acid constituting the nucleic acid-ionized lipid complex according to the present embodiment, a nucleic acid-ionized lipid complex with extremely excellent cell membrane permeability can be obtained.

[0062] As the nucleic acid constituting the nucleic acid-ionized lipid complex according to the present embodiment, various modifications may be made as long as the effects of the present invention are not impaired. Examples of such modifications include sugar chain modification, lipid modification, peptide modification, and the like.

[0063] (Ionized Lipids) Ionized lipids are lipids that exhibit charge neutrality at physiological pH and become protonated in acidic regions. Generally, ionized lipids consist of a polar head portion, a hydrophobic tail portion, and a linker portion that connects them. Because endosomes are acidic, ionized lipids taken into cells by endocytosis become cationic at the polar head portion within the endosome. It is presumed that electrostatic interactions occur between the cationic head portion of the ionized lipid in the nucleic acid-ionized lipid complex and the anionic portion of the nucleic acid, making it easier for the ionized lipid to form an inverse hexagonal structure, causing instability of the endosomal membrane, and as a result, improving the ability of the nucleic acid-ionized lipid complex to escape from the endosome.

[0064] The ionized lipids constituting the nucleic acid-ionized lipid complex according to this embodiment are not particularly limited, but cone-shaped ionized lipids (ionized lipids in which the volume of the hydrophobic tail portion is larger than that of the polar head portion) are preferred because they can further enhance endosomal escape ability. Examples of cone-shaped ionized lipids include DLin-MC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester, CAS RN: 1224606-06-7), DODMA (1,2-Dioleyloxy-3-dimethylamino-propane, CAS RN: 104162-47-2), OF-02 (3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5-piperazinedione, CAS Unsaturated ionizable lipids such as ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate), CAS RN:2036272-55-4), Lipid 5 (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1-octylnonyl ester, CAS RN:2089251-33-0), SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, CAS RN:2089251-47-6), C12-200 (1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, CAS RN:1220890-25-4), cKK-12 (3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione、CAS RN:1432494-65-9)、9A1P9(2-(dioctylamino)ethyl nonyl hydrogen phosphate、CAS RN:2760467-57-8)、AL-A12(N-dodecyl-N-(3-methoxypropyl)-1-dodecanamine、CAS RN:3037065-56-5)、AA3-DLin(piperazine-1,4-diylbis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)、CAS RN:3027565-91-6)、CIN-16645(9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester、CAS RN:1799316-64-5)、CL4H6(9-octadecenoic acid, 1,1′-[7-[4-(dipropylamino)butyl]-7-hydroxy-1,13-tridecanediyl] ester、CAS RN:2256087-35-9)、Lipid 14(((2-((4-(dimethylamino)butanoyl)oxy)ethyl)azanediyl)bis(octane-8,1-diyl) bis(2-hexyldecanoate)、CAS RN:2430034-05-0)、Lipid 29(8-[[8-[(1-ethylnonyl)oxy]-8-oxooctyl][3-[[2-(methylamino)-3,4-dioxo-1-cyclobuten-1-yl]amino]propyl]amino]-octanoic acid, 1-octylnonyl ester、CAS RN:2244716-55-8)、Lipid 222(((4-hydroxybutyl)azanediyl)bis(octane-8,1-diyl) bis(2-hexyldecanoate)、CAS RN:2985009-46-7)、TCL053(2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2-((((Z)-tetradec-9-enoyl)oxy)methyl)propane-1,3-diyl (9Z,9'Z)-bis(tetradec-9-enoate)、CAS RN:2361162-70-9)、TT3(N, 1 ,N 3 ,N 5Multi-tail ionized lipids such as -tris[3-(didodecylamino)propyl]-1,3,5-benzenetricarboxamide, CAS RN: 1821214-50-9, YSK05 (1-methyl-4,4-bis[(9Z,12Z)-9,12-octadecadien-1-yloxy]-piperidine, CAS RN: 1318793-78-0); L-319 (9-[4-(dimethylamino)-1-oxobutoxy]-heptadecanedioic acid, 1,17-di-(2Z)-2-nonen-1-yl ester, CAS RN:1351586-50-9), DLin-KC2-DMA (N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine, CAS RN:1190197-97-7), LP01 (9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester, CAS RN:1799316-64-5), 306Oi10(tetrakis(8-methylnonyl) Biodegradable ionized lipids such as 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (CAS RN: 2322290-93-5) can be used, and structural analogues of these ionized lipids can also be used. Examples of structural analogues of ionized lipids include compounds in which the number of carbon atoms or the branching state of the hydrocarbon chain portion of the ionized lipid has been altered.

[0065] In this embodiment, the ionized lipid constituting the nucleic acid-ionized lipid complex is preferably a multi-tail ionized lipid, and more preferably a multi-tail ionized lipid having a tertiary amino group in the polar head portion, because the complex with nucleic acid readily adopts an inverse hexagonal structure within endosomes, resulting in higher endosome escape ability.

[0066] The ionized lipid constituting the nucleic acid-ionized lipid complex according to this embodiment preferably has a hydrophobic tail portion having two or more saturated hydrocarbon chains, more preferably a multi-tail type ionized lipid having two or more saturated hydrocarbon chains, and even more preferably a multi-tail type ionized lipid having a tertiary amino group in the polar head portion and a hydrophobic tail portion having two or more saturated hydrocarbon chains. Having saturated hydrocarbon chains in the hydrophobic tail portion makes it easier to adopt an inverse hexagonal structure within endosomes, resulting in higher endosomal escape ability.

[0067] In the case where the ionized lipid constituting the nucleic acid-ionized lipid complex according to this embodiment has a hydrocarbon chain as its hydrophobic tail portion, it is preferable that an ester bond is inserted between one or more, preferably one to three, carbon atoms in the hydrocarbon chain. Long hydrocarbon chains may exhibit cytotoxicity, but when an ester bond is inserted into the hydrocarbon chain, the ester bond is hydrolyzed within the endosome or cytoplasm.

[0068] The ionized lipids constituting the nucleic acid-ionized lipid complex according to this embodiment are preferably multi-tail type ionized lipids having a tertiary amino group in the polar head portion and a saturated hydrocarbon chain having one or more ester bonds between carbon atoms in the hydrophobic tail portion, and more preferably multi-tail type ionized lipids selected from the group consisting of ALC-315 and SM-102.

[0069] (Complex Formation) Nucleic acids and ionized lipids can be complexed simply by bringing them into contact in a solvent in which both are soluble. Therefore, the nucleic acid-ionized lipid complex according to this embodiment can be produced simply by mixing a solution containing nucleic acids that can be introduced into cells by endocytosis with a solution containing ionized lipids. The solution containing nucleic acids that can be introduced into cells by endocytosis can be prepared by dissolving the nucleic acids in water or various buffers. The solution containing ionized lipids can be prepared by dissolving the ionized lipids in an organic solvent that is soluble in ionized lipids and is compatible with water. Examples of organic solvents that are soluble in ionized lipids and are compatible with water include dimethyl sulfoxide (DMSO). The content ratio (molar ratio) of nucleic acid to ionized lipid in the nucleic acid-ionized lipid complex according to this embodiment is not particularly limited. For example, nucleic acid (moles):ionized lipid (moles) can be 1:99 to 99:1, preferably 1:99 to 50:50, more preferably 1:99 to 10:90, and even more preferably 1:99 to 5:95.

[0070] The nucleic acid-ionized lipid complex according to this embodiment can be used as is, or mixed with a pharmacologically acceptable carrier, etc., to form a pharmaceutical composition. For example, if the nucleic acid in the nucleic acid-ionized lipid complex according to this embodiment includes a nucleic acid for suppressing the gene expression of a specific gene, such as siRNA, the pharmaceutical composition containing the nucleic acid-ionized lipid complex can be used as a pharmaceutical composition for the treatment or prevention of diseases in which the gene is a therapeutic target gene, and a therapeutic effect is obtained by suppressing the expression of the gene.

[0071] Pharmacologically acceptable carriers include various organic or inorganic carrier substances commonly used as formulation materials, for example, as excipients, lubricants, binders, and disintegrants in solid formulations; and as solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. If necessary, formulation additives such as preservatives, antioxidants, colorants, and sweeteners may also be used.

[0072] The administration route of the pharmaceutical composition containing the nucleic acid-ionized lipid complex according to this embodiment as an active ingredient is not particularly limited and can be appropriately determined considering the target cells, the desired therapeutic effect, etc. Examples of administration routes include oral, intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intraperitoneal, intrathecal, transrectal, transvaginal, ocular, and inhalation.

[0073] The target of administration of the pharmaceutical composition containing the nucleic acid-ionized lipid complex according to this embodiment is not particularly limited and may include humans, non-human mammals, birds, reptiles, amphibians, fish, etc. In vitro tissues or cultured cells of these animals may also be the target of administration.

[0074] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0075] The NMR spectrometer used for the analysis of the examples and comparative examples was the JEOL JNM-ECZ400S (400MHz). １ In 1H NMR, tetramethylsilane was detected at 0 PPM. １９ In F NMR, C ６ F ６ -162 PPM was set as the baseline value.

[0076] <RNA Synthesis> In the following experiments, RNA synthesis was performed using commercially available reagents, various phosphoramidites (acetonitrile solution, 0.1 M), and 5-ethylthio-1H-tetrazole (acetonitrile solution, 0.25 M) as an activator, with an NTS H-8 DNA / RNA synthesizer (manufactured by Nippon Techno Service Co., Ltd.).

[0077] <Cell Culture> In the following experiments, cell culture was performed as follows: Human cervical cancer HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific) supplemented with 10% FBS and 0.5% penicillin / streptomycin at 37°C in a humidified atmosphere (5% CO2 by volume). ２ Cells were cultured in a 35 mm glass-bottom dish (manufactured by IWAKI Corporation).

[0078] <Flow Cytometry> In the following experiments, flow cytometry was performed as follows: HeLa cells were placed in a 12-well plate. ５ Cells were seeded at a density of 1 cell / well and cultured. The day after seeding, the culture medium in each well was replaced with DMEM medium (1 mL) containing fluorescein-conjugated nucleic acid (2.5 μM), and incubated for 4 or 24 hours. Subsequently, the cell layer in the wells was washed twice with PBS, and the cells were detached by treatment with 0.05% (w / v) trypsin (200 μL) at 37°C for 5 minutes. The harvested cells were suspended in DMEM (600 μL). This cell suspension was separated by centrifugation (400 × g, 3 minutes), and PBS / 1% BSA (500 μL) was added. The percentage of fluorescent cells and the average fluorescence intensity of this cell suspension were analyzed using a flow cytometer ("guava easyCyte 8", Luminex).

[0079] [Example 1] R ０ However, if n1 is 2 among the groups represented by the general formula (f-1), then Rf Ｐ A structure (A1) in which a perfluorooctyl group is attached to the 5' end of the sense strand of siRNA was synthesized and complexed with an ionized lipid. As the siRNA used, a sense strand consisting of RNA represented by SEQ ID NO: 1 (5'-CUUACGCUGAGUACUUCGAAAUU-3') that targets luciferase and an antisense strand consisting of RNA represented by SEQ ID NO: 2 (5'-UUUCGAAGUACUCAGCGUAAGUU-3') was used.

[0080] <Synthesis of aTNA-N[FC10]> R ０ Among the groups represented by the general formula (f-1), n1 is 2, and Rf Ｐ A phosphoramidite (aTNA-N[FC10]) having a structure (A1) in which the group is a perfluorooctyl group was synthesized as follows.

[0081] (1) Amide condensation

[0082]

[0083] Compound 1 (0.44 g, 0.82 mmol), PyBOP (0.43 g, 0.82 mmol), and DIPEA (0.4 mL, 2.2 equivalents), dissolved in 10 mL of DMF, were dissolved in 15 mL of DMF under argon. 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoic acid (0.23 g, 0.59 mmol, 0.7 equivalents) was added to prepare the reaction solution. The resulting reaction solution was stirred at room temperature for 14 hours. The reaction mixture was then quenched with water (45 mL) and extracted twice with hexane / ethyl acetate (4:1 (volume ratio)). The organic fractions were combined, washed with water, and Na... ２ SO ４ After drying, the product was filtered and the solvent was removed under vacuum. The resulting crude product was subjected to silica gel column chromatography (hexane:ethyl acetate:Et ３ Compound 2 was obtained by purification using N = 30:20:1 (volume ratio).

[0084] (2) Amidite formation

[0085]

[0086] 93 mg, 0.31 mmol of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorus diamidite and 40 mg, 0.31 mmol of ETT were dissolved in 8 mL of dry acetonitrile. Compound 2 (160 mg, 0.20 mmol), dissolved in a THF / acetonitrile (2 mL / 2 mL) mixed solvent, was added dropwise under argon. The reaction mixture was stirred at room temperature for 24 hours, after which the solvent was evaporated under reduced pressure. The resulting crude product was purified under argon by silica gel column chromatography using degassed hexane / ethyl acetate (3:1 (volume ratio)) as the mobile phase. Compound 3 (amiditized aTNA-N[FC10]), a colorless oil, was isolated by this method.

[0087] Using the synthesized aTNA-N[FC10], a single-stranded RNA (aTNA-N[FC10]-modified RNA) was synthesized by ligating four of the structures shown below (aTNA-N1[FC10]: black circles in the formula indicate binding sites) to the 5' end of the native RNA (SEQ ID NO: 1), which is the sense strand of the siRNA.

[0088]

[0089] (3) RNA Synthesis RNA synthesis (tritil-off) was performed on a 1000 Å CPG solid-supported column (1 μmole scale). Then, under a nitrogen atmosphere, aTNA-N[FC10] amidite solution (0.1 M acetonitrile solution, 300 μL) and activator solution (300 μL) were mixed using a syringe in the presence of CPG. After 5 minutes, the resulting solution was removed from the column, and the synthesized (F17) 4-RNA (RNA with four aTNA-N[FC10] molecules tandem-linked to the 5' end of the sense strand) was recovered. The recovered RNA was purified by HPLC.

[0090] Synthesized (F17)4-mRNA was hybridized with native RNA (SEQ ID NO: 2), which is the antisense strand of siRNA, to obtain (F17)4-siRNA. Similarly, (F17)4-RNA was hybridized with RNA in which 6-fluorescein (FAM) was fused to the 3' end of native RNA (a complementary sequence to the base sequence of SEQ ID NO: 1), which is the antisense strand of siRNA. FAM This was obtained by hybridizing the native RNA (SEQ ID NO: 1), which is the sense strand of siRNA, with RNA in which FAM was attached to the 5' end of the native RNA (SEQ ID NO: 2), which is the antisense strand of siRNA. FAM I obtained it.

[0091] <Flow Cytometry> (F17) 4-siRNA in a culture medium containing HeLa cells. FAM When this was added and the cells were incubated at 37°C for 4 hours, flow cytometry confirmed that 38 times more siRNA was delivered to the cells compared to unmodified siRNA.

[0092] <Knockdown assay (1)> (F17) 4-siRNA ＦＡＭTo evaluate the gene silencing ability, HeLa-Luc cells (HeLa cells constitutively expressing luciferase protein) were used, and their expression level was assessed from the luciferase luminescence. For the luciferase assay, 0.1 μM or 2 μM of siRNA was added to the culture medium of HeLa-Luc cells, and the cells were cultured at 37°C for 6 hours. The cells were then washed with D-PBS solution and cultured for another 18 hours in EMEM medium (10% FBS, 1% NAAS, 1% P / S). Finally, the specified amount of Luciferase assay reagent (Promega) was added according to the protocol, and the chemiluminescence was measured using a plate reader (n=3). This chemiluminescence level serves as an indicator of luciferase expression.

[0093] The assay results are shown in Table 1. (F17) 4-siRNA FAM Despite a significant improvement in intracellular delivery efficiency, (F17)4-siRNA FAM Cells to which siRNA was added showed FAM Similar to the cells treated with the additive, no gene silencing was observed.

[0094]

[0095] <Knockdown assay (2)> (F17) 4-siRNA FAM The compound was complexed with the ionized lipid ALC-0315 (IL), and its gene silencing ability was investigated.

[0096] Specifically, a DMSO solution containing ionized lipid ALC-0315 (IL) was used to dissolve (F17) 4-siRNA. FAM Mix with an aqueous solution containing (F17) 4-siRNA FAM - Ionized lipid complex ((F17)4-siRNA FAM / IL)((F17)4-siRNA FAM :IL=1:60, [(F17)4-siRNA FAM A 1 μM ([IL] = 1 μM) solution was prepared. A DMSO solution containing ALC-0315 (IL) was mixed with an aqueous solution containing unmodified siRNA to prepare a siRNA-ionized lipid complex (siRNA / IL) (siRNA:IL = 1:60, [(F17)4-siRNAFAM A solution was prepared ([IL] = 1 μM, [IL] = 60 μM).

[0097] Next, HeLa-Luc cells are treated with (F17)4-siRNA. FAM Cells were cultured for 6 hours in OptiMEM medium supplemented with 100 nM of 1 / IL or siRNA / IL. The cells were then washed with D-PBS solution, and HeLa-Luc cells were cultured for a further 18 hours in EMEM medium (10% FBS, 1% NAAS, 1% P / S). After culture, the cells were treated with the specified amount of Luciferase assay reagent according to the protocol, and the chemiluminescence was measured using a plate reader (n=3). (F17)4-siRNA was used as a control. FAM The solution was added to HeLa-Luc cells to a final concentration of 100 nM, cultured in the same manner, and then a knockdown assay was performed (n=3).

[0098] (F17)4-siRNA FAM The luciferase expression level (chemiluminescence) of cells treated with (F17)4-siRNA is set to 100%. FAM Table 2 shows the relative luciferase expression levels of cells treated with / IL or siRNA / IL.

[0099]

[0100] As shown in Table 2, (F17) 4-siRNA FAM The relative luciferase expression level in cells treated with IL was 58%, and the knockdown efficiency was 100% - [(F17)4-siRNA FAM The relative luciferase expression level (%) in cells treated with siRNA / IL was 42%. In contrast, the relative luciferase expression level in cells treated with siRNA / IL was 88%, confirming that gene silencing is not induced when unmodified siRNA is complexed with ionized lipids.

[0101] <Confocal Microscopy Observation> (F17) 4-siRNA FAMHeLa-Luc cells were cultured for 6 hours in OptiMEM medium supplemented with IL to a final concentration of 100 nM. The cells were then washed with D-PBS solution and cultured for a further 18 hours in EMEM medium (10% FBS, 1% NAAS, 1% P / S). LysoTracker® Red (75 nM) (Thermo Fisher Scientific), a lysosome stain, was added to the cultured cells, and the cells were incubated for another hour. After incubation, the solution was removed from each dish, washed with PBS(-), and then DMEM (1 mL) was added. Next, each dish was placed in a confocal laser scanning microscope, and fluorescence images were acquired using green fluorescence (excitation wavelength of 488 nm and emission filter exceeding 505 nm) and red fluorescence (excitation wavelength of 577 nm and emission filter exceeding 590 nm).

[0102] (F17)4-siRNA FAM Fluorescence images of HeLa-Luc cells that have taken up / IL are shown in Figure 1. As shown in Figure 1, intracellular (F17)4-siRNA FAM Some of these show localization different from lysosomes, and (F17) 4-siRNA is released from endosomes. FAM It has been confirmed that he escaped.

[0103] <Knockdown assay (3)> (F17)4-siRNA in the presence of fucoidan, a scavenger receptor inhibitor FAM (F17) 4-siRNA is a complex of the ionized lipid ALC-0315 and FAM We investigated the gene silencing ability of IL.

[0104] (F17)4-siRNA FAMHeLa-Luc cells were cultured for 6 hours in OptiMEM medium supplemented to achieve a final concentration of 100 nM of IL and a final concentration of 0 mg / mL or 0.5 mg / mL of fucoidan. The cells were then washed with D-PBS solution and cultured for a further 18 hours in EMEM medium (10% FBS, 1% NAAS, 1% P / S). After culture, the cells were treated with the specified amount of Luciferase assay reagent according to the protocol, and the chemiluminescence was measured using a plate reader (n=3). (F17)4-siRNA was used as a control. FAM The solution was added to HeLa-Luc cells to a final concentration of 100 nM, cultured in the same manner, and then a knockdown assay was performed (n=3).

[0105] (F17)4-siRNA FAM The luciferase expression level (chemiluminescence) of cells treated with (F17)4-siRNA is set to 100%. FAM / IL only, or (F17)4-siRNA FAM Table 3 shows the relative luciferase expression levels of cells treated with IL and fucoidan.

[0106]

[0107] As shown in Table 3, (F17) 4-siRNA FAM Gene silencing induced by / IL was no longer induced in the presence of fucoidan. These results suggest that (F17)4-siRNA FAM It was suggested that / IL is introduced into cells via scavenger receptors.

[0108] [Example 2] R ０ However, if n1 is 2 among the groups represented by the general formula (f-1), then Rf Ｐ We synthesized a nucleic acid having a structure (A1) in which the group is a perfluorohexyl group.

[0109] <Synthesis of aTNA-N[FC8]> R ０ Among the groups represented by the general formula (f-1), n1 is 2, and Rf ＰA phosphoramidite (aTNA-N[FC8]) having a structure (A1) in which is a perfluorohexyl group was synthesized as follows.

[0110] (1) Amide condensation

[0111]

[0112] Compound 8 (0.44 g, 0.82 mmol) dissolved in 10 mL of DMF, PyBOP (0.43 g, 0.82 mmol), and DIPEA (0.4 mL, 2.2 equivalents) were dissolved in 15 mL of DMF under argon, and 4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononanoic acid (0.23 g, 0.59 mmol, 0.7 equivalents) was added to prepare the reaction solution. The resulting reaction solution was stirred at room temperature for 14 hours. The reaction mixture was then quenched with water (45 mL) and extracted twice with hexane / ethyl acetate (4:1 (volume ratio)). The organic fractions were combined, washed with water, and Na ２ SO ４ After drying, the product was filtered and the solvent was removed under vacuum. The resulting crude product was subjected to silica gel column chromatography (hexane:ethyl acetate:Et ３ The compound was purified by N = 30:20:1 (volume ratio) to obtain compound 9 (0.17 g, 0.22 mmol, yield 38%).

[0113] Compound 9: 1 H NMR (400 MHz, CDCl3) δ 7.39-7.21 (m, 9H), 6.84 (d, J = 5.9 Hz, 4H), 6.06 (d, J = 9.1 Hz, 1H), 4.13-4.10 (m, 1H), 3.92-3.90 (m, 1H), 3.78 (s, 6H), 3.41 (dd, J = 11.2, 5.7 Hz, 1H), 3.33 (dd, J = 9.6, 3.2 Hz, 1H), 2.90 (s, 1H), 2.53-2.44 (m, 4H), 1.12 (d, J = 6.4 Hz, 3H). 19F NMR (376 MHz, CDCl3) δ -80.7 (s, 3F), -114.4 (s, 2F), -121.8 (s, 2F), -122.8 (s, 2F), -123.4 (s, 2F), -126.0 (s, 2F).

[0114] (2) Amidite formation

[0115]

[0116] 93 mg, 0.31 mmol of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorus diamidite and ETT (40 mg, 0.31 mmol) were dissolved in 8 mL of dry acetonitrile. Compound 9 (160 mg, 0.20 mmol), dissolved in a THF / acetonitrile (2 mL / 2 mL) mixed solvent, was added dropwise under argon. The reaction mixture was stirred at room temperature for 24 hours, after which the solvent was evaporated under reduced pressure. The resulting crude product was purified under argon by silica gel column chromatography using degassed hexane / ethyl acetate (3:1 (volume ratio)) as the mobile phase. Compound 10 (amiditized aTNA-N[FC8]) (yield 38%), a colorless oil, was isolated by this method.

[0117] Compound 10: 1 H NMR (400 MHz, CDCl3) δ 7.41-7.20 (m, 9H), 6.82-6.79 (m, 4H), 5.94(d, 9.1 Hz, 1H), 4.37-4.32 (m, 1H), 4.20-4.14 (m, 1H), 3.77 (s, 6H), 3.55-3.45 (m, 4H), 3.25-3.12 (m, 2H), 2.59-2.31 (m, 6H), 1.24-0.97 (m, 15H). 19 F NMR (376 MHz, CDCl3) δ -80.7 (s, 3F), -114.5 (s, 2F), -121.8 (s, 2F), -122.8 (s, 2F), -123.4 (s, 2F), -126.0 (s, 2F).

[0118] Using the synthesized aTNA-N[FC8], a single-stranded RNA (aTNA-N4[FC8]-modified RNA) was synthesized by ligating four of the structures shown below (aTNA-N1[FC8]: black circles in the formula indicate binding sites) to the 5' end of the native RNA (SEQ ID NO: 1), which is the sense strand of the siRNA.

[0119]

[0120] The nucleic acid-ionized lipid complex according to this embodiment exhibits excellent escape ability from endosomes after being taken into cells by endocytosis, and is therefore expected to be used in the pharmaceutical field, for example, as an active ingredient in nucleic acid drugs.

Claims

1. A nucleic acid-ionized lipid complex containing a nucleic acid that can be introduced into cells by endocytosis and an ionized lipid.

2. The nucleic acid-ionized lipid complex according to claim 1, wherein the nucleic acid is introduced into the cell via a receptor present on the cell surface.

3. The nucleic acid-ionized lipid complex according to claim 2, wherein the receptor is a scavenger receptor, an asialoglycoprotein receptor, a glucagon-like peptide-1 receptor, or an integrin.

4. The following general formula (A1) or (A2) [In the formula, R ０ A nucleic acid-ionized lipid complex comprising a nucleic acid having a structure represented by [where n11 and n12 are each independently integers of 1 or more; B is a nucleic acid base; and black circles indicate bonding] and an ionized lipid.

5. The nucleic acid-ionized lipid complex according to any one of claims 1 to 4, wherein the ionized lipid has a tertiary amino group in the polar head portion.

6. The nucleic acid-ionized lipid complex according to any one of claims 1 to 4, wherein the hydrophobic tail portion of the ionized lipid has two or more saturated hydrocarbon chains.

7. The nucleic acid-ionized lipid complex according to claim 6, wherein at least one of the two or more saturated hydrocarbon chains has an ester bond inserted between carbon atoms.

8. The nucleic acid-ionized lipid complex according to any one of claims 1 to 4, wherein the ionized lipid is selected from the group consisting of ALC-315, Lipid 5, SM-102, and C12-200.

9. The aforementioned R ０ The nucleic acid-ionized lipid complex according to claim 4, wherein the alkyl group has 1 to 30 carbon atoms and is substituted with at least two fluorine atoms.

10. The aforementioned R ０ The nucleic acid-ionized lipid complex according to claim 4, wherein the group is a perfluoroalkyl group having 1 to 10 carbon atoms, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a perfluoroalkyl group having 1 to 10 carbon atoms.

11. The aforementioned R ０ The nucleic acid-ionized lipid complex according to claim 4, wherein the group is an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom.

12. The nucleic acid-ionized lipid complex according to claim 4, wherein n11 or n12 is 2 or more and 10 or less.

13. The nucleic acid-ionized lipid complex according to any one of claims 1 to 4, wherein the nucleic acid is a nucleic acid for suppressing gene expression in a cell.

14. The nucleic acid-ionized lipid complex according to claim 13, wherein the nucleic acid for suppressing gene expression in the cell is siRNA.

15. A nucleic acid-ionized lipid complex according to any one of claims 1 to 4, which does not contain lipids other than the ionized lipids.

16. A pharmaceutical composition comprising a nucleic acid-ionized lipid complex according to any one of claims 1 to 4 as an active ingredient.

17. A method for producing a nucleic acid-ionized lipid complex according to any one of claims 1 to 4, comprising mixing a solution containing a nucleic acid that can be introduced into cells by endocytosis with a solution containing an ionized lipid to complex the nucleic acid and the ionized lipid.

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