Amino lipid and lipid nanoparticles

Abstract

The purpose of the present invention is to provide: lipid nanoparticles which have excellent endosome desorption capability; and an amino lipid which can be used for the lipid nanoparticles.　The present invention includes an amino lipid having a structure represented by formula (1). "In formula (1), R1 and R2 each independently represent an alkyl group having 1 to 6 carbon atoms, or an arbitrary carbon atom in R1 and an arbitrary carbon atom in R2 may be bonded to each other to form a hetero ring containing a nitrogen atom; and R3 and R4 each independently represent an aliphatic hydrocarbon group which has 1 to 35 carbon atoms and can be substituted with a substituent group α; wherein at least one of R3 and R4 represents an aliphatic hydrocarbon group which has 5 to 35 carbon atoms and can be substituted with the substituent group α, and the substituent group α includes a hydroxyl group, an alkoxy group, a sulfanyl group, an alkylthio group, and an alkoxycarbonyl group."

Description

Aminolipids and lipid nanoparticles

[0001] The present invention relates to amino lipids and lipid nanoparticles.

[0002] A drug delivery system (DDS) is a drug discovery technology that controls the pharmacokinetics of drugs in vivo to maximize their effectiveness. A known drug delivery system is a system that uses lipid nanoparticles (LNPs). Some lipid nanoparticles contain pH-sensitive cationic lipids. pH-sensitive cationic lipids can become electrically charged or uncharged depending on changes in the pH of the body, allowing them to capture drugs, stabilize them in the blood, and disrupt endosomal membranes, thereby enabling lipid nanoparticles to deliver drugs into the cytoplasm.

[0003] As pH-sensitive cationic lipids, amino lipids are often used because of their stability in blood and low toxicity (Patent Documents 1 to 5).

[0004] International Publication No. WO 2009 / 086558 International Publication No. WO 2010 / 144740 International Publication No. WO 2017 / 075531 International Publication No. WO 2017 / 049245 International Publication No. WO 2018 / 230710

[0005] An object of the present invention is to provide lipid nanoparticles with excellent endosomal release ability and amino lipids useful therefor.

[0006] The present invention includes the following embodiments: [1] An amino lipid having a structure represented by the following formula (1): "In formula (1), R 1 and R 2 each independently represents an alkyl group having 1 to 6 carbon atoms, or R 1 and R 2 Any carbon atoms of R may be bonded to each other to form a heterocycle containing a nitrogen atom. 3 and R 4 each independently represents an aliphatic hydrocarbon group having 1 to 35 carbon atoms which can be substituted by the substituent group α. 3 and R 4At least one of the R represents an aliphatic hydrocarbon group having 5 to 35 carbon atoms which can be substituted with the substituent group α. The substituent group α represents a hydroxyl group, an alkoxy group, a sulfanyl group, an alkylthio group, and an alkoxycarbonyl group. 1 and R 2 [3] The amino lipid according to [1], wherein each of R is independently an alkyl group having 2 to 6 carbon atoms. 3 and R 4 [4] The amino lipid according to [1] or [2], wherein either one or both of the R 3 and R 4 [5] The amino lipid according to any one of [1] to [3], wherein either one or both of the R 3 and R 4 wherein either one of the amino lipids is an alkenyl group having 5 to 35 carbon atoms. [6] Lipid nanoparticles containing the amino lipid according to any of [1] to [5]. [7] Lipid nanoparticles according to [6], wherein the content of the amino lipid in the total lipids contained in the lipid nanoparticles is 20 to 80 mol %. [8] Lipid nanoparticles according to [6] or [7], further comprising one or more lipids selected from the group consisting of phospholipids, PEG lipids, and sterol lipids. [9] Lipid nanoparticles according to any of [6] to [8], which encapsulate nucleic acids.

[10] Lipid nanoparticles according to any of [6] to [9], which are used for introducing nucleic acids into cells.

[11] Lipid nanoparticles according to

[10] , wherein the cells are one type selected from the group consisting of stem cells, primary cells, and established cell lines.

[12] A method for introducing a nucleic acid into a cell, comprising: Step 1: mixing the amino lipid according to any one of [1] to [5] with a nucleic acid to obtain lipid nanoparticles encapsulating the nucleic acid; and Step 2: contacting the lipid nanoparticles obtained in Step 1 with cells.

[0007] According to the present invention, it is possible to provide lipid nanoparticles with excellent endosomal release ability and amino lipids contributing thereto. Furthermore, because the lipid nanoparticles of the present invention have excellent endosomal release ability, they can be used to transfer nucleic acids into cells into which nucleic acid transfer is difficult.

[0008] FIG. 1 is a scatter plot for the creation of an apparent pKa prediction model. FIG. 2 is a diagram showing the results of Experimental Examples 1 to 3. FIG. 3 is a diagram showing the results of Experimental Examples 4 to 8. (1) and (2) of FIG. 4 are diagrams showing the results of Experimental Examples 15 to 21. (1) and (2) of FIG. 5 are diagrams showing the results of Experimental Examples 22-1, 23-1, 24-1, 25-1, 26-1, and 27-1. (1) and (2) of FIG. 6 are diagrams showing the results of Experimental Examples 22-2, 23-2, 24-2, 25-2, 26-2, and 27-2. (1) and (2) of FIG. 7 are diagrams showing the results of Experimental Examples 22-3, 23-3, 24-3, 25-3, 26-3, and 27-3. Fig. 8 is a diagram showing the results of Experimental Examples 35 to 41. Fig. 9 is a diagram showing the results of Experimental Examples 66 to 71. Fig. 10 is a diagram showing the results of Experimental Examples 72 to 77.

[0009] As used herein, the singular forms "a", "an" and "the" include plural referents unless the singular only is intended.

[0010] In this specification, expressions expressing a numerical range such as "A to B" (A and B are real numbers satisfying A<B) are synonymous with "A or more, B or less." Furthermore, in this specification, expressions expressing a numerical range such as "A to B, or a to b" are synonymous with "A or more, B or less," "a or more, B or less," and "a or more, b or less." A, B, a, and b represent real numbers satisfying a<b<A<B.

[0011] Unless otherwise specified, each of the components exemplified in this specification, for example, the components such as lipids contained in lipid nanoparticles, can be used alone or in combination of two or more.

[0012] <Amino lipid> An amino lipid according to one embodiment of the present invention has a structure shown in formula (1) below.

[0013]

[0014] In formula (1), R 1 and R 2 each independently represents an alkyl group having 1 to 6 carbon atoms, or R 1 and R 2 Any carbon atoms of R may be bonded to each other to form a heterocycle containing a nitrogen atom. 3 and R 4 each independently represents an aliphatic hydrocarbon group having 1 to 35 carbon atoms which can be substituted by the substituent group α. 3 and R 4 At least one of the groups represents an aliphatic hydrocarbon group having 5 to 35 carbon atoms which can be substituted with the substituent group α. The substituent group α represents a hydroxyl group, an alkoxy group, a sulfanyl group, an alkylthio group, and an alkoxycarbonyl group.

[0015] R 1 and R 2 Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a 2-methylbutyl group, a neopentyl group, a 1-ethylpropyl group, an n-hexyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, and a 2-ethylbutyl group.

[0016] R 1 and R 2 is R 1 and R 2 The carbon atoms of these groups can be bonded together to form a nitrogen-containing heterocycle together with the nitrogen atom to which they are bonded. Examples of nitrogen-containing heterocycles include aziridine, azetidine, pyrrolidine, and piperidine.

[0017] R 1 and R 2is preferably an alkyl group having 2 to 6 carbon atoms, more preferably a linear alkyl group having 2 to 6 carbon atoms, and even more preferably an ethyl group.

[0018] R 3 and R 4 The aliphatic hydrocarbon group having 1 to 35 carbon atoms is a monovalent group formed by removing one hydrogen atom from any carbon atom of an aliphatic compound having 1 to 35 carbon atoms and consisting only of non-aromatic carbon and hydrogen.

[0019] Examples of the aliphatic hydrocarbon group having 1 to 35 carbon atoms include n-pentyl, isopentyl, 3-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 4-methylheptyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, methyldecyl, 1-hexyloctyl, 1-hexylnonyl, 1-heptylnonyl, 1-hexyldecyl, 1- linear or branched alkyl groups having 5 to 35 carbon atoms, such as hexyldecyl, 1-methylundecyl, 1-(1,3,3-trimethylbutyl)-2,6,6-trimethylheptyl, 10-trimethylundecyl, 3,7,11-trimethyldodecyl, 6,10,14-trimethylpentadecan-2-yl, 2,6,10,14-tetramethylpentadecyl, 3,7,11,15-tetramethylhexadecyl, 11-(tert-butoxy)-11-oxoundecanyl, 13-(tert-butoxy)-13-oxotridecanyl, 17-(tert-butoxy)-17-oxoheptadecanyl, and 2-(dodecylthio)ethanyl;

[0020] (Z)-non-8-enyl, (Z)-tridec-8-enyl, (Z)-tetradec-9-enyl, (Z)-pentadeca-8-enyl, (Z)-hexadec-9-enyl, (Z)-heptadeca-5-enyl, (Z)-octadec-6-enyl, (Z)-heptadeca-8-enyl, (Z)-octadec-9-enyl, (E)-heptadeca-8-enyl, (E)-octadec-9-enyl, (Z)-heptadeca-10-enyl, (Z)-octadec-11-enyl, (8Z,11Z)-heptadeca-8,11-dienyl, (9Z,12Z)-octadeca-9,12-dienyl, (8Z,11Z,14Z)-octadeca-8,11 linear or branched alkenyl groups having 5 to 35 carbon atoms, such as (9Z,12Z,15Z)-octadec-9,12,15-trienyl, (Z)-nonadec-10-enyl, (Z)-icosa-11-enyl, (10Z,13Z)-nonadeca-10,13-dienyl, (11Z,14Z)-icosa-11,14-dienyl, 2,6,10-trimethylundeca-1,5,9-trienyl, 3,7,11-trimethyldodeca-2,6,10-trienyl, 2,6,10,14-tetramethylpentadec-1-enyl, 3,7,11,15-tetramethylhexadec-2-enyl, and (Z)-henicos-12-enyl;

[0021] Alkynyl groups having 5 to 35 carbon atoms, such as dodec-11-ynyl, tridec-12-ynyl, pentadec-6-ynyl, hexadec-7-ynyl, pentadeca-4,6-diynyl, hexadeca-5,7-diynyl, heptadec-8-ynyl, and octadec-9-ynyl; and

[0022] Examples include alkyl groups, alkenyl groups, and alkynyl groups having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, and tert-butyl.

[0023] R 3 and R 4 It is preferable that either one or both of R is a secondary alkyl group having 5 to 35 carbon atoms. 3 and R 4 It is preferable that either one or both of R is an alkenyl group having 5 to 35 carbon atoms. 3 and R4 It is more preferable that one of R is a secondary alkyl group having 5 to 35 carbon atoms and the other is an alkenyl group having 5 to 35 carbon atoms. 3 and R 4 or R 3 and R 4 When either one or both of the above is an alkenyl group having 5 to 35 carbon atoms, the endosome release ability of the lipid nanoparticles including those of this embodiment can be further improved.

[0024] Examples of the secondary alkyl group having 5 to 35 carbon atoms include 1-hexyloctyl, 1-hexylnonyl, 1-heptylnonyl, 1-hexyldecyl, 1-methylundecyl, 1-(1,3,3-trimethylbutyl)-2,6,6-trimethylheptyl, etc. Among these, 1-hexyloctyl, 1-hexylnonyl, 1-heptylnonyl, 1-hexyldecyl, 1-heptylnonyl, and 1-methylundecyl are preferred, and 1-hexylnonyl is more preferred.

[0025] As the alkenyl group having 5 to 35 carbon atoms, (Z)-heptadeca-8-enyl and (8Z,11Z)-heptadeca-8,11-dienyl are preferred, and (Z)-heptadeca-8-enyl is more preferred.

[0026] R 3 and R 4 As for R 3 and R 4 are both (Z)-heptadec-8-enyl, or R 3 and R 4 are both (8Z,11Z)-heptadeca-8,11-dienyl, or R 3 and R 4 Both of R 3 and R 4 are both 1-(1,3,3-trimethylbutyl)-2,6,6-trimethylheptyl, or R 3 and R 4 Preferably, one of R is 1-hexylnonyl and the other is (Z)-heptadec-8-enyl, and R 3 and R4 Both of R 3 and R 4 are both 1-(1,3,3-trimethylbutyl)-2,6,6-trimethylheptyl, or R 3 and R 4 More preferably, one of R is 1-hexylnonyl and the other is (Z)-heptadec-8-enyl, 3 and R 4 It is more preferred that one of these is 1-hexylnonyl and the other is (Z)-heptadec-8-enyl.

[0027] R 3 and R 4 The aliphatic hydrocarbon group having 1 to 35 carbon atoms can be substituted with the substituent group α. "Substitutable" means that a hydrogen atom of the aliphatic hydrocarbon group is replaced with the substituent group α. The substituent group α represents a hydroxyl group, an alkoxy group, a sulfanyl group, an alkylthio group, and an alkoxycarbonyl group.

[0028] The term "apparent acid dissociation constant (hereinafter also referred to as "apparent pKa")" refers to the acid dissociation constant (pKa) of a pH-sensitive cationic lipid such as an amino lipid on the surface of a lipid nanoparticle. The apparent pKa can be calculated, for example, according to the literature (Jayaraman, M., et al., Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angewandte Chemie, 2012. 51(34): p.8529-33), by mixing 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and lipid nanoparticles in a buffer solution having a pH of 4.0 to 9.0, measuring the fluorescence intensity with a microplate reader (Ex / Em: 322 / 431 nm), and fitting to a sigmoid function. The pH at which the fluorescence intensity is at half its maximum can be calculated as the apparent pKa.

[0029] The apparent pKa can be estimated using a prediction model created by chemoinformatics analysis. The dataset used for the prediction model can be data converted into notation such as InChI, SMILES, molecular fingerprint, or MOL-File, and then converted into molecular descriptors using a library such as RDKit, Mordred, MOE, alvaDesc, PaDEL-Descriptor, or Codessa. Examples of learning algorithms used to create the prediction model include multiple linear regression, logistic regression, lasso regression, principal component analysis, ridge regression, support vector regression, random forest regression, multiple perceptron regression, convolutional neural network, and gradient boosting. The learning algorithms can be used alone or in combination of two or more.

[0030] One embodiment of the present invention relates to an amino lipid, a pH-sensitive cationic lipid that is uncharged in lipid nanoparticles at physiologically neutral pHs (pH = approximately 7.4), such as in blood, but positively charged in acidic environments (pKa = approximately 4.7 to 6.3), such as in endosomes. By producing lipid nanoparticles in an acidic buffer solution, the amino lipid can capture negatively charged drugs, such as nucleic acids, through electrostatic interactions, thereby enabling drugs to be encapsulated in the lipid nanoparticles. Furthermore, because the amino lipid in the lipid nanoparticles is uncharged in blood, nonspecific adsorption to proteins, such as red blood cells and serum albumin, due to charging can be prevented, and charging can also prevent cell membrane barrier properties and inflammation in vivo.

[0031] The amino lipid of one embodiment of the present invention is positively charged within the endosome in the lipid nanoparticle, and therefore is thought to be able to disrupt the endosomal membrane by allowing counterions to enter the endosome or increasing the ionic osmotic pressure, thereby enabling the drug to be efficiently released from the endosome.

[0032] The amino lipid of the present embodiment may include pharmaceutically acceptable salts, stereoisomers, and tautomers of the amino lipid. Examples of pharmaceutically acceptable salts include sodium salts, potassium salts, lithium salts, calcium salts, magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, and N -benzyl-phenethylamine salts, piperazine salts, tetramethylammonium salts, tris(hydroxymethyl)aminomethane salts, hydrofluoride, hydrochloride, hydrobromide, hydroiodide, nitrate, perchlorate, sulfate, phosphate, methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0033] The content of amino lipids in the total lipids contained in the lipid nanoparticles of one embodiment of the present invention is preferably 20 to 80 mol%, 30 to 75 mol%, 40 to 70 mol%, or 45 to 65 mol%. The lipid refers to a compound containing an acyl chain or isoprenoid chain having 6 or more carbon atoms.

[0034] [Method for Producing Aminolipid] The aminolipid of one embodiment of the present invention can be produced, for example, by O-acylation reaction of a glycerol derivative with a fatty acid, as shown in the following synthesis scheme 1. In synthesis scheme 1, R 1 ~R 4 is R in the above formula (1). 1 ~R 4are synonymous with each other, TsCl represents p-toluenesulfonyl chloride, TBSCl represents tert-butyldimethylchlorosilane, DMAP represents 4-dimethylaminopyridine, and TBAF represents tetrabutylammonium fluoride.

[0035]

[0036] In Synthetic Scheme 1, R 3 and R 4 When are the same aliphatic hydrocarbon group, the O-acylation reaction can be carried out without carrying out the TBS protection reaction.

[0037] In one embodiment of the present invention, R 3 and R 4Examples of fatty acids into which an aliphatic hydrocarbon group having 1 to 35 carbon atoms can be introduced include lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, 4-methyl-n-octanoic acid, nonanoic acid, isononanoic acid, monoethyl pimelate, 2,2-dimethylhexanoic acid, caprylic acid, enanthic acid, 2,2-dimethylpentanoic acid, 5-methylhexanoic acid, 3-methylpentanoic acid, 3,3-dimethyl Saturated fatty acids having 5 to 35 carbon atoms, such as butyric acid, hexanoic acid, 2,2-dimethylbutyric acid, 4-methylpentanoic acid, melissic acid, octacosanoic acid, cerotic acid, 18-(tert-butoxy)-18-oxooctadecanoic acid, nonadecanoic acid, 14-(tert-butoxy)-14-oxotetradecanoic acid, 12-(tert-butoxy)-12-oxododecanoic acid, monoethyl dodecanedioate, and undecanoic acid; palmitoleic acid, oleic acid, 8-Nonenoic acid, erucic acid, elaidic acid, (E)-dodec-2-enoic acid, 10-undecenoic acid, 10-undecynoic acid, trans-2-octenoic acid, 2-heptynoic acid, 2,2-dimethyl-4-pentenoic acid, 2-methylhexenoic acid, 4-methyl-2-pentenoic acid, trans-2-hexenoic acid, 2-methyl-4-pentenoic acid, trans-3-hexenoic acid, 5-hexenoic acid, trans-2-methyl-2-pentenoic acid, 2-methyl-2- Monounsaturated fatty acids having 5 to 35 carbon atoms, such as pentenoic acid, 5-hexynoic acid, and monomethyl itaconate; polyunsaturated fatty acids having 5 to 35 carbon atoms, such as linoleic acid, γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, 10,12-nonacosadiynoic acid, 10,12-heptacosadiynoic acid, 10,12-pentacosadiynoic acid, arachidonic acid, 2,4-heptadecadiynoic acid, 10,12-heptadecadiynoic acid, and sorbic acid;Decanoic acid, 9-decenoic acid, 4-methylnonanoic acid, citronellic acid, mono-tert-butyl succinate, 3-(dodecylthio)propionic acid, monomethyl sebacate, monoethyl glutarate, monoethyl itaconate, monoisopropyl fumarate, 3-allyloxypropionic acid, butoxyacetic acid, monoethyl maleate, butyric acid, crotonic acid, pentanoic acid, DL-2-methylbutyric acid, trans-2-pentenoic acid, 3-methoxypropionic acid fatty acids having 3 to 35 carbon atoms such as acetic acid, 4-methoxybutanoic acid, monomethyl maleate, monomethyl succinate, 3-ethoxypropionic acid, propiolic acid, 3-methylbutyric acid, 2-methyl-3-butenoic acid, 3-methylcrotonic acid, tetrolic acid, 4-pentenoic acid, methoxyacetic acid, ethoxyacetic acid, 3-(methylthio)propionic acid, pivalic acid, propionic acid, isobutyric acid, 3-(laurylthio)propionic acid, and tiglic acid; 3 and R 4 Examples of fatty acids having 5 to 35 carbon atoms into which a secondary alkyl group can be introduced include isopalmitic acid, isostearic acid, 2-hexadecyl octadecanoic acid, 2-methylhexadecanoic acid, 2-hexyl-4-pentynoic acid, 2-ethylhexanoic acid, 2-methylheptanoic acid, 2-ethylbutyric acid, 2-methylpentanoic acid, and 2-methylpalmitic acid.

[0038] In Synthesis Scheme 1, examples of amino alcohols used in the amination reaction include 2-(dimethylamino)ethanol, 2-(diethylamino)ethanol, 2-(dipropylamino)ethanol, 2-(diisopropylamino)ethanol, 2-(dibutylamino)ethanol, 2-(dipentylamino)ethanol, and 2-(dihexylamino)ethanol.

[0039] In Synthesis Scheme 1, the compounds obtained at each synthesis step can be purified by recrystallization, reprecipitation, liquid separation extraction, dialysis, or chromatography, if necessary.

[0040] <Lipid nanoparticles> The lipid nanoparticles of one embodiment of the present invention contain the amino lipid of one embodiment of the present invention. The lipid nanoparticles can further contain one or more lipids selected from the group consisting of phospholipids, PEG lipids, and sterol lipids, more preferably phospholipids, and even more preferably phospholipids, PEG lipids, and sterol lipids. The lipid nanoparticles are presumed to have a structure in which the lipid shell has a lipid membrane (lipid shell) containing the amino lipid.

[0041] [Phospholipid] Phospholipid refers to a lipid having a structure in which one or two hydroxyl groups of a phosphoric acid are ester-bonded to an alcohol. Phospholipids are zwitterionic lipids, and the hydroxyl groups of the phosphoric acid ionize to generate anions, so they exhibit amphiphilicity like surfactants and can contribute to the formation of the lipid shell of lipid nanoparticles.

[0042] Examples of phospholipids include glycerophospholipids and sphingophospholipids. Glycerophospholipids are lipids having an ester bond between glycerol and phosphoric acid, such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidic acid. Sphingophospholipids are lipids having an ester bond between sphingoid and phosphoric acid. Phosphatidylcholine is preferred as the phospholipid used in the lipid nanoparticles of one embodiment of the present invention.

[0043] Examples of phosphatidylcholines include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), didecanoylphosphatidylcholine (DDPC), dioctanoylphosphatidylcholine (DOPC), dihexanoylphosphatidylcholine (DHPC), and dibutyrylphosphatidylcholine. (DBPC), dielaidoylphosphatidylcholine, dilinoleoylphosphatidylcholine, diarachidonoylphosphatidylcholine, diicosenoylphosphatidylcholine, diheptanoylphosphatidylcholine, dicaproylphosphatidylcholine, diheptadecanoylphosphatidylcholine, dibehenoylphosphatidylcholine, eleostearoylphosphatidylcholine, hydrogenated egg phosphatidylcholine, hydrogenated soybean phosphatidylcholine, 1-palmitoyl-2-arachidonoylphosphatidylcholine phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-myristoyl-2-stearoylphosphatidylcholine Examples of such phosphatidylcholine include di-O-hexadecylphosphatidylcholine, trans-dielaidoylphosphatidylcholine, dipalmiteroyl-phosphatidylcholine, n-octadecyl-2-methylphosphatidylcholine, n-octadecylphosphatidylcholine, 1-laurylpropanediol-3-phosphocholine, erythro-N-lignoceroylsphingophosphatidylcholine, and palmitoyl-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine.

[0044] Examples of phosphatidylserines include distearoylphosphatidylserine (DSPS), dimyristoylphosphatidylserine (DMPS), dilauroylphosphatidylserine (DLPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine, lysophosphatidylserine, eleostearoylphosphatidylserine, and 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidylserine.

[0045] Phosphatidylinositols include, for example, dipalmitoylphosphatidylinositol, distearoylphosphatidylinositol, and dilauroylphosphatidylinositol.

[0046] Phosphatidylglycerols include, for example, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, lysophosphatidylglycerol, hydrogenated soy phosphatidylglycerol, hydrogenated egg phosphatidylglycerol, and cardiolipin (diphosphatidylglycerol).

[0047] Examples of phosphatidylethanolamines include dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, didecanoylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysophosphatidylethanolamine, N-(7-nitro-2,1,3-benzoxydiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine, eleostearoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine, and 1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine.

[0048] Phosphatidic acids include, for example, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dimyristoyl phosphatidic acid, and dioleyl phosphatidic acid.

[0049] Sphingophospholipids include sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, ceramide ciliatin, ceramide phosphorylethanolamine, and ceramide phosphorylglycerol.

[0050] The content of phospholipids in all lipids contained in the lipid nanoparticles of one embodiment of the present invention is, from the viewpoint of stabilizing the lipid shell of the lipid nanoparticles, typically 0 to 40 mol%, preferably 1 to 35 mol%, more preferably 2 to 30 mol%, even more preferably 3 to 25 mol%, particularly preferably 4 to 20 mol%, and especially preferably 5 to 15 mol%.

[0051] [PEG lipid] PEG lipid refers to a lipid having a polyethylene glycol (PEG) chain. Due to the hydrophilicity of the PEG chain, PEG lipid forms a hydration layer around the lipid nanoparticles, which suppresses aggregation of the lipid nanoparticles, prevents nonspecific adsorption of the lipid nanoparticles to proteins, etc., avoids capture and degradation of the lipid nanoparticles by phagocytes, etc., and improves blood retention. The weight-average molecular weight of the PEG lipid is usually 100 to 5,000. The weight-average molecular weight is a value measured by gel permeation chromatography using polystyrene as a standard.

[0052] Examples of PEG lipids include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate, PEGylated ceramide (PEG-cer), or ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl) ) carbamate or PEG dialkoxypropyl carbamates such as 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, N-[methoxy-poly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA), and 1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6-dioxaotanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG).

[0053] The content of PEG lipid in all lipids contained in the lipid nanoparticles of one embodiment of the present invention is typically 0.5 to 15 mol%, preferably 0.8 to 10 mol%, more preferably 1 to 8 mol%, even more preferably 1.3 to 5 mol%, and particularly preferably 1.5 to 3 mol%.

[0054] [Sterol lipids] Sterol lipids are lipids that have a side chain and a hydroxyl group on a steroid skeleton, and are thought to contribute to the physical stability of the lipid membrane by incorporating into the hydrocarbon chain of the lipid membrane of lipid nanoparticles.

[0055] Examples of sterol lipids include cholesterol, cholesterol succinate, dihydrocholesterol, lanosterol, dihydrolanosterol, desmosterol, stigmasterol, sitosterol, campesterol, brassicasterol, zymosterol, ergosterol, campesterol, fucosterol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, epicholesterol, dehydroergosterol, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, and cholesterol valerate. cholesteryl, cholesterol hemisuccinate, 3βN-(N',N'-dimethylaminoethane)-carbamoylcholesterol, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, coprostanol, cholesterol esters, cholesteryl phosphorylcholine, and 3,6,9-trioxaoctan-1-ol-cholesteryl-3e-ol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate.

[0056] The content of sterol lipids in all lipids contained in the lipid nanoparticles of one embodiment of the present invention is, from the viewpoint of stabilizing the lipid shell of the lipid nanoparticles, typically 0 to 70 mol%, preferably 1 to 60 mol%, more preferably 5 to 55 mol%, even more preferably 10 to 50 mol%, and particularly preferably 15 to 45 mol%.

[0057] [Other Constituents of Lipid Nanoparticles] Other components contained in lipid nanoparticles include, for example, pH-sensitive cationic lipids (excluding the amino lipids of one embodiment of the present invention), hydrophilic polymer-modified lipids (excluding PEG lipids), glycolipids, and fatty acids.

[0058] Examples of pH-sensitive cationic lipids (excluding the amino lipid of one embodiment of the present invention) include 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MoChol), 4-(2-aminoethyl)-morpholino-cholesterol-2,3-dimethylhemisuccinate (DmC4Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-2,2-dimethylhemimalonate (DmC3Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemimalonate (C3Mo2), [(3-morpho [1-Methyl-2-(2-morpholin-4-yl-ethylcarbamoyl)propyl]-carbamic acid cholesteryl ester (Chol-CN-Mo3), [1-Methyl-2-(2-morpholin-4-yl-ethylcarbamoyl)propyl]-carbamic acid cholesteryl ester (Chol-DMC3N-Mo2), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-dipal Mitoyl-3-trimethylammonium-propane (DPTAP), 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), palmitoyloleoyl-3-trimethylammonium-propane (POTAP), 1,2-dioleoyl-3-dimethylhydroxyethyl-ammonium-propane (DORI), 1,2-dimyristoyl-3-dimethylhydroxyethyl-ammonium-propane (DMRI), 1,2-dipalmitoyl-3-dimethylhydroxyethyl-ammonium-propane (DPR I), 1,2-distearoyl-3-dimethylhydroxyethyl-ammonium-propane (DSRI), palmitoyloleoyl-3-dimethylhydroxyethyl-ammonium-propane (PORI), 1,2-dioleoyl-3-methyldihydroxyethylammonium-propane (DOMDHEAP), 1,2-dimyristoyl-3-methyldihydroxyethylammonium-propane (DMMDHEAP), 1,2-dipalmitoyl-3-methyldihydroxyethylammonium-propane (DPMDHEAP), 1,2-Distearoyl-3-methyldihydroxyethylammonium-propane (DSMDHEAP), palmitoyloleoyl-3-methyldihydroxyethyl-ammonium-propane (POMDHEAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), 1,2-distearoyl-3-dimethyl ammonium-propane (DSDAP), 1,2-dioleoyl-3-methylhydroxyethylammonium-propane (DOMHEAP), 1,2-dioleoyl-3-dihydroxyethylammonium-propane (DODHEAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-MC3-DMA), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldokanoate (ALC-0315), and heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).

[0059] Hydrophilic polymer-modified lipids (excluding PEG lipids) include lipids having hydrophilic polymers. Examples of hydrophilic polymers include vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-vinylpyridine, 4-vinylpyridine, N-vinyl succinimide, N-vinyl formamide, N-vinyl-N-methyl formamide, N-vinyl acetamide, N-vinyl-N-methyl acetamide, 2-hydroxyethyl methacrylate, acrylamide, methacrylamide, N,N-dimethyl acrylamide, N-iso-propyl acrylamide, diacetone acrylamide, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine, glutamine, N-methyl glycine, methylene glycol, n-propylene glycol, iso-propylene glycol, and hydroxypropylene glycol.

[0060] The lipid nanoparticles may contain glycolipids for the purpose of structural stabilization of the lipid nanoparticles. Examples of glycolipids include diglycosyl diglyceride, glycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, sulfoxyribosyl diglyceride, digalactosyl glyceride, digalactosyl dilauroyl glyceride, digalactosyl dimyristoyl glyceride, digalactosyl dipalmitoyl glyceride, digalactosyl distearoyl glyceride, galactosyl glyceride, and galactosyl dilauroyl glyceride. Glycerolipids such as galactosylglyceride, galactosyl dimyristoylglyceride, galactosyl dipalmitoylglyceride, galactosyl distearoylglyceride, and digalactosyldiacylglycerol; and glycosphingolipids such as ceramides (cerebrosides), galactosylceramide, lactosylceramide, digalactosylceramide, gangliosides, sulfatides, ceramide oligohexosides, and globosides.

[0061] Examples of fatty acids include saturated or unsaturated fatty acids such as caprylic acid, pelargonic acid, capric acid, undecylenic acid, lauric acid, tridecylenic acid, myristic acid, pentadecylenic acid, palmitic acid, margaric acid, stearic acid, nonadecylenic acid, arachidic acid, dodecenoic acid, tetradecenoic acid, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid, and docosapentaenoic acid.

[0062] Ligands can be attached to the surface of the lipid nanoparticles of one embodiment of the present invention to impart specific functions to the lipid nanoparticles. Examples of the ligands include proteins such as antibodies and antibody fragments, aptamers, and small molecular weight compounds that can specifically bind to target receptors.

[0063] Methods for binding a ligand to the surface of lipid nanoparticles according to one embodiment of the present invention include a method in which a group capable of binding to a ligand, such as a maleimide group or an N-hydroxysuccinimidyl group, is introduced to the end of the lipid, preferably a PEG lipid, and the ligand is bound to the group capable of binding to the ligand, followed by production of lipid nanoparticles, or a method in which a ligand is bound to the surface of lipid nanoparticles after production of the lipid nanoparticles.

[0064] The lipid nanoparticles of one embodiment of the present invention can encapsulate a nucleic acid as a pharmaceutical (payload). Examples of nucleic acids include RNAs such as dsRNA, siRNA, miRNA, shRNA, ptgsRNA, DsiRNA, mRNA, circular RNA, circular mRNA, rRNA, tRNA, snRNA, snoRNA, gRN, sgRN, and aptamers; DNAs such as cDNA, plasmid DNA, synthetic DNA, modified DNA, vector DNA, ssDNA, dsDNA, circular DNA, and linear DNA; and DNAs or RNAs such as ASO.

[0065] The lower limit of the nucleic acid encapsulation rate in the lipid nanoparticles is 60% or more, 65% or more, 70% or more, or 75% or more, and the upper limit is 100% or less, less than 100%, 99% or less, or 95% or less. The nucleic acid encapsulation rate refers to the encapsulation rate of the nucleic acid contained in the lipid nanoparticles (nucleic acid encapsulation rate), and can be measured by fluorimetry. The nucleic acid encapsulation rate can be calculated using the following formula (2). Examples of nonionic surfactants in formula (2) include Triton. TM X-100 is an example.

[0066] {(amount of nucleic acid in the presence of a nonionic surfactant)−(amount of nucleic acid in the absence of a nonionic surfactant)} ÷ (amount of nucleic acid in the presence of a nonionic surfactant)×100(%)...Equation (2)

[0067] Lipid nanoparticles encapsulating nucleic acids can be used for nucleic acid transfer into cells. The cells to be transferred with nucleic acids are not particularly limited, and examples include cells derived from animals such as humans, pigs, dogs, rats, and mice. Preferred animal cells include humans and pigs. Examples of such cells include somatic cells such as hepatocytes, spleen cells, muscle cells, nerve cells, and cardiomyocytes; immune cells such as T cells, B cells, NK cells, monocytes, macrophages, and dendritic cells; epithelial cells such as skin cells and intestinal epithelial cells; cancer cells; and germ cells; stem cells, primary cells, and established cell lines. Preferred cells are stem cells, primary cells, or established cell lines.

[0068] Stem cells are cells that have the ability to self-renew and can differentiate into one or more specific cell types, and include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), somatic stem cells, etc. Specific examples of stem cells include human embryonic stem cells, human iPS cells, mouse embryonic stem cells, mouse iPS cells, epiblast stem cells, ES-like cells, mouse mesenchymal stem cells, neural stem cells, hematopoietic stem cells, skeletal muscle stem cells, hair follicle stem cells, spermatogonial stem cells, ovarian stem cells, amniotic fluid stem cells, dental pulp stem cells, inducible neural stem cells, muscle stem cells, umbilical cord blood-derived stem cells, adipose-derived stem cells, and epithelial stem cells.

[0069] Primary cells are cells directly isolated from normal or tumor tissues of a living organism, and are not cell lines. Primary cells include primary cells derived from normal tissues such as umbilical vein endothelial cells, normal skin fibroblasts, primary hepatocytes, bronchial epithelial cells, mammary epithelial cells, aortic endothelial cells, alveolar epithelial cells, epidermal keratinocytes, cardiac fibroblasts, bronchial epithelial cells, skeletal muscle cells, cardiac myocytes, and renal cortical epithelial cells; and primary cells derived from tumor tissues such as brain tumors, breast cancers, colon cancers, pancreatic cancers, prostate cancers, liver cancers, lung cancers, ovarian cancers, and bladder cancers.

[0070] An established cell line is a cell that has the property of being able to be passaged indefinitely (immortalized), and includes cells derived from cancer cells or cells that have been artificially immortalized by, for example, introducing an immortalizing gene. Examples of established cell lines include HeLa cells, HepG2 cells, SH-SY5Y cells, Jurkat cells, HEK293 cells, MCF-7 cells, A549 cells, NIH-3T3 cells, RAW264.7 cells, K562 cells, CHO-K1 cells, U-2OS cells, PC-3 cells, THP-1 cells, NK-92 cells, bEND.3 cells, and Neuro-2a cells.

[0071] The method for introducing the nucleic acid in the lipid nanoparticles into the cells is not particularly limited as long as it can bring the lipid nanoparticles containing the nucleic acid into contact with the cells. In the case of in vitro, for example, a method of adding a dispersion of lipid nanoparticles containing the nucleic acid to a liquid or solid medium containing the cells and culturing the cells can be mentioned.

[0072] In the case of in vivo, for example, a dispersion of lipid nanoparticles encapsulating nucleic acids can be administered to a subject by intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, intraperitoneal injection, portal vein injection, intranasal administration, or the like to bring the dispersion into contact with cells in the living body.

[0073] In the case of ex vivo, for example, an organ, tissue, or cell is collected from a living body and then cultured with lipid nanoparticles encapsulating nucleic acid. For example, a method is exemplified in which T cells are collected from a subject, then cultured with lipid nanoparticles encapsulating nucleic acid, and then the T cells are brought into contact with the lipid nanoparticles encapsulating nucleic acid.

[0074] After contact with a cell, the lipid nanoparticles encapsulating the nucleic acid are taken up into the cell and fuse with the membrane of the endosome within the cell, thereby releasing the encapsulated nucleic acid into the cell and introducing the nucleic acid into the cell.

[0075] The lipid nanoparticles according to one embodiment of the present invention have excellent endosomal detachment ability, and therefore can deliver nucleic acids to cells that are difficult to deliver nucleic acids to, in addition to the above-mentioned in vivo cells, such as stem cells, primary cells, or established cell lines. Furthermore, due to the excellent nucleic acid delivery function of the lipid nanoparticles, they are useful not only as a transfection reagent for cells in vitro, but also for the above-mentioned ex vivo gene delivery, particularly when delivering a gene encoding a chimeric antigen receptor to T cells used in CAR-T therapy.

[0076] In the lipid nanoparticles of one embodiment of the present invention, the ratio (N / P ratio) of the number of nitrogen atoms (N) of the pH-sensitive cationic lipid to the number of phosphorus atoms (P) derived from the nucleic acid at pH 6.5 is typically 2 to 15, preferably 2.5 to 14, more preferably 3 to 13, and even more preferably 3.5 to 12.0.

[0077] [Production of lipid nanoparticles] Examples of methods for producing lipid nanoparticles include the thin film method, simple hydration method, reverse phase evaporation (REV) method, alcohol dilution method, and microfluidic device method. To control the size of lipid nanoparticles, extrusion (extrusion filtration) can be performed under high pressure through a membrane filter with a uniform pore size.

[0078] The lipid nanoparticles can be provided as a dispersion. Examples of the dispersion medium used in the dispersion include aqueous dispersion media such as buffers such as phosphate buffer, Tris buffer, Tris-buffered saline, Meth buffer, Hepes buffer, citrate buffer, and phosphate-buffered saline, as well as saline and cell culture media. The concentration of the lipid nanoparticles contained in the dispersion is typically 0.01 to 100 mg / mL.

[0079] In order to stabilize the lipid nanoparticles, the dispersion liquid may contain monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, maltose, and sucrose; trisaccharides such as raffinose and melezinose; polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol; and polyhydric alcohols such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and 1,3-butylene glycol.

[0080] To store a dispersion containing lipid nanoparticles for a long period of time, it is preferable to eliminate electrolytes from the aqueous solvent medium as much as possible from the viewpoint of physical stability such as suppressing aggregation. Furthermore, from the viewpoint of chemical stability of lipids, it is preferable to set the pH of the aqueous dispersion medium to a weakly acidic to near-neutral range (pH 3.0 to 8.0). Furthermore, it is preferable to remove dissolved oxygen from the dispersion by nitrogen bubbling or the like.

[0081] The lipid nanoparticles in the dispersion can be extracted from the dispersion by freeze-drying or spray-drying. When freeze-drying or spray-drying is performed, the freeze-drying or spray-drying can be performed stably by including the monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, and / or polyhydric alcohols in the dispersion.

[0082] The average particle size of the lipid nanoparticles in the dispersion medium is, for example, 10 to 300 nm, 15 to 200 nm, 20 to 150 nm, 25 to 125 nm, or 30 to 100 nm. The average particle size herein refers to the average particle size (Z-average harmonic mean diameter) determined by the cumulant method. The average particle size can be calculated by calculating the Stokes-Einstein diffusion coefficient from the autocorrelation function measured by dynamic light scattering and converting it to particle size. The polydispersity index (PDI) of the lipid nanoparticles in the dispersion medium is 0.001 to 0.2, 0.005 to 0.15, or 0.01 to 0.1. The polydispersity index (PDI) of the lipid nanoparticles in the dispersion medium can be measured by dynamic scattering.

[0083] <Method for introducing nucleic acid into cells> One embodiment of the present invention is a method for introducing nucleic acid into cells, comprising: step 1 of mixing the amino lipid with a nucleic acid to obtain lipid nanoparticles encapsulating the nucleic acid; and step 2 of contacting the lipid nanoparticles obtained in step 1 with cells.

[0084] [Step 1] In step 1, the amino lipid and nucleic acid are not particularly limited and can be mixed, for example, as follows. The amino lipid is dissolved in alcohol, such as ethanol, along with one or more lipids selected from the phospholipids, PEG lipids, and sterol lipids as optional components to prepare a lipid solution of any concentration. The desired nucleic acid is dissolved in a buffer solution such as phosphate buffer, phosphate-buffered saline, Tris buffer, Tris-buffered saline, Meth buffer, Hepes buffer, citrate buffer, or acetate buffer to prepare a nucleic acid solution of any concentration. The prepared lipid solution and nucleic acid solution can be mixed using known mixing methods, such as a micromixer, flow reactor, ultrasonic mixer, or magnetic stirrer. The ratio of lipid nanoparticles to nucleic acid can be set within the above-mentioned N / P ratio range.

[0085] [Step 2] The meanings of the terms used in Step 2, in which lipid nanoparticles encapsulating nucleic acids are introduced into cells, are as described above. The nucleic acids are released into the cells from the lipid nanoparticles that have been taken up into the cells in Step 2, and the nucleic acids are introduced into the cells.

[0086] [Kit] Another embodiment of the present invention is a kit containing an amino lipid having the structure shown in the above formula (1). In addition to the amino lipid, the kit may also include a buffer solution for dissolving the amino lipid, a container or dispensing device such as a tube or syringe, an instruction manual, etc. The kit can be used to produce the lipid nanoparticles.

[0087] Hereinafter, the embodiments of the present invention will be described in more detail based on examples, but the embodiments of the present invention are not limited to these examples. Unless otherwise specified, the experiments shown in the examples were carried out at room temperature (approximately 10 to 35°C).

[0088] The abbreviations of the compounds used in the examples are as follows: CDI: 1,1-carbonyldiimidazole, DBU: 1,8-diazabicyclo[5.4.0]-7-undecene, DMAP: 4-(dimethylamino)pyridine, DMSO: dimethyl sulfoxide, DMF: N,N-dimethylformamide, THF: tetrahydrofuran, TBAF: tetra-n-butylammonium fluoride, TBDMSCl: tert-butyldimethylsilyl chloride

[0089] <Measurement Methods> In the examples, each physical property was measured as follows. [Measurement of Particle Size and Polydispersity Index (PDI)] Particle size and polydispersity index (PDI) were measured by dynamic scattering (Zeta Sizer ADVANCE (Malvern Panalytical)). The average particle size was calculated using the Stokes-Einstein equation by detecting fluctuations in scattered light that reflect the diffusion coefficient using the dynamic scattering method.

[0090] [Measurement of nucleic acid encapsulation rate] The nucleic acid encapsulation rate was measured using Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the product manual. TM After treatment with Fluorescence Intensity Test Kit (Invitrogen), the nucleic acid encapsulation rate was measured by fluorimetry using a Quant-iT RiboGreen RNA Assay kit (Invitrogen). The nucleic acid encapsulation rate was calculated using the above formula (2).

[0091] [Measurement of apparent pKa (actual value)] A buffer solution containing 20 mM Tris (trishydroxymethylaminomethane), 20 mM phosphate, 20 mM acetic acid, and 150 mM sodium chloride (pH 4.0-9.0) was prepared and dispensed into a 96-well plate. The lipid nanoparticle solution was mixed with the TNS solution and added to each well of the 96-well plate. The fluorescence intensity of each well was measured using a microplate reader (SPARK (Tecan)) (λex = 322 nm, λem = 431 nm), and the apparent pKa was calculated by applying sigmoidal fitting to the pH at which the fluorescence intensity reached half its maximum.

[0092] [Measurement of apparent pKa (predicted value)] Based on academic literature, the composition and performance of lipid nanoparticles (particle size, nucleic acid encapsulation rate, IC50, ED50, pKa, PDI, etc.) and molecular descriptors (AAM, PEOE, VSA, etc.) converted from the lipid structure in SMAILS notation were used as a data set, and principal component analysis and multivariate regression analysis were performed to create an apparent pKa prediction model (R 2 = 0.76). A scatter plot of the apparent pKa prediction model is shown in Figure 1.

[0093] <Preparation of amino lipids> As amino lipids, the amino lipids of Examples 1 to 5 shown in Table 1 and Comparative Examples 1 to 4 shown in Table 2 were prepared. Compounds 1 to 5 were produced by the method described below. MC3 was the reagent "D-Lin-MC3-DMA" manufactured by MedChemExpress, ALC-0315 was the reagent "ALC-0315" manufactured by MedChemExpress, SM-102 was the reagent "SM-102" manufactured by MedChemExpress, and DODAP was the reagent "DODAP" manufactured by MedChemExpress. In addition, the structures of compounds such as amino lipids were 1 Identification was performed by 1 H NMR (AVANCE III 600, manufactured by Bruker, or JNM-ECZL600R, manufactured by JEOL Ltd.).

[0094]

[0095]

[0096] Example 1 1-(2-(diethylamino)ethoxy)-3-((2-hexyldecanoyl)oxy)propan-2-yl oleate (compound 1) was produced according to the synthesis scheme 2 shown below.

[0097]

[0098] (1) Synthesis of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate: In a flask, trimethylamine (38.6 g) and DMAP (1.2 g) were added to a solution of 2,2-dimethyl-1,3-dioxolane-4-methanol (TCI) (25.2 g) in acetonitrile (194 mL), and the mixture was stirred at room temperature for 10 minutes. p-TSCl (40.0 g) was gradually added to the flask, and the mixture was stirred at room temperature for 6 hours. The solution after stirring was concentrated using a rotary evaporator. Ethyl acetate was added, and the mixture was separated and washed with an aqueous sodium hydroxide solution (0.5 M) and saturated saline. Anhydrous sodium sulfate was added to the organic layer for dehydration, and the organic layer was purified by silica gel chromatography (ethyl acetate as the developing solvent) to produce (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (54.6 g). The compound obtained １ The results of H-NMR measurement are shown below.

[0099] １ H-NMR (600MHz, CDCl3) δ (ppm): 1.30 (s, 3H), 1.33 (s, 3H), 2.44 (s, 3H), 3.75 (dd, J = 5.16, 8.77 Hz, 1H), 3.95-4.04 (m, 3H), 4.25-4.27 (m, 1H), 7.34 (d, J = 7.98Hz, 2H), 7.78 (d, J = 8.34Hz, 2H)

[0100] ​(2) Synthesis of 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylethan-1-amine: In a flask, a solution of diethylaminoethanol (16.3 g) in DMF (38.0 mL) was added dropwise to a suspension of 60% sodium hydride (7.0 g) in DMF (38.0 mL) under ice-cooling. After stirring at room temperature for 1 hour, a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl-4-methylbenzenesulfonate (38.0 g) in DMF (76.0 mL) was added dropwise. After stirring at 140°C for 2 hours, the mixture was cooled to room temperature by exothermic cooling. The solution was added to ice-cold water, and extraction was performed with ethyl acetate. The organic phase was washed with saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration, and the solvent was then removed using a rotary evaporator to produce 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylethan-1-amine (22.9 g). １ The results of H-NMR measurement are shown below.

[0101] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 1.00 (t, J = 7.11Hz, 6H), 1.33 (s, 3H), 1.39 (s, 3H), 2.53-2.57 (m, 4H), 2.62-2.65 (m, 2H), 3.4 4 (dd, J=5.52, 9.90Hz, 1H), 3.50-3.58 (m, 3H), 3.69-3.72 (m, 1H), 4.01-4.04 (m, 1H), 4.23-4.25 (m, 1H)

[0102] ​(3) Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diol: In a flask, 35% aqueous hydrochloric acid (20 mL) was added to a solution of 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylethan-1-amine (10.0 g) in THF (100 mL), and the mixture was stirred at room temperature for 3 hours. After concentration using a rotary evaporator, the mixture was neutralized with aqueous sodium hydroxide and extracted with ethyl acetate. The organic layer was washed with saturated saline. Anhydrous sodium sulfate was added to the gas layer for dehydration, and the layer was purified by silica gel chromatography (a mixture of ethyl acetate and methanol as the developing solvent) to produce 3-(2-(diethylamino)ethoxy)propane-1,2-diol (4.0 g). The compound obtained １ The results of H-NMR measurement are shown below.

[0103] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 1.01 (t, J = 7.17Hz, 6H), 2.54-2.59 (m, 6H), 3.49 (dd, J = 6.5 7, 10.53Hz, 1H), 3.53-3.62 (m, 5H), 3.77-3.79 (m, 1H), 5.24 (br, 1H)

[0104] (4) Synthesis of 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-ol: In a flask, imidazole (0.3 g) was added to a solution of 3-(2-(diethylamino)ethoxy)propane-1,2-diol (0.8 g) in DMF (7.7 mL), and the mixture was stirred at room temperature for 10 minutes. Then, TBDMSCl (0.6 g) was added, and the mixture was stirred at 60°C for 5 hours. Ethyl acetate and aqueous sodium hydroxide solution (0.5 M) were added, and the mixture was subjected to separation and extraction. The organic layer was separated and washed with saturated saline. Anhydrous sodium sulfate was added to the air layer to dehydrate it, and then the mixture was purified by silica gel chromatography (developing solvent: a mixture of heptane and ethyl acetate) to produce 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-ol (1.0 g). The compound obtained １ The results of H-NMR measurement are shown below.

[0105] ​​１ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.04 (s, 6H), 0.87 (s, 9H), 1.03 (t, J=7.22Hz, 6H), 2.55-2.62 (m, 6H), 3.47 (dd, J=6.51, 10.58Hz, 1H), 3.55-3.63 (m, 5H), 3.74-3.76 (m, 1H)

[0106] (5) Synthesis of 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-yl oleate: In a flask, oleic acid (0.49 g) and CDI (0.28 g) were dissolved in ethyl acetate (2.5 mL) and stirred at room temperature for 1 hour. A solution of 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-ol (0.5 g) and DBU (0.05 g) in ethyl acetate (2.5 mL) was added, and the mixture was stirred at room temperature for 8 hours. The mixture was washed with purified water, aqueous sodium hydroxide (0.5 M), and saturated saline. Anhydrous sodium sulfate was added to the air layer to dehydrate it, and then it was purified by silica gel chromatography (developing solvent: a mixture of heptane, ethyl acetate, and methanol) to produce 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-yl oleate (0.8 g). １ The results of H-NMR measurement are shown below.

[0107] １ H-NMR (600MHz, DMSO-d6) δ (ppm): 0.04 (s, 6H), 0.85-0.88 (m, 12H), 1.03 (t, J = 7.20Hz, 6H), 1.18-1.71 (m, 22H), 1.96-2.08 (m, 4H), 2.30-2.35 (m, 2H), 2.54-2.68 (m, 6H), 3.51-3.78 (m, 5H), 3.81-3.85 (m, 1H), 4.50-4.54 (m, 1H), 5.29-5.39 (m, 2H)

[0108] ​(6) Synthesis of 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl oleate: 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-yl oleate (0.8 g) was added to a flask with TBAF (1 mol / L in THF) (2.7 g) and stirred at room temperature for 5 hours. After concentration, the mixture was dissolved in ethyl acetate and washed with aqueous sodium hydroxide solution (0.5 M) and saturated saline solution. Anhydrous sodium sulfate was added to the organic layer for dehydration, and the mixture was purified by silica gel chromatography (developing solvent: a mixture of heptane, ethyl acetate, and methanol) to produce 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl oleate (0.5 g). １ The results of H-NMR measurement are shown below.

[0109] １ H-NMR (600MHz, DMSO-d6) δ (ppm): 0.85 (s, 3H), 1.03 (t, J = 7.22Hz, 6H), 1.18-1.71 (m, 22H), 1.96-2.08 (m, 4H), 2.30-2.35 (m, 2H), 2.54-2.68 (m, 6H), 3.50-3.69 (m, 6H), 4.50-4.54 (m, 1H), 5.29-5.39 (m, 2H)

[0110] (7) Synthesis of 1-(2-(diethylamino)ethoxy)-3-((2-hexyldecanoyl)oxy)propan-2-yl oleate (Compound 1) In a flask, isopalmitic acid (0.3 g) and CDI (0.2 g) were dissolved in ethyl acetate (2.5 mL) and stirred at room temperature for 1 hour. A solution of 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl oleate (0.5 g) and DBU (0.03 g) in ethyl acetate (2.5 mL) was added, and the mixture was stirred at room temperature for 8 hours. The mixture was separated and washed with purified water, aqueous sodium hydroxide solution (0.5 M), and saturated saline. Anhydrous sodium sulfate was added to the organic layer for dehydration, and then the organic layer was purified by silica gel chromatography (developing solvent: a mixture of heptane, ethyl acetate, and methanol) to produce Compound 1 (0.7 g). １ The results of H-NMR measurement are shown below. ​

[0111] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.86-0.88 (m, 9H), 1.17 (br, 6H), 1.23-1.60 (m, 46H), 1.97-2.04 (m, 4H), 2.24-2.35 (m, 3H), 2.81 (br, 6 H), 3.54-3.61 (m, 2H), 3.72 (br, 2H), 4.08-4.15 (m, 1H), 4.30-4.35 (m, 1H), 5.18-5.24 (m, 1H), 5.31-5.37 (m, 2H)

[0112] Example 2 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diylbis(2-hexyldecanoate) (Compound 2) In a flask, isopalmitic acid (2.8 g) and CDI (1.8 g) were dissolved in ethyl acetate (5 mL) and stirred at room temperature for 1 hour. A solution of 3-(2-(diethylamino)ethoxy)propane-1,2-diol (1.0 g) and DBU (0.3 g) in ethyl acetate (5 mL) was added, and the mixture was stirred at room temperature for 8 hours. Thereafter, the reaction solution was separated and washed with purified water, an aqueous sodium hydroxide solution (0.5 M), and saturated saline. Anhydrous sodium sulfate was added to the organic layer for dehydration, and the organic layer was filtered and then purified by silica gel chromatography (developing solvent: a mixture of heptane, ethyl acetate, and methanol) to produce Compound 2 (3.3 g). １ The results of H-NMR measurement are shown below.

[0113] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.87 (t, J = 7.26, 12H), 1.25-1.61 (m, 54H), 2.29-2.35 (m, 2H), 2.90 (br, 6H), 3.55- 3.62 (m, 2H), 3.75 (br, 2H), 4.07 (br, 1H), 4.35 (dd, J = 4.08, 12.00Hz, 1H), 5.18-5.23 (m, 1H)

[0114] ​​Example 3 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diylbis(2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoic acid) (Compound 3) Compound 3 was produced in the same manner as in Example 2, except that isostearic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0115] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.77-1.59 (m, 70H), 2.12-2.20 (m, 2H), 2.89 (br, 6H), 3.57-3.65 (m , 2H), 3.76 (br, 2H), 4.05-4.17 (m, 1H), 4.23-4.37 (m, 1H), 5.17-5.20 (m, 1H)

[0116] Example 4 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diyl dioleate (Compound 4) Compound 4 was produced in the same manner as in Example 2, except that oleic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0117] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.87 (t, J = 7.20Hz, 6H), 1.16 (br, 6H), 1.24-1.62 (m, 44H), 1.89-2.02 (m, 8H), 2.30 (br, 4H), 2.79 (br, 6H), 3.55-3.61 (m, 2H), 3.71 (br, 2H), 4.12 (br, 1H), 4.30 (dd, J=3.78, 11.70Hz, 1H), 5.18-5.21 (m, 1H), 5.31-5.37 (m, 4H)

[0118] Example 5 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diyl(9Z,12Z)-bis(octadeca-9,12-dienoate) (Compound 5) Compound 5 was produced in the same manner as in Example 2, except that linoleic acid was used instead of isopalmitic acid. １ ​​The results of H-NMR measurement are shown below.

[0119] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.88 (t, J = 7.26Hz, 6H), 1.15 (br, 6H), 1.25-1.62 (m, 32H), 2.04 (m, 8H), 2.30 (m, 4H), 2.74-2.83 (m, 10H), 3.55-3.62 (m, 2H), 3.71 (br, 2H), 4.12 (br, 1H), 4.30 (dd, J=3.78, 11.64, 1H), 5.18-5.21 (m, 1H), 5.29-5.40 (m, 8H)

[0120] <Production of lipid nanoparticles encapsulating siRNA targeting mouse blood coagulation factor VII mRNA>

[0121] Example 6 The amino lipid (Compound 1) produced in Example 1, phospholipid (1,2-Distearoyl-sn-glycero-3-PC, catalog number "COATSOME MC-8080", Yuka Sangyo Co., Ltd., hereinafter referred to as "DSPC"), sterol lipid (cholesterol, catalog number "C8667", Sigma-Aldrich Co., Ltd., hereinafter referred to as "Chol"), and PEG lipid (1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene, product name "SUNBRIGHT GM-020", Yuka Sangyo Co., Ltd., hereinafter referred to as "PEG-DMG") were dissolved in ethanol at a molar ratio of AL (amino lipid):DSPC:Chol:PEG-DMG = 50:5:45:0.5 to a total lipid concentration of 8 mM, to prepare a lipid solution.

[0122] ​An siRNA targeting mouse blood coagulation factor VII mRNA (a hybridization of a nucleic acid having the base sequence shown in SEQ ID NO: 1 and a nucleic acid having the base sequence shown in SEQ ID NO: 2; hereinafter, also referred to as "FVII siRNA") was dissolved in acetate buffer (25 mM, pH 4) to a concentration of 40 μg / mL to prepare a FVII siRNA solution. A lipid solution (0.5 mL) and 1.5 mL of the FVII siRNA solution were mixed in a flow reactor. The mixed solution was dialyzed against phosphate-buffered saline (PBS) to produce a dispersion containing lipid nanoparticles (LNP-FVII-1) encapsulating FVII siRNA.

[0123] SEQ ID NO: 1 sense strand (5'-3'): GGAucAucucAAGucuuACT*T SEQ ID NO: 2 antisense strand (5'-3'): GuAAGAcuuGAGAuGAuccT*T In SEQ ID NOs: 1 and 2, lowercase sequences are 2'-fluoro modified nucleic acids, and * indicates a phosphorothioate bond.

[0124] The physical properties of the obtained lipid nanoparticles were measured by the above-mentioned methods, and the results are shown in Table 3.

[0125]

[0126] Examples 7 and 8, Comparative Examples 5 and 6 LNP-FVII-2 (Example 7), LNP-FVII-3 (Example 8), LNP-FVII-MC3 (Comparative Example 5), and LNP-FVII-DODAP (Comparative Example 6) were produced in the same manner as in Experimental Example 6, except that Compound 2 produced in Example 2, Compound 3 produced in Example 3, MC3 in Comparative Example 1, or DODAP in Comparative Example 4 was used in the molar ratios shown in Table 4 instead of Compound 1 in Example 6 as the amino lipid.

[0127]

[0128] The physical properties of the lipid nanoparticles of Examples 7 and 8 and Comparative Examples 5 and 6 were measured by the above-mentioned methods. The results are shown in Table 3.

[0129] <In Vivo Evaluation of Lipid Nanoparticles Encapsulating siRNA> [Experimental Example 1] The following experiment was conducted to evaluate the endosomal release ability based on the delivery of a drug contained in the lipid nanoparticles to the cytoplasm. The LNP-FVII-1 produced in Example 6 was diluted with phosphate-buffered saline (PBS, pH 7.4) to give a FVII siRNA concentration of 0.03 mg / kg body weight and administered via the tail vein to mice (C57BL / 6N, 6 weeks old, female, CLEA Japan, n=2). Subsequently, the mice were euthanized 24 hours later, and the livers were removed and frozen in liquid nitrogen.

[0130] The frozen liver was homogenized using a Multi-Bead Shocker (Yasui Kikai) at 2500 rpm for 15 seconds, and an RNA solution was obtained according to the product manual for the nucleic acid extraction kit (NucleoSpin (registered trademark) RNA (Takara)).

[0131] Next, a commercially available kit (TaqMan) was used for the reverse transcription reaction. TM A reaction solution was prepared using a MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). The reverse transcription reaction using a thermal cycler (SimpliAmp Thermal Cycler (Thermo Fisher Scientific)) was carried out at 16°C for 30 minutes, followed by 42°C for 30 minutes, and then 85°C for 5 minutes to obtain the desired cDNA solution.

[0132] Furthermore, the obtained cDNA solution was subjected to a commercial kit (TaqMan TM A reaction solution was prepared using Universal Master Mix, no. UNG (Thermo Fisher Scientific). The real-time PCR reaction conditions using a real-time PCR device (7500 Fast Real-Time PCR System (Thermo Fisher Scientific)) were 95°C for 1 minute, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute, to quantify the amount of FVII siRNA in the liver. The results are shown in Table 5 and Figure 2.

[0133]

[0134] [Experimental Examples 2 to 3] The amount of FVII siRNA in the liver was quantified in the same manner as in Experimental Example 1, except that LNP-FVII-2 produced in Example 7 or LNP-FVII-3 produced in Example 8 was used instead of LNP-FVII-1 as the lipid nanoparticle. The results are shown in Table 5 and Figure 2.

[0135] Experimental Example 4 The following experiment was conducted to evaluate the endosomal detachment ability based on the knockdown rate (KD rate) of siRNA targeting mouse blood coagulation factor VII mRNA. The LNP-FVII-1 produced in Example 6 was diluted with phosphate-buffered saline (PBS, pH 7.4) to give a FVII siRNA concentration of 0.03 mg / kg body weight and administered via the tail vein to mice (C57BL / 6N, 6-week-old, female, CLEA Japan, n=3). Subsequently, 24 hours later, blood was collected from the inferior vena cava of the mice under isoflurane anesthesia. Subsequently, the blood samples were centrifuged at 3,000 rpm at 4°C for 5 minutes to obtain plasma samples. Next, the blood coagulation factor VII concentration in the plasma sample and the blood coagulation factor VII concentration in the plasma sample from the group not administered lipid nanoparticles were measured using a BIOPHEN FVII kit (Hyphen Biomed), and the knockdown rate was calculated based on the following formula (3). The results are shown in Table 6 and Figure 3.

[0136] Knockdown rate (%) = (1 - (blood coagulation factor VII concentration in plasma samples from lipid nanoparticle-administered group / blood coagulation factor VII concentration in plasma samples from lipid nanoparticle-unadministered group)) × 100 ... (3)

[0137]

[0138] [Experimental Examples 5 to 8] Except for using LNP-FVII-2 produced in Example 7, LNP-FVII-3 produced in Example 8, LNP-FVII-MC3 produced in Comparative Example 5, or LNP-FVII-DODAP produced in Comparative Example 6 instead of LNP-FVII-1 as lipid nanoparticles, the blood coagulation factor FVII concentration in plasma samples and the blood coagulation factor FVII concentration in plasma samples from the lipid nanoparticle-untreated group were measured in the same manner as above, as Experimental Examples 5 to 8, respectively, and the knockdown rate was calculated. The results are shown in Table 6 and FIG. 3.

[0139] <Production of lipid nanoparticles encapsulating mRNA> [Example 9] The amino lipid (Compound 1) produced in Example 1 was dissolved in ethanol at a molar ratio of DSPC:Chol:PEG-DMG = 60:10:30:1.5 to give a total lipid concentration of 8 mM, to prepare a lipid solution.

[0140] NanoLuc ＴＭ NLuc mRNA was prepared by performing an in vitro transcription reaction on pDNA encoding luciferase (NLuc) (Promega).

[0141] NLuc mRNA was dissolved in acetate buffer (25 mM, pH 4.0) to a concentration of 40 μg / mL to prepare an NLuc mRNA solution. 0.5 mL of the lipid solution and 1.5 mL of the NLuc mRNA solution were mixed in a flow reactor. The mixed solution was dialyzed against phosphate-buffered saline (PBS, pH 7.4) to produce a dispersion containing lipid nanoparticles (LNP-NLuc-1) encapsulating NLuc mRNA.

[0142] Example 10, Comparative Examples 7 to 10 In Example 9, LNP-NLuc-2 (Example 10), LNP-NLuc-MC3 (Comparative Example 7), LNP-NLuc-SM-102 (Comparative Example 8), LNP-NLuc-ALC-0315 (Comparative Example 9), and LNP-NLuc-DODAP (Comparative Example 10) were produced in the same manner as in Example 9, except that Compound 2 produced in Example 2, MC3 in Comparative Example 1, SM-102 in Comparative Example 2, ALC-0315 in Comparative Example 3, or DODAP in Comparative Example 4 was used in the molar ratios shown in Table 7 instead of Compound 1 as the amino lipid.

[0143]

[0144] The average particle size, polydispersity index (PDI), and nucleic acid encapsulation rate of each lipid nanoparticle produced above were measured using the above-mentioned measurement methods. The results are shown in Table 8.

[0145]

[0146] In vitro evaluation of mRNA-encapsulated lipid nanoparticles: HeLa cells were obtained from the JCRB Cell Bank (cell number: JCRB9004). For in vitro evaluation, HeLa cells were cultured in Eagle's minimum essential medium (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum and penicillin / streptomycin.

[0147] Experimental Example 9 Cell viability was measured using the MultiTox-Fluor Multiplex Cytotoxicity Assay Kit (Promega) according to the product manual. HeLa cells (1.0E+4 cells / well) were seeded in a 96-well plate. 100 μL of medium containing the lipid nanoparticles LNP-NLuc-1 prepared in Example 9 was added to each well so that the amount of NLuc mRNA added per well was 50 ng or 10 ng. The mixture was then incubated for 24 hours (37°C, 5% CO2 atmosphere). The MultiTox-Fluor Multiplex Cytotoxicity Assay reagent was then added, followed by an additional 1 hour of incubation (37°C, 5% CO2 atmosphere). The fluorescence intensity of each well was measured using a microplate reader (λex = 400 nm, λem = 505 nm) (n = 5). The cell viability was calculated as the mean ± standard deviation, with the wells containing PBS(-) added as a control, with a viability of 100%, and the wells containing digitonin added as a control, with a viability of 0%. The evaluation results are shown in Table 9.

[0148]

[0149] Experimental Examples 10 to 14 Cell viability was calculated in the same manner as in Experimental Example 9, except that LNP-NLuc-2 produced in Example 10, LNP-NLuc-MC3 produced in Comparative Example 7, LNP-NLuc-SM-102 produced in Comparative Example 8, LNP-NLuc-ALC-0315 produced in Comparative Example 9, or LNP-NLuc-DODAP produced in Comparative Example 10 was used instead of LNP-NLuc-1 as the lipid nanoparticle. The results are shown in Table 9.

[0150] Experimental Example 15 The following experiment was conducted to evaluate the endosomal escape ability of lipid nanoparticles based on the intracellular delivery of drugs contained in the lipid nanoparticles using a luciferase assay. The luciferase assay was performed using the Nano-Glo Luciferase Assay System (registered trademark, Promega) according to the product manual. HeLa cells (1.0E+4 cells / well) were seeded in a 96-well plate, and 100 μL of medium containing the lipid nanoparticles LNP-NLuc-1 produced in Example 9 was added to each well so that the amount of NLuc mRNA added per well was 50 ng or 10 ng. The mixture was then incubated for 24 hours (37°C, 5% CO2 atmosphere). Nano-Glo Luciferase Assay Reagent (registered trademark) was then added, and the luminescence intensity (CPS) of each well was measured using a microplate reader (n=5). The results are shown in Table 10 and Figure 4.

[0151]

[0152] Experimental Examples 16 to 21 The luminescence intensity was measured in the same manner as in Experimental Example 15, except that LNP-NLuc-2 produced in Example 10, LNP-NLuc-MC3 produced in Comparative Example 7, LNP-NLuc-SL-102 produced in Comparative Example 8, LNP-NLuc-ALC-0315 produced in Comparative Example 9, LNP-NLuc-DODAP produced in Comparative Example 10, or PBS was used instead of LNP-NLuc-1 as the lipid nanoparticle. The results are shown in Table 10 and FIG. 4.

[0153] <In Vivo Evaluation of Lipid Nanoparticles Encapsulating mRNA> [Experimental Examples 22-1 to 22-3] The following experiments were conducted to evaluate the endosomal release ability of lipid nanoparticles based on the intraorgan delivery of drugs contained in the lipid nanoparticles using a luciferase assay. LNP-NLuc-1 produced in Example 9 was diluted with phosphate-buffered saline (PBS, pH 7.4) to give an Nluc mRNA concentration of 0.05 mg / kg body weight or 0.5 mg / kg body weight and administered via the tail vein to mice (C57BL / 6N, 6-week-old, female, CLEA Japan, n=5). 24 hours after administration, the mice were euthanized, and the organs were collected, frozen in liquid nitrogen, and temporarily stored.

[0154] Each stored organ was thawed on ice, Glo Lysis Buffer (Promega) was added, and the organ was disrupted using a homogenizer. The organ solution was centrifuged at 13,000 rpm at 4°C for 10 minutes to collect the supernatant. The amount of luciferase-derived luminescence was measured for the supernatant using the Nano-Glo Luciferase Assay System (registered trademark, Promega), and the BCA was analyzed. TM The amount of protein was measured using a Protein Assay Kit (Thermo Fisher Scientific). From these results, the luminescence per protein (CPS) of each organ (liver (Experimental Example 22-1), spleen (Experimental Example 22-2), and pancreas (Experimental Example 22-3)) was calculated. The results are shown in Table 11 and FIG. 5.

[0155]

[0156] [Experimental Examples 23-1 to 27-3] The luminescence intensity was measured in the same manner as in Examples 22-1 to 22-3, except that LNP-NLuc-2 produced in Example 10, LNP-NLuc-MC3 produced in Comparative Example 7, LNP-NLuc-SM-102 produced in Comparative Example 8, or LNP-NLuc-ALC-0315 produced in Comparative Example 9 was used instead of LNP-NLuc-1 as the lipid nanoparticle. The results are shown in Table 11 and Figures 5 to 7.

[0157] <Study on the composition of lipid nanoparticles> [Examples 11 to 17] LNP-NLuc-4-1 (Example 11), LNP-NLuc-4-2 (Example 12), LNP-NLuc-4-3 (Example 13), LNP-NLuc-4-4 (Example 14), LNP-NLuc-4-5 (Example 15), LNP-NLuc-4-6 (Example 16), and LNP-NLuc-1-5 (Example 17) were produced in the same manner as in Example 9, except that compound 4 produced in Example 4 or compound 1 in Example 1 was used in the molar ratios shown in Table 12 instead of compound 1 in Example 9 as the amino lipid.

[0158]

[0159] The physical properties of the lipid nanoparticles of Examples 11 to 17 were measured by the above-mentioned methods. The results are shown in Table 13.

[0160]

[0161] [Experimental Examples 28 to 34] Cell viability was calculated in the same manner as in Experimental Example 9, except that LNP-NLuc-4-1 to LNP-NLuc-4-6 produced in Examples 11 to 16, or LNP-NLuc-1-5 produced in Example 17 were used instead of LNP-NLuc-1 as lipid nanoparticles. The results are shown in Table 14.

[0162]

[0163] [Experimental Examples 35 to 41] The luminescence intensity was measured in the same manner as in Experimental Example 15, except that LNP-NLuc-4-1 to LNP-NLuc-4-6 produced in Examples 11 to 16, or LNP-NLuc-1-5 produced in Example 17 were used instead of LNP-NLuc-1 as the lipid nanoparticles. The results are shown in Table 15 and FIG. 8.

[0164]

[0165] <Preparation of Amino Lipids 2> As amino lipids, the amino lipids shown in Examples 18 to 39 in Tables 16 to 20 were prepared in the same manner as in the section <Preparation of Amino Lipids>. Compounds 7, 21 to 41 were produced by the methods described below. The structures of compounds such as amino lipids are shown below. 1Identification was performed by 1 H NMR (AVANCE III 600, manufactured by Bruker, or JNM-ECZL600R, manufactured by JEOL Ltd.).

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Example 18 Synthesis of 18-di-tert-butyl O'1,O1-(3-(2-(diethylamino)ethoxy)propane-1,2-diyl)di(octadecanedioate) (Compound 21) Compound 21 was produced in the same manner as in Example 2, except that 18-(tert-butoxy)-18-oxooctadecanoic acid was used instead of isopalmitic acid. 1 The results of H-NMR measurement are shown below.

[0172] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 1.09 (br, 6H), 1.23-1.26 (m, 48H), 1.42 (s, 18H), 1.52-1.62 (m, 8H), 2.16-2.31 (m, 8H), 2.68-2.75 (m, 6H), 3.54-3.64 (m, 4H), 4.07-4.14 (m, 1H), 4.28-4.31 (m, 1H), 5.17-5.20 (m, 1H)

[0173] Example 19 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diyl ditetradecanoate (Compound 22) Compound 22 was produced in the same manner as in Example 2, except that myristic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0174] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.86 (t, J=6Hz, 6H), 1.05 (t, J=6Hz, 6H), 1.24-1.30 (m, 40H), 1.56-1.62 (m, 4H), 2.27-2.31 (m, 4H), 2.62-2.69 (m, 6H), 3.53-3.62 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.32 (m, 1H), 5.17-5.20 (m, 1H)

[0175] Example 20 Synthesis of 14-di-tert-butyl O'1,O1-(3-(2-(diethylamino)ethoxy)propane-1,2-diyl)di(tetradecanedioate) (Compound 23) Compound 23 was produced in the same manner as in Example 2, except that 14-(tert-butoxy)-14-oxotetradecanoic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0176] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 1.08 (br, 6H), 1.23-1.26 (m, 32H), 1.42 (s, 18H), 1.52-1.59 (m, 8H), 2.16-2.19 (m, 4H), 2.26-2.30 (m, 4H), 2.66-2.73 (m, 6H), 3.52-3.62 (m, 4H), 4.10-4.13 (m, 1H), 4.28-4.31 (m, 1H), 5.18-5.19 (m, 1H)

[0177] Example 21 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diyl(13Z,13'Z)-bis(docos-13-enoate) (Compound 24) Compound 24 was produced in the same manner as in Example 2, except that erucic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0178] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.86-0.88 (m, 6H), 1.08 (br, 6H), 1.25-1.31 (m, 56H), 1.58-1.60 (m, 4H), 2.00 (br, 8H), 2.27-2.31 (m, 4H), 2.66-2.72 (m, 6H), 3.56-3.60 (m, 4H), 4.11-4.15 (m, 1H), 4.29-4.32 (m, 1H), 5.18-5.20 (m, 1H), 5.31-5.36 (m, 4H)

[0179] Example 22 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diylbis(2-methylhexadecanoate) (Compound 25) Compound 25 was produced in the same manner as in Example 2, except that 2-methylpalmitic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0180] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.85-0.87 (m, 6H), 1.07 (br, 6H), 1.12-1.13 (m, 6H), 1.24 (br, 48H), 1.34 (br, 2H), 1.62 (br, 2H), 2.41 (br, 2H), 2.66-2.72 (m, 6H), 3.55-3.61 (m, 4H), 4.09-4.15 (m, 1H), 4.30-4.34 (m, 1H), 5.19 (br, 1H)

[0181] Example 23 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diyldiundecanoate (Compound 7) Compound 7 was produced in the same manner as in Example 2, except that undecanoic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0182] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.84-0.87 (m, 6H), 1.05 (t, J=12Hz, 6H), 1.24-1.27 (m, 28H), 1.57-1.60 (m, 4H), 2.26-2.30 (m, 4H), 2.61-2.69 (m, 6H), 3.53-3.58 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.31 (m, 1H), 5.18-5.20 (m, 1H)

[0183] Example 24 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diylbis(decanoic acid) (Compound 26) Compound 26 was produced in the same manner as in Example 2, except that decanoic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0184] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.84-0.87 (m, 6H), 1.05 (t, J=12Hz, 6H), 1.24-1.27 (m, 24H), 1.57-1.59 (m, 4H), 2.26-2.30 (m, 4H), 2.61-2.69 (m, 6H), 3.53-3.58 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.31 (m, 1H), 5.18-5.20 (m, 1H)

[0185] Example 25 Synthesis of 3-(2-(diethylamino)ethoxy)propane-1,2-diylbis(3-(dodecylthio)propanoate) (Compound 27) Compound 27 was produced in the same manner as in Example 2, except that 3-(laurylthio)propionic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0186] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.84-0.87 (m, 6H), 1.08 (br, 6H), 1.24-1.34 (m, 36H), 1.52-1.56 (m, 4H), 2.50 (t, J=12Hz, 4H), 2.58-2.75 (m, 14H), 3.56-3.61 (m, 4H), 4.16-4.19 (m, 1H), 4.32-4.35 (m, 1H), 5.20-5.22 (m, 1H)

[0187] Example 26 Synthesis of 12-di-tert-butyl O'1,O1-(3-(2-(diethylamino)ethoxy)propane-1,2-diyl)di(dodecanedioate) (Compound 28) Compound 28 was produced in the same manner as in Example 2, except that 12-(tert-butoxy)-12-oxododecanoic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0188] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 1.06 (br, 6H), 1.24 (br, 24H), 1.42 (s, 18H), 1.52-1.58 (m, 8H), 2.15-2.18 (m, 4H), 2.26-2.29 (m, 4H), 2.66-2.71 (m, 6H), 3.52-3.60 (m, 4H), 4.10-4.13 (m, 1H), 4.27-4.30 (m, 1H), 5.17-5.18 (m, 1H)

[0189] Example 27 Synthesis of 1-(2-(diethylamino)ethoxy)-3-((2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl)oxy)propan-2-yl oleate (Compound 29) Compound 29 was produced in the same manner as Compound 1, except that isostearic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0190] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.81-0.88 (m, 27H), 1.09 (br, 6H), 1.25-1.41 (m, 30H), 1.59 (br, 2H), 1.95-2.03 (br, 4H), 2.11-2.17 (m, 1H), 2.26-2.28 (m, 2H), 2.70-2.77 (m, 6H), 3.54-3.63 (m, 4H), 4.08-4.13 (m, 1H), 4.25-4.32 (m, 1H), 5.19 (br, 1H), 5.29-5.35 (m, 2H)

[0191] Example 28 Synthesis of 1-(2-(diethylamino)ethoxy)-3-(tetradecanoyloxy)propan-2-yl oleate (Compound 30) Compound 30 was produced in the same manner as Compound 1, except that myristic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0192] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.86 (t, J=6Hz, 6H), 1.06 (br, 6H), 1.24-1.28 (m, 40H), 1.59-1.60 (m, 4H), 1.99-2.00 (m, 4H), 2.27-2.31 (m, 4H), 2.63-2.70 (m, 6H), 3.53-3.59 (m, 4H), 4.10-4.14 (m, 1H), 4.29-4.32 (m, 1H), 5.18-5.20 (m, 1H), 5.29-5.36 (m, 2H)

[0193] Example 29 Synthesis of 1-(tert-butyl) 14-(3-(2-(diethylamino)ethoxy)-2-(oleyloxy)propyl)tetradecanedioate (Compound 31) Compound 31 was produced in the same manner as Compound 1, except that 14-(tert-butoxy)-14-oxotetradecanoic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0194] １ ​​H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.86 (t, J=6Hz, 3H), 1.08 (t, J=12Hz, 6H), 1.23-1.31 (m, 36H), 1.42 (s, 9H), 1.52-1.60 (m, 6H), 1.97-2.00 (m, 4H), 2.18 (t, J=12Hz, 2H), 2.27-2.31 (m, 4H), 2.66-2.73 (m, 6H), 3.54-3.63 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.31 (m, 1H), 5.17-5.20 (m, 1H), 5.29-5.35 (m, 2H)

[0195] Example 30 Synthesis of 3-(2-(diethylamino)ethoxy)-2-(oleyloxy)propyl (Z)-docos-13-enoate (Compound 32) Compound 32 was produced in the same manner as Compound 1, except that erucic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0196] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.87 (t, J=12Hz, 6H), 1.09 (t, J=6Hz, 6H), 1.25-1.32 (m, 48H), 1.57-1.62 (m, 4H), 1.98-2.01 (m, 8H), 2.27-2.31 (m, 4H), 2.68-2.74 (m, 6H), 3.54-3.63 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.32 (m, 1H), 5.18-5.21 (m, 1H), 5.30-5.36 (m, 4H)

[0197] Example 31 Synthesis of 3-(2-(diethylamino)ethoxy)-2-(oleyloxy)propyl(9Z,12Z)-octadeca-9,12-dienoate (Compound 33) Compound 33 was produced in the same manner as Compound 1, except that linoleic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.​

[0198] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.85-0.88 (m, 3H), 1.07 (t, J=12Hz, 6H), 1.25-1.36 (m, 34H), 1.56-1.62 (m, 4H), 1.98-2.05 (m, 8H), 2.27-2.31 (m, 4H), 2.65-2.76 (m, 8H), 3.54-3.65 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.31 (m, 1H), 5.17-5.21 (m, 1H), 5.29-5.39 (m, 6H)

[0199] Example 32 Synthesis of 1-(2-(diethylamino)ethoxy)-3-(palmitoyloxy)propan-2-yl oleate (Compound 34) Compound 34 was produced in the same manner as Compound 1, except that palmitic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0200] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.87 (t, J=6Hz, 3H), 1.09 (t, J=6Hz, 6H), 1.24-1.34 (m, 44H), 1.58-1.61 (m, 4H), 1.98-2.01 (m, 4H), 2.27-2.31 (m, 4H), 2.68-2.75 (m, 6H), 3.54-3.64 (m, 4H), 4.11-4.14 (m, 1H), 4.29-4.32 (m, 1H), 5.17-5.21 (m, 1H), 5.30-5.36 (m, 2H)

[0201] Synthesis of 3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl oleate (Compound 35) 3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl oleate was synthesized according to the following synthesis scheme 3.

[0202] ​​

[0203] (8) Synthesis of 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-yl 2-hexyldecanoate: This was produced in the same manner as 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-yl oleate, except that isopalmitic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below.

[0204] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.02 (s, 3H), 0.05 (s, 3H), 0.84-0.86 (m, 15H), 1.05 (t, J=6Hz, 6H), 1.22-1.31 (m, 20H), 1.57-1.61 (m, 4H), 2.25-2.29 (m, 1H), 2.62-2.69 (m, 6H), 3.37-3.42 (m, 1H), 3.54-3.71 (m, 4H), 3.93-3.98 (m, 1H), 4.97-5.00 (m, 1H)

[0205] (9) Synthesis of 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl 2-hexyldecanoate 11-ethyl-2,2,3,3-tetramethyl-4,8-dioxa-11-aza-3-silatridecan-6-yl 2-hexyldecanoate was prepared in the same manner as in the preparation of 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl oleate. １ The results of H-NMR measurement are shown below.

[0206] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.84-0.86 (m, 6H), 1.05-1.09 (m, 6H), 1.20-1.28 (m, 20H), 1.39-1.43 (m, 2H), 1.55-1.59 (m, 2H), 2.31-2.36 (m, 1H), 2.65 (br, 6H), 3.60-3.73 (m, 4H), 3.91-3.95 (m, 1H), 4.08-4.12 (m, 1H), 4.97-5.00 (m, 1H)

[0207] (10) Synthesis of 3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl oleate (Compound 35) Compound 35 was produced in the same manner as Compound 1, except that 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl 2-hexyldecanoate was used instead of 1-(2-(diethylamino)ethoxy)-3-hydroxypropan-2-yl oleate and oleic acid was used instead of isopalmitic acid. １ The results of H-NMR measurement are shown below.

[0208] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.85-0.87 (m, 9H), 1.06 (br, 6H), 1.24-1.31 (m, 40H), 1.40-1.43 (m, 2H), 1.54-1.60 (m, 4H), 1.98-2.03 (m, 4H), 2.26-2.34 (m, 3H), 2.64-2.70 (m, 6H), 3.52-3.59 (m, 4H), 4.10-4.14 (m, 1H), 4.30-4.35 (m, 1H), 5.17-5.23 (m, 1H), 5.31-5.36 (m, 2H)

[0209] ​Example 34 Synthesis of 1-(tert-butyl) 14-(3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl)tetradecanedioate (Compound 36) Compound 36 was produced in the same manner as Compound 35, except that 14-(tert-butoxy)-14-oxotetradecanoic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below.

[0210] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.84-0.86 (m, 6H), 1.08 (br, 6H), 1.23-1.25 (m, 38H), 1.42 (s, 9H), 1.52-1.60 (m, 6H), 2.16-2.19 (m, 2H), 2.25-2.33 (m, 3H), 2.66-2.73 (m, 6H), 3.51-3.61 (m, 4H), 4.10-4.13 (m, 1H), 4.29-4.34 (m, 1H), 5.16-5.21 (m, 1H)

[0211] Example 35 Synthesis of 1-(tert-butyl) 12-(3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl) dodecanedioate (Compound 37) Compound 37 was produced in the same manner as Compound 35, except that 12-(tert-butoxy)-12-oxododecanoic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below.

[0212] １ H-NMR (600MHz, CDCl ３ ​​) δ (ppm): 0.85 (t, J=12Hz, 6H), 1.09 (t, J=6Hz, 6H), 1.23-1.25 (m, 32H), 1.40-1.44 (m, 11H), 1.52-1.61 (m, 6H), 2.16-2.33 (m, 5H), 2.68-2.75 (m, 6H), 3.52-3.64 (m, 4H), 4.09-4.13 (m, 1H), 4.29-4.34 (m, 1H), 5.16-5.22 (m, 1H)

[0213] Example 36 Synthesis of 1-(tert-butyl) 18-(3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl) octadecandioate (Compound 38) Compound 38 was produced in the same manner as Compound 35, except that 18-(tert-butoxy)-18-oxooctadecanoic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below.

[0214] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.86 (t, J=12Hz, 6H), 1.11 (br, 6H), 1.23-1.27 (m, 44H), 1.40-1.43 (m, 11H), 1.53-1.61 (m, 6H), 2.16-2.34 (m, 5H), 2.71-2.77 (m, 6H), 3.52-3.65 (m, 4H), 4.10-4.13 (m, 1H), 4.29-4.34 (m, 1H), 5.17-5.23 (m, 1H)

[0215] Example 37 Synthesis of 3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl(9Z,12Z)-octadeca-9,12-dienoate (Compound 39) Compound 39 was produced in the same manner as Compound 35, except that linoleic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below.

[0216] １ H-NMR (600MHz, CDCl​​３ ) δ (ppm): 0.85-0.88 (m, 9H), 1.08 (t, J=6Hz, 6H), 1.25-1.35 (m, 34H), 1.41-1.44 (m, 2H), 1.54-1.61 (m, 4H), 2.01-2.05 (m, 4H), 2.26-2.35 (m, 3H), 2.66-2.76 (m, 8H), 3.52-3.62 (m, 4H), 4.10-4.13 (m, 1H), 4.29-4.35 (m, 1H), 5.18-5.23 (m, 1H), 5.29-5.39 (m, 4H)

[0217] Example 38 Synthesis of 3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl (Z)-docos-13-enoate (Compound 40) Compound 40 was produced in the same manner as Compound 35, except that erucic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below.

[0218] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.85-0.88 (m, 9H), 1.09 (t, J=6Hz, 6H), 1.24-1.31 (m, 48H), 1.40-1.43 (m, 2H), 1.54-1.62 (m, 4H), 1.98-2.03 (m, 4H), 2.26-2.34 (m, 3H), 2.68-2.75 (m, 6H), 3.54-3.63 (m, 4H), 4.11-4.13 (m, 1H), 4.30-4.35 (m, 1H), 5.17-5.23 (m, 1H), 5.31-5.36 (t, J=6Hz, 2H)

[0219] Example 39 Synthesis of 3-(2-(diethylamino)ethoxy)-2-((2-hexyldecanoyl)oxy)propyl tetradecanoate (Compound 41) Compound 41 was produced in the same manner as Compound 35, except that myristic acid was used instead of oleic acid. １ The results of H-NMR measurement are shown below. ​

[0220] １ H-NMR (600MHz, CDCl ３ ) δ (ppm): 0.85-0.87 (m, 9H), 1.09 (t, J=6Hz, 6H), 1.24-1.27 (m, 40H), 1.40-1.43 (m, 2H), 1.54-1.62 (m, 4H), 2.26-2.35 (m, 3H), 2.69-2.76 (m, 6H), 3.52-3.64 (m, 4H), 4.10-4.13 (m, 1H), 4.29-4.34 (m, 1H), 5.17-5.23 (m, 1H)

[0221] <Production of lipid nanoparticles encapsulating mRNA 2> [Examples 40 to 61, Comparative Examples 11 to 12] In Example 9, instead of the amino lipid (compound 1) produced in Example 1, the amino lipids (compounds 7, 21 to 41) produced in Examples 18 to 39 above were used in the molar ratios shown in Table 21. Each lipid nanoparticle was produced by the same method as in Example 9.

[0222]

[0223] The average particle size, polydispersity index (PDI), nucleic acid encapsulation rate, and apparent pKa of each lipid nanoparticle prepared above were measured using the above-mentioned measurement methods. The results are shown in Table 22.

[0224]

[0225] <In vitro evaluation 2 of lipid nanoparticles encapsulating mRNA> [Experimental Examples 42 to 65] In Experimental Examples 9 and 15, cell viability and luciferase assays were performed in the same manner as in Experimental Examples 9 and 15, except that the lipid nanoparticles shown in Table 23 were used instead of the LNP-NLuc-1 used in Experimental Examples 9 and 15. The results of each experiment are shown in Table 23.

[0226]

[0227] ​<Production of lipid nanoparticles encapsulating EGFP mRNA> [Example 62] The amino lipid (Compound 1) produced in Example 1: DSPC:Chol:PEG-DMG in a molar ratio of 60:10:30:1.5 was dissolved in ethanol to give a total lipid concentration of 8 mM, to prepare a lipid solution.

[0228] EGFP mRNA was prepared by performing an in vitro transcription reaction on pDNA encoding EGFP.

[0229] EGFP mRNA was dissolved in acetate buffer (25 mM, pH 4.0) to a concentration of 40 μg / mL to prepare an NLuc mRNA solution. 0.5 mL of the lipid solution and 1.5 mL of the EGFP mRNA solution were mixed in a flow reactor. The mixed solution was dialyzed against phosphate-buffered saline (PBS, pH 7.4) to produce a dispersion containing lipid nanoparticles (LNP-EGFP-1) encapsulating EGFP mRNA.

[0230] [Examples 63-64, Comparative Examples 13-14] In Example 62, LNP-EGFP-2 (Example 63), LNP-EGFP-4 (Example 64), LNP-EGFP-SM-102 (Comparative Example 13), and LNP-EGFP-ALC-0315 (Comparative Example 14) were produced in the same manner as in Example 62, except that Compound 2, Compound 4, SM-102, and ALC-0315 were used in the molar ratios (mol%) shown in Table 24 instead of Compound 1 as the amino lipid.

[0231]

[0232] The average particle size, polydispersity index (PDI), and nucleic acid encapsulation rate of each lipid nanoparticle produced above were measured using the above-mentioned measurement methods. The results are shown in Table 25.

[0233]

[0234] <Production of a transfection reagent containing EGFP mRNA> [Production Example 1] Lipofectamine MessengerMAX (Thermo Fisher Scientific) was used as the transfection reagent. According to the product manual, the Lipofectamine MessengerMAX reagent and an EGFP mRNA solution were mixed to produce a dispersion containing a transfection reagent (LMM-EGFP) containing EGFP mRNA.

[0235] In vitro evaluation of lipid nanoparticles encapsulating EGFP mRNA. HepG2 cells, Jurkat E6.1 cells, and SH-SY5Y cells were obtained from the ECACC cell bank. The basal media for HepG2 cells, Jurkat E6.1 cells, and SH-SY5Y cells were DMEM (low glucose) (Sigma-Aldrich), RPMI 1640 (Sigma-Aldrich), and a 1:1 mixture of Ham's F12 (Gibco) and EMEM (Sigma-Aldrich) containing 1% MEM non-essential amino acid solution (Gibco). For in vitro evaluation, 10% heat-inactivated fetal bovine serum and penicillin / streptomycin were further added to complete the medium, and cell culture was performed.

[0236] [Experimental Examples 66-71] Cell viability was evaluated using the MultiTox-Fluor Multiplex Cytotoxicity Assay Kit (Promega) using the lipid nanoparticles produced in Examples 62-64 and Comparative Examples 13-14 instead of the lipid nanoparticles of Example 9, except that the amount of EGFR mRNA added per well was 10 ng, 100 ng, or 500 ng. The results obtained as Experimental Examples 66-70 are shown in Table 26 and Figure 9. Similarly, the lipid nanoparticles of Example 9 were evaluated using the dispersion produced in Production Example 1 instead of the lipid nanoparticles of Example 9, except that the amount of EGFR mRNA added per well was 100 ng. The results obtained as Experimental Example 71 are shown in Table 26 and Figure 9. In FIG. 9, the three bar graphs of Experimental Examples 66 to 70 show the results when, from the left, the amounts of EGFR mRNA added were 10 ng, 100 ng, and 500 ng.

[0237]

[0238] [Experimental Example 72] Evaluation of transfection efficiency The transfection efficiency was evaluated according to the following procedure: The cells were seeded in a 96-well plate and incubated for 24 hours (37°C, 5% CO ２ To 100 μL of medium containing cells (SH-SY5Y cells: 1.4E+4 cells / well, HepG2 cells: 2.8E+4 cells / well, Jurkat E6.1 cells: 2.8E+4 cells / well) incubated at 37°C for 24 hours (37°C, 5% CO ２ (Under the atmosphere).

[0239] The adherent cells were then washed with PBS and detached by trypsin / EDTA treatment, and collected by centrifugation. The culture medium was then centrifuged to collect the floating cells. After centrifugation, each cell was washed with flow cytometry buffer and subjected to fluorescence analysis (λex = 488 nm, λem = 525 ± 30 nm) (n = 5) using a flow cytometer. The percentage of EGFP-positive cells in the gated cell population was used as the transfection efficiency, and the mean ± standard deviation was calculated. The evaluation results are shown in Table 27 and Figure 10.

[0240] [Experimental Examples 73 to 77] Instead of LNP-EGFP-1 as lipid nanoparticles, LNP-EGFP-2 produced in Example 63, LNP-EGFP-4 produced in Example 64, LNP-EGFP-SM-102 produced in Comparative Example 13, and LNP-EGFP-ALC-0315 produced in Comparative Example 14 were used. The transfection efficiency was calculated as Experimental Examples 73 to 76 in the same manner as in Experimental Example 72, except that the lipid nanoparticles of Example 9 were replaced with the dispersion produced in Production Example 1, and the amount of EGFP mRNA added per well was 100 ng. The transfection efficiency was calculated as Experimental Example 77 in the same manner as in Experimental Example 72. The results are shown in Table 27 and FIG. 10. In FIG. 10, the three bar graphs of Experimental Examples 72 to 76 show the results when, from the left, the amounts of EGFR mRNA added were 10 ng, 100 ng, and 500 ng.

[0241]

[0242] <Screening of Amino Lipids Using Apparent pKa Prediction Model> According to the apparent pKa prediction model, the amino lipids shown in Tables 28 to 30 are predicted to have excellent endosomal escape ability.

[0243]

[0244]

[0245]

Claims

1. An amino lipid having the structure shown in formula (1) below. "In formula (1), R 1 and R 2 each independently represents an alkyl group having 1 to 6 carbon atoms, or R 1 and R 2 Any carbon atoms of R may be bonded to each other to form a heterocycle containing a nitrogen atom. 3 and R 4 each independently represents an aliphatic hydrocarbon group having 1 to 35 carbon atoms which can be substituted by the substituent group α. 3 and R 4 At least one of the groups represents an aliphatic hydrocarbon group having 5 to 35 carbon atoms which can be substituted by the substituent group α. The substituent group α represents a hydroxyl group, an alkoxy group, a sulfanyl group, an alkylthio group, and an alkoxycarbonyl group.

2. The above R 1 and R 2 The amino lipid according to claim 1, wherein each of the alkyl groups is independently an alkyl group having 2 to 6 carbon atoms.

3. The above R 3 and R 4 The amino lipid according to claim 1, wherein either one or both of the above is a secondary alkyl group having 5 to 35 carbon atoms.

4. The above R 3 and R 4 2. The amino lipid according to claim 1, wherein either one or both of the above is selected from the group consisting of 1-hexylnonyl and 1-(1,3,3-trimethylbutyl)-2,6,6-trimethylheptyl.

5. The above R 3 and R 4 The amino lipid according to claim 1, wherein either one of the above is an alkenyl group having 5 to 35 carbon atoms.

6. Lipid nanoparticles containing the amino lipid according to any one of claims 1 to 5.

7. The lipid nanoparticles according to claim 6, wherein the content of the amino lipid in all lipids contained in the lipid nanoparticles is 20 to 80 mol %.

8. The lipid nanoparticle of claim 6, further comprising one or more lipids selected from the group consisting of phospholipids, PEG lipids, and sterol lipids.

9. The lipid nanoparticle of claim 6, which encapsulates a nucleic acid.

10. The lipid nanoparticle of claim 6, which is used for introducing nucleic acids into cells.

11. The lipid nanoparticles of claim 10, wherein the cells are one selected from the group consisting of stem cells, primary cells, and established cell lines.

12. A method for introducing nucleic acid into cells, comprising: Step 1: mixing the amino lipid according to any one of claims 1 to 5 with nucleic acid to obtain lipid nanoparticles encapsulating the nucleic acid; and Step 2: bringing the lipid nanoparticles obtained in Step 1 into contact with cells.

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