Lipid nanoparticles

Abstract

Provided are lipid nanoparticles that enable sufficient uptake of lipid nanoparticles into cells and efficient expression of a substance encapsulated in the lipid nanoparticles. The present invention provides lipid nanoparticles containing an amphiphilic compound, wherein the amphiphilic compound has: (i) a moiety (I) that contains a constituent unit (A) derived from a monomer (a) which has two or more hydroxyl groups in the molecules thereof, and in which, among carbon atoms in the constituent unit, the number of carbon atoms constituting the side chains is 2-10; and (ii) a hydrocarbon group having 8 or more carbon atoms.

Description

lipid nanoparticles

[0001] The present invention relates to lipid nanoparticles.

[0002] In recent years, active efforts have been made to develop preparations based on drug delivery systems (i.e., drug delivery systems (DDS)) (DDS preparations).

[0003] Lipid nanoparticles (LNPs) are attracting attention as drug carriers for DDS of nucleic acid medicines because they can deliver nucleic acids into cells. To maintain the lipid nanoparticles in the blood for a certain period of time, lipids modified with polyethylene glycol (PEG) have been used in lipid nanoparticles encapsulating nucleic acids (see, for example, Patent Document 1).

[0004] Special Publication No. 2020-532528

[0005] An object of the present invention is to provide lipid nanoparticles that can sufficiently uptake lipid nanoparticles into cells and improve the efficiency of uptake of encapsulated substances into cells.

[0006] Preferred configurations of the lipid nanoparticles of the present invention are those described in (1) to (17) below.

[0007] (1) A lipid nanoparticle comprising an amphipathic compound, the amphipathic compound having (i) a moiety (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms.

[0008] (2) The lipid nanoparticles according to (1), in which nucleic acids are encapsulated within the lipid nanoparticles.

[0009] (3) The structural unit (A) is represented by the following chemical formula (1):

[0010]

[0011] In formula (1), R １ represents a hydrogen atom or a methyl group, and X represents —C(═O)—O—, —C(═O)—NH—, —O—, or —CH２ O- or -CH ２ CH ２ The lipid nanoparticle according to (1) or (2), wherein the structural unit is represented by:

[0012] (4) The lipid nanoparticle according to (3), wherein the proportion of the structural unit (A) in the portion (I) is 80 to 100% by mass.

[0013] (5) The lipid nanoparticle according to any one of (1) to (4), wherein the modification rate (%) with the amphipathic compound, calculated by the following formula, is 0.01% or more and less than 40%: Modification rate (%) = {the amphipathic compound (mol) / total lipids contained in the lipid nanoparticle (mol)} × 100 (%).

[0014] (6) The lipid nanoparticle according to any one of (1) to (5), which contains a cationic lipid.

[0015] (7) The lipid nanoparticle according to (6), wherein the molar ratio of the cationic lipid to the total lipid present in the lipid nanoparticle is 0.5 to 75 mol%.

[0016] (8) The lipid nanoparticle according to any one of (1) to (7), which contains a phospholipid.

[0017] (9) The lipid nanoparticle according to (8), further comprising a sterol, wherein the molar ratio of the phospholipid to the sterol is phospholipid:sterol = 1:0.1 to 1:40.

[0018] (10) The lipid nanoparticle according to any one of (1) to (9), having a particle diameter of 10 to 300 nm.

[0019] (11) A pharmaceutical comprising the lipid nanoparticles according to any one of (1) to (10).

[0020] (12) A method for producing lipid nanoparticles comprising a phospholipid, a sterol, a cationic lipid, and an amphipathic compound, wherein the amphipathic compound has (i) a moiety (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms, the method comprising: mixing the phospholipid, the sterol, the cationic lipid, and the amphipathic compound with a solvent; and contacting the resulting mixture with an encapsulation substance to obtain lipid nanoparticles encapsulating the encapsulation substance.

[0021] (13) The method for producing lipid nanoparticles according to (12), wherein the encapsulated substance is a nucleic acid.

[0022] (14) The structural unit (A) is represented by the following chemical formula (1):

[0023]

[0024] In formula (1), R １ represents a hydrogen atom or a methyl group, and X represents —C(═O)—O—, —C(═O)—NH—, —O—, or —CH ２ O- or -CH ２ CH ２ The method for producing lipid nanoparticles according to (12) or (13), wherein the structural unit is represented by:

[0025] (15) The method for producing lipid nanoparticles according to (14), wherein the proportion of the structural unit (A) in the portion (I) is 80 to 100% by mass.

[0026] (16) The method for producing lipid nanoparticles according to any one of (12) to (15), wherein the molar ratio of cationic lipids to all lipids present in the lipid nanoparticles is 0.5 to 75 mol%.

[0027] (17) The method for producing lipid nanoparticles according to any one of (12) to (16), wherein the molar ratio of the phospholipid to the sterol is phospholipid:sterol = 1:0.1 to 1:40.

[0028] 1 is a diagram showing the results of cell introduction of lipid nanoparticles encapsulating mRNA. 2 is a diagram showing the results of cell introduction of lipid nanoparticles encapsulating siRNA. 3 is a diagram showing the results of encapsulation efficiency of siRNA into lipid nanoparticles. 4 is a diagram showing the nucleic acid recovery rates of Examples 21 to 42. 5 is a diagram showing the relative light output and particle size of Examples 21 to 28 compared to Comparative Example 15. 6 is a diagram showing the relative light output and particle size of Examples 21 to 28 compared to Comparative Example 15 under different pH conditions. 7 is a diagram showing the relative light output and particle size of Examples 24 and Comparative Example 15 of Examples 29 to 36. 8 is a diagram showing the relative light output and particle size of Example 24 and Comparative Example 15 of Examples 37 to 42. 9 is a diagram showing the relative light output of Example 24 compared to Comparative Example 15 in different cell types.

[0029] One aspect of the present invention is a lipid nanoparticle comprising an amphipathic compound, wherein the amphipathic compound has (i) a moiety (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms.

[0030] According to this embodiment, it is possible to improve the efficiency of intracellular uptake of encapsulated substances in lipid nanoparticles. As described above, lipid nanoparticles require modification, but when lipid nanoparticles are modified with PEG, there is a problem that the intracellular delivery efficiency is reduced compared to unmodified lipid nanoparticles (for example, see Figure 1, Comparative Example 15, and Comparative Example 8, described below). However, lipid nanoparticles modified with the amphiphilic compound of this embodiment have improved intracellular delivery efficiency compared to unmodified lipid nanoparticles (for example, see Figure 1, Comparative Example 15, and Example 8, described below). Therefore, the lipid nanoparticles of this embodiment are very useful as carriers of encapsulated substances that need to be delivered into cells to exert their medicinal effects, such as nucleic acid drugs. Furthermore, by containing cationic lipids in the lipid nanoparticles, lipid nanoparticles taken up into cells can easily escape from endosomes, facilitating the transport of the lipid nanoparticles into the cytoplasm. Furthermore, the lipid nanoparticles of this embodiment also have good storage stability.

[0031] The mechanism by which the lipid nanoparticles of this embodiment have such excellent intracellular delivery properties is unknown, but it is thought that when the amphiphilic compound and each lipid (phospholipid, sterol, and cationic lipid) of this form form lipid nanoparticles, they form a structure that is easily taken up into cells, resulting in improved efficacy of the encapsulated substance.

[0032] Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following embodiments. Furthermore, a combination of two or more of the individual preferred embodiments of the present invention described below is also a preferred embodiment of the present invention.

[0033] In this specification, the range "X to Y" means "X or more and Y or less," and "weight" and "mass" are treated as synonyms. In addition, in this specification, "(meth)acrylate" means acrylate or methacrylate, and "(meth)acrylic" means acrylic or methacrylic, and acrylate and methacrylate may be used alone or in combination. Furthermore, unless otherwise specified, operations and measurements of physical properties, etc. are performed under conditions of room temperature (20 to 25°C) and relative humidity of 40 to 50% RH.

[0034] <Lipid nanoparticles> The lipid nanoparticles of this embodiment contain an amphipathic compound. Furthermore, the lipid nanoparticles of this embodiment usually contain phospholipids as a component constituting the lipid nanoparticles. The lipid nanoparticles of this embodiment preferably contain a cationic lipid. By containing a cationic lipid, when the encapsulated substance is anionic, the cationic lipid and the encapsulated substance form a complex (aggregate), making it easier for the encapsulated substance to be packed within the lipid nanoparticles. Furthermore, by containing a cationic lipid, the lipid nanoparticles can be efficiently transferred from the endosome into the cytoplasm, making it easier for the function (pharmaceutical effect) of the encapsulated substance (particularly nucleic acid) to be exerted.

[0035] The particle diameter of the lipid nanoparticles is greater than 0 nm and equal to or less than 1000 nm. As used herein, "particle diameter" refers to the average particle diameter measured by dynamic light scattering. The particle diameter can be measured using a particle diameter measuring device that utilizes dynamic light scattering. The particle diameter of the lipid nanoparticles is preferably 10 nm or more, and from the viewpoints of improving cellular uptake, ease of sterile filtration, and improved storage stability, it is more preferably 10 to 300 nm, even more preferably 30 to 250 nm, even more preferably 30 to 200 nm, and particularly preferably 30 to 180 nm.

[0036] Each component constituting the lipid nanoparticles will be explained below.

[0037] <Amphipathic Compound> The number average molecular weight (Mn) of the amphipathic compound is preferably 1000 or more, more preferably 2000 or more, and even more preferably 3000 or more, from the viewpoint of suppressing aggregation of lipid nanoparticles in an aqueous solvent and promoting uniform uptake into cells. Furthermore, the number average molecular weight (Mn) of the amphipathic compound is preferably 90,000 or less, more preferably 30,000 or less, and even more preferably 15,000 or less, from the viewpoint of excretion from the body. The number average molecular weight (Mn) of the amphipathic compound is preferably 1,000 to 90,000, more preferably 2,000 to 30,000, and even more preferably 3,000 to 15,000. The number average molecular weight (Mn) can be determined by the measurement method described in the Examples below.

[0038] The amphiphilic compound according to this embodiment has a moiety (I) including a structural unit (A) having a predetermined structure, and a hydrocarbon group having 8 or more carbon atoms. Hereinafter, this amphiphilic compound will also be simply referred to as an "amphiphilic compound."

[0039] These components will be described in detail below.

[0040] (Moiety (I)) The amphiphilic compound according to the present invention first has a moiety (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit. In a preferred embodiment, the moiety (I) is composed of a polymer having a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit. This polymer is also referred to as a "polymer constituting moiety (I)" hereinafter. Herein, "structural unit (Q) derived from monomer (P)" (P represents any appropriate symbol, and the notation (P) may be omitted) typically means a unit in which one of the polymerizable unsaturated double bonds in "monomer (P)" (or simply "monomer") is opened by polymerization, resulting in unit (Q) (Q represents any appropriate symbol, and the notation (Q) may be omitted) constituting at least a part of the polymer. The "structural unit derived from monomer (P)" may be a structural unit formed by a different manufacturing method, as long as it has the same structure as the structural unit formed by polymerization of monomer (P) (or simply "monomer") as described above (in the specific example shown below, the structural unit represented by general formula (Q)). For example, structural unit (A) may be formed by polymerizing monomer (a) in which the hydroxyl group is protected, and then subjecting the resulting structural unit to a deprotection treatment. For example, a structural unit derived from glycerin (meth)acrylate may be formed by polymerizing (2-methyl-2-ethyl-1,3-dioxolan-4-yl)methyl (meth)acrylate or glycidyl ether (meth)acrylate, followed by hydrolysis.

[0041] The monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the constituent unit is preferably a vinyl monomer, more preferably a (meth)acrylic monomer. Furthermore, the monomer (a) may be a monofunctional monomer or a polyfunctional monomer, but preferably contains a monofunctional monomer, and more preferably consists of a monofunctional monomer.

[0042] Here, as the (meth)acrylic monomer for monomer (a), (meth)acrylates such as glycerin mono(meth)acrylate (also known as 2,3-dihydroxypropyl(meth)acrylate), 1,2-dihydroxyethyl(meth)acrylate, 2,2-dihydroxyethyl(meth)acrylate, dihydroxybutyl(meth)acrylate, trimethylolpropane mono(meth)acrylate, pentaerythritol mono(meth)acrylate, and dipentaerythritol mono(meth)acrylate are preferably used. Among these, glycerin monoacrylate (GLMA) and / or glycerin monomethacrylate (GLMMA) are preferred due to their industrial availability and high reactivity. By polymerizing these (meth)acrylic monomers (a), the ethylenic double bond contained in the monomer (a) is cleaved to produce the structural unit (A). Note that only one type of monomer (a) may be used alone, or two or more types may be used in combination.

[0043] Furthermore, among the carbon atoms of the structural unit (A) derived from monomer (a), the number of carbon atoms constituting the side chain is 2 to 10, but in this specification, the "side chain" refers to the portion other than the main chain. "The number of carbon atoms constituting the side chain is 2 to 10" refers to the (total) number of carbon atoms in the entire side chain (all (four) groups bonded to carbon atoms in the main chain). The "main chain" refers to the chain of continuously bonded carbon atoms in a polymer formed by linking structural units, that has the largest number of carbon atoms. As mentioned above, among the carbon atoms of the structural unit derived from monomer (a), the number of carbon atoms constituting the side chain is 2 to 10, but this number is preferably 3 to 8, and more preferably 4 to 6. Furthermore, the side chain possessed by the structural unit derived from monomer (a) may be an unsubstituted or substituted alkyl group having 2 to 10 carbon atoms, and the substituent may be a hydroxyl group, and the substituent may contain two or more hydroxyl groups.

[0044] Here, the structural unit (A) preferably contains a structural unit represented by the following chemical formula (1).

[0045]

[0046] In formula (1), R １ represents a hydrogen atom or a methyl group, and is preferably a methyl group. X represents -C(=O)-O-, -C(=O)-NH-, -O-, or -CH ２ O- or -CH ２ CH ２ It represents —O—, and is preferably —C(═O)—O—.

[0047] Among the constitutional units represented by the above chemical formula (1), R １ is a hydrogen atom, and X is -C(=O)-O-, it is derived from glycerin monoacrylate (GLMA) as the monomer (a). １ is a methyl group, and X is —C(═O)—O— is derived from glycerin monomethacrylate (GLMMA) as the monomer (a).

[0048] The proportion of structural units (A) derived from monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting the side chain among the carbon atoms of the structural units, in the moiety (I) constituting the amphiphilic compound is, for example, 1 to 100% by mass, preferably 20 to 100% by mass, more preferably 50 to 100% by mass, even more preferably 60 to 100% by mass, still more preferably 80 to 100% by mass, particularly preferably 90 to 100% by mass, and most preferably 100% by mass. When the proportion of structural units (A) is within the above range, the effects of the present invention can be achieved.

[0049] When the moiety (I) constituting the amphiphilic compound contains a structural unit other than the structural unit (A) (hereinafter also referred to simply as "structural unit (B)"), the structural unit (B) can be derived from any radically polymerizable monomer (hereinafter, a monomer that becomes the structural unit (B) by copolymerization is also referred to as "monomer (b)"). When the moiety (I) constituting the amphiphilic compound contains the structural unit (B), the proportion of the structural unit (B) in the moiety (I) is, for example, 99% by mass or less, preferably 80% by mass or less, more preferably 50% by mass or less, even more preferably 40% by mass or less, even more preferably 20% by mass or less, and particularly preferably 10% by mass or less. The moiety (I) constituting the amphiphilic compound does not have to contain the structural unit (B).

[0050] Examples of the monomer (b) include, other than the monomer (a), hydroxyl group-containing (meth)acrylates, polyoxyalkylene group-containing unsaturated monomers, alkoxyalkyl (meth)acrylates, vinyl monomers, cyclic compounds, etc. These monomers (b) may also be used alone or in combination of two or more.

[0051] Examples of the hydroxyl group-containing (meth)acrylate include hydroxyalkyl (meth)acrylates in which the hydroxyalkyl group has 2 to 4 carbon atoms, such as 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate.

[0052] Examples of the polyoxyalkylene group-containing unsaturated monomer include a monomer represented by the following chemical formula (2).

[0053]

[0054] In formula (2), R ２ , R ３ and R ４ each independently represents a hydrogen atom or a methyl group, R ５ represents an alkylene group having 2 to 18 carbon atoms, and R ６ represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms (preferably an alkyl group or an alkenyl group), Y represents an alkylene group having 1 to 5 carbon atoms, a —CO— group, or R２ R ４ C=CR ３ When the - group is a vinyl group, it represents a direct bond, and m represents -(R ５ O)-groups (the average number of moles of oxyalkylene groups per mole of the polyoxyalkylene group-containing unsaturated monomer), and represents a number of 1 to 300. ５ O) ｍ There are two or more types of R ５ When the group is composed of two or more R ５ O may be bonded in any of random, block or alternating forms.

[0055] Examples of polyoxyalkylene group-containing unsaturated monomers include unsaturated alcohol polyalkylene glycol adducts, polyalkylene glycol ester monomers, and (alkoxy)polyalkylene glycol monomaleic acid esters.

[0056] The unsaturated alcohol polyalkylene glycol adduct is a compound in which a polyalkylene glycol chain is added to an alcohol having an unsaturated group. Examples of the unsaturated alcohol polyalkylene glycol adduct include polyethylene glycol monovinyl ether, polyethylene glycol monoallyl ether, polyethylene glycol mono(2-methyl-2-propenyl) ether, polyethylene glycol mono(2-butenyl) ether, polyethylene glycol mono(3-methyl-3-butenyl) ether, polyethylene glycol mono(3-methyl-2-butenyl) ether, polyethylene glycol mono(2-methyl-3-butenyl) ether, polyethylene glycol mono(2-methyl-2-butenyl) ether, polyethylene glycol mono(1,1-dimethyl-2-propenyl) ether, polyethylene polypropylene glycol mono(3-methyl-3-butenyl) ether, and methoxypolyethylene glycol mono(3-methyl-3-butenyl) ether.

[0057] The polyalkylene glycol ester monomer is a monomer in which an unsaturated group and a polyalkylene glycol chain are bonded via an ester bond.

[0058] The polyalkylene glycol ester monomer is preferably, for example, an ester of an alkoxypolyalkylene glycol in which 1 to 300 moles of an oxyalkylene group having 2 to 18 carbon atoms is added to an alcohol, and (meth)acrylic acid. Among the alkoxypolyalkylene glycols, those containing an oxyethylene group as the main component are preferred. Examples of the alcohol include aliphatic alcohols having 1 to 30 carbon atoms such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, octanol, 2-ethyl-1-hexanol, nonyl alcohol, lauryl alcohol, cetyl alcohol, and stearyl alcohol; alicyclic alcohols having 3 to 30 carbon atoms such as cyclohexanol; and unsaturated alcohols having 3 to 30 carbon atoms such as (meth)allyl alcohol, 3-buten-1-ol, and 3-methyl-3-buten-1-ol. Examples of the esterified products include methoxy polyethylene glycol mono(meth)acrylate, methoxy(polyethylene glycol polypropylene glycol) mono(meth)acrylate, methoxy(polyethylene glycol polybutylene glycol) mono(meth)acrylate, methoxy(polyethylene glycol polypropylene glycol polybutylene glycol) mono(meth)acrylate, etc. Among the polyalkylene glycol ester monomers, for example, (alkoxy)polyalkylene glycol mono(meth)acrylates such as methoxy polyethylene glycol monomethacrylate are preferred.

[0059] Examples of the alkoxyalkyl (meth)acrylate include alkoxyalkyl (meth)acrylates in which the alkoxy group has 1 to 4 carbon atoms and the alkyl group has 1 to 4 carbon atoms, such as methoxymethyl (meth)acrylate, methoxyethyl (meth)acrylate, methoxypropyl (meth)acrylate, ethoxymethyl (meth)acrylate, ethoxyethyl (meth)acrylate, and ethoxypropyl (meth)acrylate. These alkoxyalkyl (meth)acrylates may be used alone or in combination of two or more.

[0060] Examples of the vinyl monomer include (meth)acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, tert-butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, n-lauryl (meth)acrylate, n-stearyl (meth)acrylate, diaminomethyl (meth)acrylate, diaminoethyl (meth)acrylate, dimethylamino (meth)acrylate, diethylamino (meth)acrylate, glycidyl (meth)acrylate, styrene, aziridines, 2-(meth)acryloyloxymethyl phosphorylcholine, 2-(meth)acryloyloxyethyl phosphorylcholine, tetrahydrofurfuryl (meth)acrylate, isopropylacrylamide, vinyl alcohol, vinylformamide, vinylisobutylacrylamide, (meth)acrylamide, dimethylacrylamide, vinylacetamide, and N-vinylpyrrolidone.

[0061] Examples of the cyclic compound include lactides such as L-lactide, lactones such as ε-caprolactone, trimethyl carbonate, cyclic amino acids, and morpholine-2,5-dione.

[0062] When the moiety (I) is composed of a polymer containing the structural unit (A) (hereinafter, also referred to as simply the polymer or the above-mentioned polymer), the polymer may have a block copolymer configuration obtained by bonding together polymers of the same type or different types.

[0063] The number-average molecular weight (Mn) of the polymer is preferably 1,000 or more, more preferably 2,000 or more, and even more preferably 3,000 or more, from the viewpoint of improving the expression level of nucleic acid through uniform dispersion of particles. Furthermore, the number-average molecular weight (Mn) of the polymer is preferably 90,000 or less, more preferably 30,000 or less, and even more preferably 15,000 or less, from the viewpoint of excretion from the body. The number-average molecular weight (Mn) of the polymer may be 1,000 to 90,000, 2,000 to 30,000, or 3,000 to 15,000. In the Examples described below, the number-average molecular weight (Mn) of the amphiphilic compound is measured, and therefore the Mn value of the portion (I) (polymer) can be calculated by subtracting the molecular weights of portions other than the portion (I) (polymer) from this measured value. If an accurate value can be calculated, the Mn value may be determined by a similar method using only the portion (I) (polymer).

[0064] The molecular weight distribution of the polymer (value of [weight average molecular weight (Mw) / number average molecular weight (Mn)]) is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, even more preferably 1 to 1.5, and even more preferably 1 to 1.3, from the viewpoint of the modifying ability of the amphiphilic compound (polymer) to the lipid nanoparticles.

[0065] The polymer constituting the portion (I) can be obtained by polymerizing a monomer composition containing the monomer (a) and, if necessary, the monomer (b). Examples of methods for polymerizing the monomer composition include living radical polymerization methods typified by radical polymerization, atom transfer radical polymerization, and reversible addition-fragmentation chain transfer (RAFT) polymerization, ionic polymerization, ring-opening polymerization, coordination polymerization, and polycondensation methods, but the present invention is not limited to these examples.

[0066] A solvent may be used when polymerizing the monomer composition. Examples of the solvent include organic solvents such as aromatic solvents such as benzene, toluene, and xylene; alcoholic solvents such as methanol, ethanol, isopropanol, n-butanol, and tert-butanol; halogen-containing solvents such as dichloroethane and dichloromethane; ether solvents such as diethyl ether, propylene glycol methyl ether, dipropylene glycol methyl ether, ethyl cellosolve, and butyl cellosolve; ester solvents such as ethyl acetate, butyl acetate, and cellosolve acetate; ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and diacetone alcohol; and amide solvents such as dimethylformamide, as well as water. Among these, alcoholic solvents and / or water are preferred from the standpoint of reactivity. These solvents may be used alone or in combination of two or more. The amount of solvent may be determined appropriately taking into consideration the polymerization conditions, the composition of the monomer composition, the concentration of the resulting polymer, and the like.

[0067] When polymerizing the monomer composition, a chain transfer agent can be used to adjust the molecular weight of the polymer or to introduce a hydrocarbon group (including a hydrocarbon group having 8 or more carbon atoms, as described below).

[0068] The amount of the chain transfer agent is not particularly limited and may be appropriately determined depending on the type of monomer contained in the monomer composition, polymerization conditions such as polymerization temperature, the target molecular weight of the polymer, and the like.

[0069] When polymerizing the monomer composition, a polymerization initiator can be used.

[0070] Examples of the polymerization initiator include a radical polymerization initiator, a living radical polymerization initiator, etc. These polymerization initiators may be used alone or in combination of two or more.

[0071] The amount of the polymerization initiator may be appropriately determined depending on the desired physical properties of the resulting polymer.

[0072] The polymerization conditions for polymerizing the monomer composition may be appropriately set depending on the polymerization method, and are not particularly limited.

[0073] The polymer constituting the moiety (I) can be obtained by polymerizing the monomer composition as described above. The resulting polymer may have a functional group at its terminal. If the polymer constituting the moiety (I) has a functional group at its terminal, it can easily modify a drug or link to a specific hydrocarbon group (described later) via the functional group. However, the polymer constituting the moiety (I) does not need to have a functional group at its terminal. The amphiphilic compound according to the present invention has a hydrocarbon group having 8 or more carbon atoms and the moiety (I) (including a polymer) in the molecule. When the polymer constituting the moiety (I) has a functional group at its terminal, the functional group may be present at only one terminal of the polymer, or may be present at both terminals. Furthermore, the functional group present at the terminal of the polymer may be located on the side where the moiety (I) is bonded to the specific hydrocarbon group (described later), or on the opposite side.

[0074] The functional group that the polymer constituting the portion (I) may have is preferably an anionic functional group, a cationic functional group, a nonionic functional group, or an amphoteric functional group. The functional group is preferably a reactive functional group. Suitable reactive functional groups include an -SH group, a group represented by the formula: -COOM (M represents a hydrogen atom or an alkali metal atom), a hydroxyl group, an allyl group, an epoxy group, an aldehyde group, and an -NH ２ group, CONH- group, etc. Examples of M include alkali metal atoms such as sodium atom and potassium atom. When the polymer has functional groups at its terminals, the number of functional groups is not particularly limited, but is preferably 1 to 6, more preferably 1 to 4, and even more preferably 1 to 2.

[0075] In order to introduce a functional group into the terminal of the polymer constituting the moiety (I), a functional group-containing compound for introducing a functional group into the polymer can be used. Examples of the functional group-containing compound for introducing a functional group into the terminal of the polymer include alkali metal thioacetate salts such as sodium thioacetate and potassium thioacetate, thiol-based chain transfer agents such as cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and sodium salts and potassium salts thereof; RAFT agents such as 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid; 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-methyl-N-[1,1-bis(hydroxymethyl)-2-hydroxymethyl]-2-propanol; Examples of the polymerization initiator include a polymerization initiator having a functional group introduced therein, such as 2,2'-azobis[N-(2-hydroxyethyl)-2-methoxypropanamide], 2,2'-azobis(2-methyl-2-propenylpropanamide), 2,2'-bis(2-imidazolin-2-yl)[2,2'-azobispropane]dihydrochloride, 2,2'-azobis(propane-2-carboxamidine)dihydrochloride, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine], 2,2'-azobis[2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane dihydrochloride, cyclohexanone peroxide, and acetylacetone peroxide. These functional group-containing compounds may be used alone or in combination of two or more. The functional group-containing compounds described above include those corresponding to the chain transfer agents and polymerization initiators described above. The functional group-containing compounds corresponding to the chain transfer agents and polymerization initiators may be used for only one of the purposes of the chain transfer agent or polymerization initiator and the functional group-containing compound, or may be used for both purposes.

[0076] Furthermore, when a living polymerization initiator is used as the polymerization initiator, a functional group may be introduced at the end of a polymer prepared using the living polymerization initiator by reacting a functional group-containing compound with a halogen atom present at the end of the polymer. Examples of functional group-containing compounds that can react with such halogen atoms to introduce a functional group at the end of the polymer include amine compounds such as ethylenediamine and propyldiamine, dithiol compounds such as ethanedithiol, propanedithiol, and hexadecanedithiol, allyl mercaptan, and thiol compounds such as cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and sodium and potassium salts thereof.

[0077] The amount of the functional group-containing compound for introducing a functional group into the terminal of the polymer is not particularly limited and may be appropriately set depending on the types of monomers (structural units) constituting the polymer, polymerization conditions such as polymerization temperature, the targeted molecular weight of the polymer, etc. When obtaining a polymer having a number average molecular weight of several thousand to several tens of thousands, the amount of the chain transfer agent is preferably 0.1 to 20 parts by mass, and more preferably 0.5 to 15 parts by mass, per 100 parts by mass of the monomer.

[0078] Examples of methods for introducing a functional group into the terminal of the polymer constituting the moiety (I) include: (1) a method of obtaining a polymer by polymerizing a monomer composition in the presence of a polymerization initiator into which the functional group has been introduced; (2) a method of obtaining a polymer by polymerizing a monomer composition in the presence of a chain transfer agent into which the functional group has been introduced; and (3) a method of reacting a halogen atom present at the terminal of the polymer with a functional group-containing compound, but the present invention is not limited to these examples.

[0079] (Hydrocarbon Group) The amphiphilic compound according to the present invention is characterized in that it also has a hydrocarbon group having 8 or more carbon atoms, in addition to the above-mentioned "moiety (I) including a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule." The specific structure of the hydrocarbon group is not particularly limited.

[0080] As an example, the hydrocarbon group is preferably a group contained in an organic compound that has the ability to form molecular aggregates through hydrophobic interactions in an aqueous solution. Examples of such organic compounds include hydrocarbons, hydrophobic polymers, lipids, and other organic molecules. Preferably, the organic compound is a hydrocarbon and / or a lipid, more preferably a hydrocarbon.

[0081] Examples of the hydrocarbon include aliphatic hydrocarbons having 8 to 50 carbon atoms and aromatic hydrocarbons having 8 to 50 carbon atoms. That is, in a preferred embodiment of the present invention, the hydrocarbon group has 8 to 50 carbon atoms, more preferably 8 to 40, even more preferably 8 to 30, and particularly preferably 8 to 20 carbon atoms. Preferably, the hydrocarbon is an aliphatic hydrocarbon having 8 to 50 carbon atoms (the hydrocarbon group is an aliphatic hydrocarbon group having 8 to 50 carbon atoms).

[0082] Examples of aliphatic hydrocarbons having 8 to 50 carbon atoms include linear alkanes such as octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and icosane, branched alkanes thereof, and cyclic alkanes thereof, with linear alkanes being preferred.

[0083] Examples of aromatic hydrocarbons having 8 to 50 carbon atoms include 2-phenylethane, 1,3,5-trimethylbenzene, naphthalene, anthracene, fluorescein, and positional isomers thereof.

[0084] Examples of the hydrophobic polymer include polymers obtained by polymerizing, as a main component, vinyl monomers that can be the constituent units of the above-mentioned moiety (I) and have a hydrocarbon group having 8 or more carbon atoms in the side chain.

[0085] "Lipid" refers to an organic compound that has a hydrophobic moiety (e.g., a long-chain fatty acid, a hydrocarbon chain, or a sterol group), is poorly soluble in water, and readily dissolves in organic solvents. Lipids usually have hydrophilic groups such as hydroxyl groups, carboxylic acids (carboxyl groups), and amino groups. In this case, the hydrophobic moiety preferably has 8 or more carbon atoms. Therefore, the amphipathic compounds of the present invention are also included in the category of lipids. "Lipids" can be further broadly classified into phospholipids, glycolipids (glyceroglycolipids and sphingoglycolipids), sphingolipids, sterols, neutral lipids, saturated or unsaturated fatty acids, etc.

[0086] Phospholipids are broadly classified into glycerophospholipids and sphingophospholipids. Representative glycerophospholipids include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidic acid (PA). On the other hand, a representative sphingophospholipid is sphingomyelin. Specific examples of phospholipids include the lipids listed in (a) to (g) below.

[0087] (a) Phosphatidylcholines Specific examples of phosphatidylcholines include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), didecanoylphosphatidylcholine (DDPC), dioctanoylphosphatidylcholine, dihexanoylphosphatidylcholine (DHPC), dibutyrylphosphatidylcholine (DBPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), didecanoylphosphatidylcholine (DDPC), dioctanoylphosphatidylcholine, dihexanoylphosphatidylcholine (DHPC), dibutyrylphosphatidylcholine (DBPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DL ... Lydoylphosphatidylcholine, Dilinoleoylphosphatidylcholine, Diarachidonoylphosphatidylcholine, Diicosenoylphosphatidylcholine (DEPC), Diheptanoylphosphatidylcholine, Dicaproylphosphatidylcholine, Diheptadecanoylphosphatidylcholine, Dibehenoylphosphatidylcholine, Eleostearoylphosphatidylcholine, Hydrogenated Egg Phosphatidylcholine (HEPC), Hydrogenated Soy Phosphatidylcholine (HSPC), 1-Palmitoyl-2-arachidyl 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 include tearoylphosphatidylcholine, 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.

[0088] (b) Phosphatidylserines Specific examples of phosphatidylserines include distearoylphosphatidylserine (DSPS), dimyristoylphosphatidylserine (DMPS), dilauroylphosphatidylserine (DLPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), lysophosphatidylserine, eleostearoylphosphatidylserine, and 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidylserine.

[0089] (c) Phosphatidylinositols Specific examples of phosphatidylinositols include dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), and dilauroylphosphatidylinositol (DLPI).

[0090] (d) Phosphatidylglycerols Specific examples of phosphatidylglycerols include dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), lysophosphatidylglycerol, hydrogenated soybean phosphatidylglycerol (HSPG), hydrogenated egg phosphatidylglycerol (HEPG), and cardiolipin (diphosphatidylglycerol).

[0091] (e) Phosphatidylethanolamines (cephalins) Specific examples of phosphatidylethanolamines (cephalins) include dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), didecanoylphosphatidylethanolamine (DDPE), N-glutarylphosphatidylethanolamine (NGPE), lysophosphatidylethanolamine, N-(7-nitro-2,1,3-benzoxydiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine, eleostearoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine, and 1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine. In Production Example 5 described below, distearoyl N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (COATSOME (registered trademark) FE-8080MA3, manufactured by NOF Corporation), a distearoylphosphatidylethanolamine (DSPE) derivative having a maleimide group, is used as the lipid raw material. The maleimide group can react with a thiol group that can be introduced at the end of the polymer that constitutes moiety (I), thereby bonding to the polymer that constitutes moiety (I). In this way, phosphatidylethanolamines (and other lipid derivatives) that have a functional group such as a maleimide group or succinimide group for linking to moiety (I), together with a hydrocarbon group having various numbers of carbon atoms of 8 or more, can also be used in a similar manner.

[0092] (f) Phosphatidic Acids Specific examples of phosphatidic acids include dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA), dimyristoylphosphatidic acid (DMPA), and dioleylphosphatidic acid (DOPA).

[0093] (g) Sphingophospholipids Specific examples of sphingophospholipids include sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, ceramide ciliatin, ceramide phosphorylethanolamine, and ceramide phosphorylglycerol.

[0094] The most representative sterol is cholesterol. In addition to cholesterol, other sterols include, for example, cholesterol succinate, dihydrocholesterol, lanosterol, dihydrolanosterol, desmosterol, stigmasterol, sitosterol, campesterol, brassicasterol, zymosterol, ergosterol, campesterol, fucosterol, 22-ketosterol, 20-hydroxycholesterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, epicholesterol, Examples include dehydroergosterol, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, 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.

[0095] Neutral lipids include, for example, diglycerides (e.g., diolein, dipalmitolein) and mixed caprylin-caprin diglycerides, triacylglycerols (e.g., triolein, tripalmitolein, trimyristolein, trilaurin, tricaprin, tricaprylin, tricaproin, etc.), squalene, and tocopherol.

[0096] The saturated fatty acids and unsaturated fatty acids include saturated or unsaturated fatty acids having 5 to 30 carbon atoms.

[0097] Other organic molecules (whether natural compounds or chemically synthesized compounds) can also be used as a source of hydrocarbon groups having 8 or more carbon atoms in the amphiphilic compound according to the present invention, provided that they have such hydrocarbon groups.

[0098] In a preferred embodiment of the present invention, the hydrocarbon group having 8 or more carbon atoms (preferably 8 to 50 carbon atoms) is preferably bonded to the moiety (I) containing the structural unit (A) directly or via a divalent linking group, or is present as part of a lipid (e.g., a phospholipid) that is bonded to the moiety (I) containing the structural unit (A) directly or via a divalent linking group.

[0099] Like the polymer constituting moiety (I), the hydrocarbon group having 8 or more carbon atoms may also have a functional group at its terminal. When the hydrocarbon group having 8 or more carbon atoms has a functional group at its terminal, it becomes possible to modify a medicine or the like via the functional group. However, the hydrocarbon group having 8 or more carbon atoms does not have to have a functional group at its terminal. When the hydrocarbon group having 8 or more carbon atoms has a functional group at its terminal, the functional group may be present at only one terminal of the hydrocarbon group having 8 or more carbon atoms, or may be present at both terminals. Furthermore, the functional group present at the terminal of the hydrocarbon group having 8 or more carbon atoms may be located on the side where the hydrocarbon group having 8 or more carbon atoms is bonded to moiety (I), or on the opposite side.

[0100] From the viewpoint of excretion from the body and the like, the molecular weight of the hydrocarbon group having 8 or more carbon atoms is preferably 100 to 5000, more preferably 150 to 2000, and even more preferably 150 to 1000. Here, the proportion of the molecular weight of the "hydrocarbon group having 8 or more carbon atoms" in the molecular weight of the amphiphilic compound is preferably 0.2 to 20%, more preferably 0.5 to 15%, and even more preferably 1 to 10%.

[0101] Furthermore, in the present invention, when a hydrocarbon group having 8 or more carbon atoms is present in an amphipathic compound as part of a lipid, the molecular weight of the lipid is preferably 100 to 2000, more preferably 150 to 1000. Here, when a hydrocarbon group having 8 or more carbon atoms is present in an amphipathic compound as part of a lipid, the proportion of the molecular weight of the lipid in the molecular weight of the amphipathic compound is preferably 2 to 50%, more preferably 5 to 35%, and even more preferably 10 to 20%.

[0102] The divalent linking group is a group that links the moiety (I) with a hydrocarbon group having 8 or more carbon atoms, and its structure is not particularly limited, and it may be included as part of the structure of the moiety (I). Specific examples of such divalent linking groups include -S-, -S-C(=S)-, -S-C(=S)-S-, and -S-C(=S)-N(-R ａ )-, -S-C(=S)-O-, -S-R ｂ -C(=O)-O-, -SR ｂ -C(=O)-N(-R ａ ) -, -S-R ｂ -O-, -S-R ｂ -O-C(=O)-, -O-, -O-C(=O)-, -N(-R ａ )-C(=O)- and divalent linking groups containing these. ａ is a hydrogen atom or a hydrocarbon group having 1 to 30 carbon atoms. ｂ is a hydrocarbon group having 1 to 30 carbon atoms. A residue obtained by removing one hydrogen atom and one hydrocarbon group having 8 or more carbon atoms from a lipid (including modified lipids) is also one preferred form of the divalent linking group. The divalent linking group preferably has a molecular weight of 5,000 or less, more preferably 2,000 or less, and even more preferably 1,000 or less.

[0103] Here, examples of a method for linking a hydrocarbon group having 8 or more carbon atoms to moiety (I) (the polymer constituting moiety (I)) via a divalent linking group include a method in which a monomer composition is polymerized in the presence of a chain transfer agent and / or a polymerization initiator to obtain the polymer constituting moiety (I), and the chain transfer agent or polymerization initiator contains, in the molecule, a structure capable of constituting a moiety containing a hydrocarbon group having 8 or more carbon atoms. In particular, by polymerizing the monomer composition using a chain transfer agent (such as octanethiol, decanethiol, dodecanethiol, hexadecanethiol, or octadecanethiol) having a thiol group (—SH group) bonded to a hydrocarbon group having 8 or more carbon atoms (preferably 8 to 50 carbon atoms, more preferably 8 to 40 carbon atoms, even more preferably 8 to 30 carbon atoms, and particularly preferably 8 to 20 carbon atoms), it is possible to obtain an amphiphilic compound in which the hydrocarbon group having 8 or more carbon atoms is bonded to moiety (I) via a thioether bond (—S—) (see Production Examples 1 to 4 described below). In a preferred embodiment of the present invention, the amphiphilic compound is obtained by polymerizing a monomer composition containing a vinyl monomer (preferably a meth(acrylic monomer, more preferably glycerin mono(meth)acrylate) and a chain transfer agent having a thiol group (—SH group) bonded to a hydrocarbon group having 8 or more carbon atoms.

[0104] Another method for linking a hydrocarbon group having 8 or more carbon atoms to moiety (I) (the polymer constituting moiety (I)) via a divalent linking group involves first polymerizing a monomer composition in the presence of a chain transfer agent to obtain the polymer constituting moiety (I), and then performing RAFT polymerization using a reversible addition-fragmentation chain transfer (RAFT) agent as the chain transfer agent to introduce a thiol group (—SH group) into the end of the polymer constituting moiety (I). Subsequently, a compound having a hydrocarbon group having 8 or more carbon atoms and a functional group (e.g., a maleimide group) capable of reacting with the thiol group (—SH group) is reacted with the thiol group (—SH group). This allows the hydrocarbon group having 8 or more carbon atoms to be linked to moiety (I) via a thioether bond (—S—) (see Production Example 5 described below). This method is particularly useful when the hydrocarbon group having 8 or more carbon atoms is present in an amphiphilic compound as part of a lipid or other organic molecule.

[0105] In the amphiphilic compound according to the present invention, the polymer constituting the moiety (I), or the polymer in the case where a hydrocarbon group having 8 or more carbon atoms is present in the compound as part of the polymer, may have a crosslinked structure.

[0106] <Modification rate with amphipathic compound> In one embodiment, the modification rate (% (mol)) of the lipid nanoparticle with the amphipathic compound is 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, or 1% or more. In one embodiment, the modification rate (%) of the lipid nanoparticle with the amphipathic compound is less than 40%, 30% or less, particularly 20% or less, 18% or less, 16% or less, 15% or less, 12% or less, or 10% or less. In one embodiment, the modification rate (%) of the lipid nanoparticle with the amphipathic compound may be 5% or less (e.g., 0.005 to 5%, or 0.01 to 5%), or less than 5% (e.g., 0.005% or more but less than 5%, or 0.01% or more but less than 5%). In one embodiment, the modification rate (% (mol)) of the lipid nanoparticles with the amphipathic compound is 0.005% or more and less than 40%, 0.01% or more and less than 40%, 0.05% or more and less than 40%, 0.1 to 30%, 0.1 to 20%, 0.2 to 18%, 0.3 to 16%, 0.4 to 15%, 0.5 to 12%, 0.6 to 10%, 0.7 to 10%, 0.8 to 10%, 1 to 10%, 0.005 to 5%, or 0.005% or more and less than 5%. When the modification rate is equal to or greater than the above lower limit, the effect of improving lipid nanoparticle introduction into cells by the amphipathic compound is more easily obtained. Furthermore, when the modification rate is equal to or greater than the above lower limit, the encapsulation efficiency of the encapsulated substance (e.g., nucleic acid) is improved. Furthermore, the blood retention of the lipid nanoparticles is improved. When the modification rate is below the upper limit, the particle stability of the lipid nanoparticles is improved, the amphiphilic compound is more likely to improve the introduction of lipid nanoparticles into cells, and the function is more likely to be exerted (for example, when the encapsulated substance is a nucleic acid, high expression efficiency is more likely to be obtained).

[0107] The modification rate of the lipid nanoparticles with the amphipathic compound can be calculated by the following formula: Modification rate (%) = {amphipathic compound (mol) / total (mol) of each lipid contained in the lipid nanoparticle} × 100 (%).

[0108] Furthermore, the total amount of each lipid contained in the lipid nanoparticle is, for example, the total amount of phospholipids, cationic lipids, amphipathic compounds, and sterols. Therefore, in a preferred embodiment of the present invention, the modification rate (A) (%) shown below is 0.01% or more and less than 40%. In one embodiment, the modification rate (A) (% (mol%)) may be 0.005% or more and less than 40%, 0.01% or more and less than 40%, 0.05% or more and less than 40%, 0.1 to 30%, 0.1 to 20%, 0.2 to 18%, 0.3 to 16%, 0.4 to 15%, 0.5 to 12%, 0.6 to 10%, 0.7 to 10%, 0.8 to 10%, 1 to 10%, 0.005 to 5%, or 0.005% or more and less than 5%.

[0109] Modification rate (A) (%) = {amphiphilic compound (mol) / total (mol) of phospholipid, cationic lipid, amphiphilic compound, and sterol contained in lipid nanoparticle} × 100 (%).

[0110] In this specification, the amount of amphipathic compound and the total amount of lipid in the lipid nanoparticles correspond to the amount of each lipid added during the manufacturing process, and the amount of amphipathic compound and the total amount of lipid can be measured by extracting the lipid nanoparticles using an organic solvent (e.g., ethanol or methanol) or a surfactant (e.g., Triton (registered trademark) X100 or Tween 80) and then using HPLC.

[0111] <Phospholipids> Examples of phospholipids include the lipids described in the section on amphipathic compounds above.

[0112] The phospholipids used in the lipid nanoparticles may be used alone or in combination of two or more.

[0113] From the viewpoint of particle formability, the phospholipids used in the lipid nanoparticles preferably include phosphatidylcholines and phosphatidylethanolamines.

[0114] Specific examples of phosphatidylcholines include those listed above. The phosphatidylcholines may be used alone or in combination of two or more.

[0115] From the viewpoint of particle formability, the phospholipids used in the lipid nanoparticles preferably contain phosphatidylcholines, and preferably contain hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristylphosphatidylcholine (DMPC), or a mixture thereof, more preferably contain distearoylphosphatidylcholine (DSPC), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), or a mixture thereof, even more preferably contain distearoylphosphatidylcholine (DSPC) and / or hydrogenated soybean phosphatidylcholine (HSPC), and may consist solely of distearoylphosphatidylcholine (DSPC) and / or hydrogenated soybean phosphatidylcholine (HSPC).

[0116] In addition, it is also preferable to use a phospholipid having an unsaturated structure, since this allows the function of the encapsulated substance to be easily exerted (for example, when the encapsulated substance is a nucleic acid, high expression efficiency is likely to be obtained). Examples of phospholipids having an unsaturated structure include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), and dioleylphosphatidic acid (DOPA). One type of phospholipid used in lipid nanoparticles is a combination of phosphatidylcholines and phospholipids having an unsaturated structure, preferably distearoylphosphatidylcholine (DSPC), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), or a mixture thereof, with a phospholipid having an unsaturated structure, and more preferably distearoylphosphatidylcholine (DSPC) and / or hydrogenated soybean phosphatidylcholine (HSPC) with dioleoylphosphatidylethanolamine (DOPE). When a combination of phosphatidylcholines (excluding phospholipids having an unsaturated structure) and a phospholipid having an unsaturated structure is used, the mass ratio of the phosphatidylcholines (excluding phospholipids having an unsaturated structure) to the phospholipid having an unsaturated structure is, for example, 1:0.05-50 (molar ratio), or may be 1:0.1-10 (molar ratio).

[0117] The ratio (mol%) of phospholipids to the total lipids present in the lipid nanoparticles is, for example, 1 to 80 mol%, 2 to 70 mol%, or 2 to 60 mol%, and in one embodiment, 3 to 30 mol%, 5 to 20 mol%, or 8 to 18 mol%. Only one type of the above phospholipids may be used, or two or more types may be used in combination. When multiple phospholipids are used, it is preferable that the phospholipids as a whole have the above ratio. Furthermore, the total lipids preferably refer to the sum of phospholipids, cationic lipids, amphipathic compounds, and sterols.

[0118] <Cationic lipid> When the substance encapsulated in the lipid nanoparticle is a negatively charged substance (e.g., nucleic acid, etc.), combining it with a cationic lipid makes it possible to encapsulate the substance when the dispersion medium of the lipid nanoparticle is hydrophilic, and also makes it possible to reduce the particle size of the resulting lipid nanoparticle.

[0119] Cationic lipids are roughly divided into a hydrophobic portion and a hydrophilic portion. The hydrophobic portion contains a hydrophobic group such as a fatty acid group or a sterol group, while the hydrophilic portion contains a cationic group such as an amino group. Known cationic lipid compositions include those containing one hydrophobic group per hydrophilic group (hereinafter referred to as "single-chain cationic lipid") and those containing two hydrophobic groups per hydrophilic group (hereinafter referred to as "double-chain cationic lipid").

[0120] Examples of cationic lipids include, but are not limited to, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), (4-hydroxybutyl)azanediyl)di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate, (9Z,12Z) -3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecan-9,12-dienoate (LP01), di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-L-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3β-N-(N',N',dimethyl-aminoethane)-carbamoylcholesterol (DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium nium (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminoum trifluoroacetate (DOSPA), 1,2-distearoyl-3-trimethylammonium propane (DSTAP), 9,12-octadecadienoic acid, (9Z,12Z)-1,1',1',1'-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilo-di-4,1-butanediyl)] ester (OF-C4-Deg-Lin), 1,1'-[[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (CKK-E12), bis(2-(octyldisulfanyl)ethyl)3,3'-((4-methyl-11-oxo) -12-oxa-15,16-dithia-4,8-diazatetracosyl)azanediyl)dipropionate (306-O12B-3), tetrakis(2-(octyldisulfanyl)ethyl)3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetril))tetrapropionate (306-O12B), 2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2-(((Z)-tetradec-9-enoyl)oxy)methyl)propane-1,3-diyl (9Z ,9'Z)-bis(tetradec-9-enoate (TCL053), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5-piperazinedione (OF-02), 1-methyl-4,4-bis[(9Z,12Z)-9,12-octadecadien-1-yloxy]-piperazine (YSK05), N1,N16-didodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13-tetraazahexadecane diamide (98N12-5), alkyl-modified polyamidoamine (PAMAM) dendrimer (G0-C14), N1,N3,N5-tris[3-(didodecylamino)propyl]-1,3,5-benzenetricarbonamide (TT3), 1,2-dimyristoyl-3-trimethylammonium propane chloride (DMTAP), 1-[2-[bis(2-hydroxytridecyl)amino]ethoxy]ethylamino]tridecan-2-ol (C13-112-tri-tail), bis(2-(tetradecylthio)ethyl)-3,3'-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (93-O17S), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-({8-[(3β)-co- (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), stearamidopropyl dimethylamine; 1,2-dioleoyl-3-trimethylammonium propane chloride (DOTAP chloride), 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), dioctadecyldimethylammonium chloride (DODAC), dioctadecyldimethylammonium chloride (DDAB), stearyltrimethylammonium chloride, etc. These cationic lipids may be used alone or in combination of two or more. Among them, the cationic lipid is preferably a tertiary amine, and more preferably a compound having no unsaturated bond in the molecule and having multiple ester bonds, such as heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) and / or (4-hydroxybutyl)azanediyl)di(hexane-6,Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) is even more preferred.

[0121] The cationic lipid is preferably a cationic lipid having two or more hydrophobic moieties each having 8 or more carbon atoms (double-chain cationic lipid).

[0122] Furthermore, cationic lipids are, for example, neutral in charge at physiological pH (around pH 7.4) and protonated (up to H) in the acidic range (for example, pH 6.5 or less). ＋ ) lipid molecules (ionized lipids). Such ionized lipids are usually tertiary amines. The cationic lipid being an ionic lipid is a preferred embodiment in terms of the ease with which an anionic encapsulated substance (especially nucleic acid) can be functionalized and the retention in blood. Using such compounds, uptake into cells can also be controlled.

[0123] These cationic lipids may be used alone or in combination of two or more.

[0124] In one embodiment, the molar ratio of the cationic lipid to the total lipids present in the lipid nanoparticles (preferably the sum of phospholipids, cationic lipids, amphipathic compounds, and sterols) is 0.5 to 80 mol%, 0.5 to 75 mol%, 5 to 75 mol%, 5 to 70 mol%, or 10 to 70 mol%. In one embodiment, the molar ratio of the cationic lipid to the total lipids present in the lipid nanoparticles (preferably the sum of phospholipids, cationic lipids, amphipathic compounds, and sterols) may be 10 to 80 mol%, 15 to 80 mol%, 20 to 80 mol%, 25 to 75 mol%, or 40 to 75 mol%. When the cationic lipid content is above the above-mentioned lower limit, migration from the endosome to the cytoplasm is facilitated, and the encapsulation efficiency of the anionic encapsulated substance (particularly nucleic acid) is improved and / or the function is easily expressed. When the content is below the above-mentioned upper limit, particle formation is facilitated.

[0125] <Sterol> The lipid nanoparticles of this embodiment preferably contain a sterol.

[0126] Sterols include cholesterol, cholesterol succinate, dihydrocholesterol, lanosterol, dihydrolanosterol, desmosterol, stigmasterol, sitosterol, campesterol, brassicasterol, zymosterol, ergosterol, campesterol, fucosterol, 22-ketosterol, 20-hydroxycholesterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, epicholesterol, and dehydrocholesterol. Examples of sterols include droergosterol, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol hemisuccinate, 3βN-(N',N'-dimethylaminoethane)-carbamoylcholesterol, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, coprostanol, cholesterol ester, cholesteryl phosphorylcholine, and 3,6,9-trioxaoctan-1-ol-cholesteryl-3e-ol. One type of sterol may be used alone, or two or more types may be used in combination. Of these, cholesterol is preferred.

[0127] In one embodiment, the content of sterol relative to the total lipids (preferably the sum of phospholipids, cationic lipids, amphipathic compounds, and sterols) is 5 to 80 mol%, 10 to 75 mol%, 15 to 70 mol%, or 20 to 65 mol%. When the sterol content is within the above range, lipid nanoparticles can be easily formed and the particle size can be appropriate.

[0128] In one embodiment, the lipid nanoparticles contain phospholipids and sterols, and the molar ratio of the phospholipids to the sterols is phospholipid:sterol = 1:0.1 to 1:40, 1:1 to 1:10, or 1:2 to 1:5. Such a molar ratio facilitates the formation of lipid nanoparticles having an appropriate particle size. In one embodiment, the lipid nanoparticles contain phospholipids and sterols, and the molar ratio of the phospholipids to the sterols is phospholipid:sterol = 1:0.05 to 1:40, 1:0.1 to 1:40, 1:0.15 to 1:15, 1:0.15 to 1:5, 1:0.15 or greater but less than 1:1, or 1:0.5 or greater but less than 1:1. Such a molar ratio improves intracellular uptake efficiency and facilitates high expression efficiency, particularly when the encapsulated substance is a nucleic acid.

[0129] In one embodiment, the modification rate (%) of the lipid nanoparticles with the amphipathic compound is 0.005% or more and less than 40%, 0.005 to 30%, 0.005 to 20%, 0.05 to 15%, 0.05 to 10%, or 0.005 to 5% (0.01 to 5%, 0.005% or more and less than 5%, or 0.01% or more and less than 5%), and the molar ratio of phospholipid to sterol is phospholipid:sterol=1:0.1 to 1:40, 1:0.15 to 1:15, 1:0.15 to 1:5, or 1:0.15 or more and less than 1.

[0130] Furthermore, in one embodiment, the modification rate (%) of the lipid nanoparticles with the amphiphilic compound is 0.005% or more and less than 40%, 0.005 to 30%, 0.005 to 20%, 0.05 to 15%, 0.05 to 10%, or 0.005 to 5% (0.01 to 5%, 0.005% or more and less than 5%, or 0.01% or more and less than 5%), and the molar ratio of the phospholipid to the sterol is phospholipid:sterol=1:0.05 to 1:40, 1: The molar ratio of the cationic lipid to the total lipids present in the lipid nanoparticles (preferably the total of phospholipids, cationic lipids, amphipathic compounds, and sterols) is 10 to 80 mol%, 15 to 80 mol%, 20 to 80 mol%, 25 to 75 mol%, or 40 to 75 mol%. In this configuration, the efficiency of uptake into cells is improved, and high expression efficiency is likely to be obtained, particularly when the encapsulated substance is a nucleic acid.

[0131] <Encapsulated substance encapsulated inside lipid nanoparticles> The encapsulated substance encapsulated inside lipid nanoparticles is not particularly limited, but is preferably an anionic compound because it forms a complex with the cationic lipid and is easily introduced into cells. Examples of anionic compounds include nucleic acids; pharmaceuticals such as benzalkonium chloride, bethanechol chloride, and pyridostigmine bromide; magnesium ascorbyl phosphate; and sodium ascorbyl phosphate.

[0132] The amount of nucleic acid encapsulated in the lipid nanoparticle is, for example, 0.01 to 20% by mass, or alternatively 0.05 to 15% by mass, or alternatively 0.1 to 10% by mass.

[0133] Among these, nucleic acids are preferred as the encapsulated substance because they are more likely to exert their effects. That is, one aspect of the present invention is lipid nanoparticles that contain the above-mentioned amphiphilic compound and encapsulate nucleic acids. Examples of nucleic acids include DNA, mRNA, siRNA, microRNA, gapmers, ribozymes, aptamers, artificial nucleic acids, CpG oligodeoxynucleotides, and antisense DNA / RNA.

[0134] When the encapsulated substance is a nucleic acid, in one embodiment, the total number of amine groups (N) of the cationic lipid relative to the total number of nucleic acid phosphate groups (P) (hereinafter also referred to simply as the N / P ratio) is 0.01 to 55, 0.01 to 50, 0.03 to 50, 0.05 to 25, 1 to 25, 4 to 25, or 4 to 10. When the encapsulated substance is a nucleic acid, in one embodiment, the total number of amine groups (N) of the cationic lipid relative to the total number of nucleic acid phosphate groups (P) (hereinafter also referred to simply as the N / P ratio) is 0.01 to 55, 0.03 to 55, 0.05 to 55, 1 to 55, 2 to 55, 4 to 55, 10 to 55, or 20 to 55. Such an N / P ratio is preferable because it allows the cationic lipid nanoparticles and nucleic acid to efficiently form a complex, thereby improving the encapsulation efficiency of the encapsulated substance.

[0135] When the encapsulated substance is a nucleic acid, in one embodiment, the molar ratio of cationic lipid to nucleic acid is, for example, 2,000 to 200,000, and may be 5,000 to 100,000.

[0136] <Other Components> The lipid nanoparticles may contain other components. Examples of other components include water, saline, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, carboxymethylcellulose sodium salt, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, phosphate-buffered saline, biodegradable polymers, serum-free media, surfactants, oils and fats, pH adjusters, and physiological pH buffer solutions acceptable in vivo. These additives may be used alone or in combination of two or more.

[0137] <Method for producing lipid nanoparticles> The method for producing lipid nanoparticles is not particularly limited, and conventionally known methods can be used. Specifically, when the encapsulated substance is nucleic acid, a method can be used in which a lipid solution is obtained by dissolving a phospholipid, a cationic lipid, a sterol, and an amphipathic compound in an organic solvent, and the lipid solution is mixed with a nucleic acid solution in which nucleic acid is dissolved in an aqueous solvent to obtain a dispersion of lipid nanoparticles.

[0138] Examples of aqueous solvents include water and buffer solutions. Examples of buffer solutions include phosphate buffered saline (PBS), acetate buffer solution, citrate buffer solution, borate buffer solution, tartrate buffer solution, Tris buffer solution, MES buffer solution, and HEPES buffer solution. The aqueous solvents may be used alone or in combination of two or more. In particular, when the encapsulated substance is a nucleic acid, the aqueous solvent is preferably a buffer solution.

[0139] The nucleic acid concentration in the aqueous solvent is, for example, 0.1 to 10,000 μg / mL, or may be 0.5 to 1,000 μg / mL, or may be 0.5 to 100 μg / mL. The pH of the aqueous solvent is preferably 3.5 to 4.5. When the aqueous solvent is added, the temperature of the aqueous solvent is preferably 20 to 45° C., and preferably 25 to 40° C.

[0140] The organic solvent is preferably a polar organic solvent, such as ethanol, isopropanol, butanol, chloroform, etc. The organic solvent may be used alone or in combination of two or more. Among them, ethanol is preferably used from the viewpoint of particle formability.

[0141] The lipid concentration in the organic solvent is, for example, 0.01 to 100 mg / mL, may be 0.05 to 10 mg / mL, or may be 0.05 to 1 mg / mL.

[0142] The lipid solution and the nucleic acid solution can be mixed using a microfluidic mixing system (e.g., Asia microfluidic system (Syrris) or Nanoassemblr (Precision Nanosystems)). When mixing is performed using a microfluidic mixing system, the flow rate is, for example, 0.1 to 20 mL / min. The mixing temperature is, for example, 4 to 45°C, and may be 10 to 35°C.

[0143] The resulting lipid particles are treated by dialysis or filtration, preferably by diafiltration, to remove the organic solvent. After treatment, the aqueous solution is converted to a buffer solution at a neutral pH, between pH 6.8 and pH 7.5, preferably pH 7.2, such as a PBS buffer. The resulting aqueous solution is preferably sterilized before storage or use, such as by filtration through a 0.22 μm filter.

[0144] By mixing the lipid solution with the nucleic acid solution, the hydrophilic portions of the amphipathic compound and phospholipid are positioned on the surface of the particle, creating a hydrophobic environment inside the particle, allowing the complex of hydrophobic nucleic acid and cationic lipid nanoparticles to be encapsulated.

[0145] <Form> Another aspect of the present invention is a dispersion in which the lipid nanoparticles are dispersed in a dispersion medium. This dispersion makes it easy to use in pharmaceutical applications, which are suitable for such applications. The dispersion medium is preferably an aqueous solvent. The aqueous solvent is not particularly limited, and examples thereof include water (sterilized water), saline, phosphate-buffered saline (PBS), citrate buffer, Tris buffer, carbonate buffer, and acetate buffer.

[0146] The lipid nanoparticles may also be in the form of a powder obtained by freeze-drying or the like after removing the dispersion or the dispersion medium.

[0147] <Uses of Lipid Nanoparticles> The lipid nanoparticles of this embodiment are excellent in intracellular uptake, and can easily deliver desired components or compounds into cells. Therefore, the lipid nanoparticles of this embodiment are preferably lipid nanoparticles for delivering encapsulated substances into cells.

[0148] Another embodiment of the present invention is a composition for intracellular delivery, comprising the amphipathic compound, a nucleic acid, a phospholipid, a sterol, and a cationic lipid. The contents of each component in the composition are the same as those described for the lipid nanoparticles.

[0149] Furthermore, the lipid nanoparticles of this embodiment are excellent in intracellular uptake and can therefore be used particularly for pharmaceutical purposes. That is, another embodiment of the present invention is a medicine comprising the lipid nanoparticles.

[0150] The pharmaceutical may consist of the lipid nanoparticles of the present disclosure, or may be a composition further containing other components. Examples of such other components include water, saline, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, carboxymethylcellulose sodium salt, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, phosphate-buffered saline, biodegradable polymers, serum-free media, surfactants acceptable as pharmaceutical additives, and physiological pH buffer solutions acceptable in vivo. These additives may be used alone or in combination.

[0151] Furthermore, the pharmaceutical containing the lipid nanoparticles of the present disclosure is preferably used as a liquid formulation or a gel formulation.

[0152] In addition to the lipid nanoparticles of the present disclosure, the liquid formulation preferably contains water, saline, phosphate buffered saline, citrate-phosphate buffer, or the like.

[0153] In addition to the lipid nanoparticles of the present disclosure, the gel formulation preferably contains a neutralized anionic polymer such as polyacrylic acid, a thickener such as carboxypolymethylene and carboxymethylcellulose, Pemulen, a polymeric emulsifier, or polycarbophil, a lower alcohol such as ethanol or isopropanol, and water.

[0154] Furthermore, lipid nanoparticles or pharmaceuticals (compositions) containing lipid nanoparticles can be used in either in vitro or in vivo tests. Furthermore, the method of administering lipid nanoparticles and the like to a living body is preferably parenteral administration, i.e., local administration such as intra-articular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intrathecal administration, intracisternal administration, intraventricular administration, or intradermal administration. They can also be used in ex vivo treatments, in which cells or tissues are removed from an organism for treatment or therapy and then returned to the original organism. Intravenous or intraperitoneal administration of the (pharmaceutical) composition can also be performed by bolus injection.

[0155] The lipid nanoparticles or pharmaceuticals containing lipid nanoparticles of the present disclosure are expected to be pharmaceuticals that have high intracellular transport and good retention in the blood.

[0156] The present invention will now be described in more detail based on examples, but the present invention is not limited to these examples.

[0157] [Measurement of Number Average Molecular Weight of Polymer] The number average molecular weight of the polymers produced in Production Examples 1 to 5 described below was measured by gel permeation chromatography (GPC) under the following measurement conditions.

[0158] [Conditions for measuring the number average molecular weight of polymers (polymers obtained in Production Examples 1 to 5)] Measuring instrument: Tosoh Corporation, product number: HLC-8320GPC Molecular weight column: Tosoh Corporation, product number: TSKgel SuperAWM-H and SuperAW2500, two columns connected in series Eluent: dimethylformamide with 10 mmol / L lithium bromide added Standard substance for calibration curve: polystyrene Preparation of measurement solution: A solution with a polymer concentration of 0.2% by mass is prepared by dissolving the polymer in dimethylformamide, and the filtrate is used after filtering the solution.

[0159] [Production of Amphiphilic Compound (Polymer)] An amphiphilic compound having a polymeric form was produced by the following method.

[0160] (Production Example 1) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monoacrylate, 0.098 g of 1-octadecanethiol, 0.021 g of 2,2'-azobis(isobutyronitrile), 2.25 g of ethanol, and 0.25 g of water. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 80°C for 3 hours. The resulting reaction solution was added dropwise to diethyl ether and purified to obtain polyglycerin monoacrylate containing an alkyl group (n-octadecyl group) at the terminal. The number-average molecular weight of the resulting amphiphilic compound (polymer) was 4100. The molecular weight of the hydrocarbon group (n-octadecyl group) having 8 or more carbon atoms was 253.5.

[0161] (Production Example 2) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monoacrylate, 0.049 g of 1-octadecanethiol, 0.014 g of 2,2'-azobis(isobutyronitrile), 1.8 g of ethanol, and 0.2 g of water. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 80°C for 3 hours. The resulting reaction solution was added dropwise to diethyl ether and purified to obtain polyglycerin monoacrylate containing an alkyl group (n-octadecyl group) at the terminal. The number-average molecular weight of the resulting amphiphilic compound (polymer) was 8,600. The molecular weight of the hydrocarbon group (n-octadecyl group) having 8 or more carbon atoms was 253.5.

[0162] (Production Example 3) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monomethacrylate, 0.045 g of 1-octadecanethiol, 0.013 g of 2,2'-azobis(isobutyronitrile), 1.6 g of ethanol, and 0.4 g of n-butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 80°C for 3 hours. The solution portion of the resulting reaction mixture was added dropwise to diethyl ether for purification, yielding polyglycerin monomethacrylate containing an alkyl group (n-octadecyl group) at the terminal. The number-average molecular weight of the resulting amphiphilic compound (polymer) was 4100. The molecular weight of the hydrocarbon group (n-octadecyl group) having 8 or more carbon atoms was 253.5.

[0163] (Production Example 4) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monomethacrylate, 0.045 g of 1-octadecanethiol, 0.013 g of 2,2'-azobis(isobutyronitrile), 1.6 g of ethanol, and 0.4 g of n-butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 80°C for 3 hours. The precipitated portion of the resulting reaction solution was dissolved in 1.0 g of ethanol, and the solution was further purified by dropping it into diethyl ether, yielding polyglycerin monomethacrylate containing an alkyl group (n-octadecyl group) at the terminal. The number-average molecular weight of the resulting amphiphilic compound (polymer) was 14,000. The molecular weight of the hydrocarbon group (n-octadecyl group) having 8 or more carbon atoms was 253.5.

[0164] (Production Example 5) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monomethacrylate, 0.022 g of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 0.016 g of 2,2'-azobis(2,4-dimethylvaleronitrile), 0.8 g of ethanol, and 0.2 g of n-butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 50°C for 30 minutes. The resulting reaction solution was added dropwise to diethyl ether for purification. 0.2 g of the resulting polymer, 0.25 g of propylamine, and 1.0 g of water were charged, stirred overnight at room temperature, and lyophilized to obtain polyglycerin monomethacrylate having a thiol group introduced at the end. The number-average molecular weight of the resulting polymer was 5,200.

[0165] Subsequently, 0.0165 g of the polymer obtained above, 0.0276 g of distearoyl N-(3-maleimido-1-oxopropyl)-L-α-phosphatidylethanolamine (COATSOME® FE-8080MA3, manufactured by NOF Corporation), 75 μL of triethylamine, 0.06 g of chloroform, and 0.4 g of methanol were placed in a 10 mL screw tube. The mixture was then allowed to react overnight at room temperature, and then purified by dropwise addition to diethyl ether to obtain polyglycerol monomethacrylate containing lipids at its terminals. The number-average molecular weight of the resulting amphiphilic compound (polymer) was 6,000. Of this, the molecular weight of the lipid portion, which is the source of the hydrocarbon groups having 8 or more carbon atoms, was approximately 954.

[0166] [Materials for nucleic acid encapsulation tests] <Nucleic acid> siRNA and mRNA were used as nucleic acids. Lusiferase siRNA was synthesized by Hokkaido System Science Co., Ltd. using a sequence (sense strand: CUUACGCUGAGUACUUCGAdTdT, antisense strand: UCGAAGUACUCAGCGUAAGdTdT) described in a known literature (prior literature JP 2010-77091 A) and annealed. Lusiferase mRNA was purchased from TriLink BioTechnologies.

[0167] <Cationic lipids> Stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) SM-102 (Funakoshi Co., Ltd.).

[0168] [Analysis of Nucleic Acid Encapsulation Efficiency] The nucleic acid encapsulation efficiency and encapsulation amount into lipid particles were determined by Ribogreen Assay using Quant-iT™ RiboGreen™ (ThermoFisher SCIENTIFIC). Specifically, lipid particle suspensions were diluted with HEPES buffer A (10 mM HEPES, 300 mM sucrose, pH 7.4) and HEPES buffer B (HEPES buffer A + 0.1% Triton X-100), respectively. To each of these diluted solutions (100 μL), an equivalent amount of Quant-iT RiboGreen RNA reagent diluted 2000-fold with TE buffer was added, and the fluorescence intensity (excitation wavelength 485 nm, fluorescence wavelength 520 nm) was measured using a 96-well plate. The encapsulation efficiency (%) was calculated using the following formula:

[0169] Encapsulation efficiency (%) = (B - A) ÷ B × 100 A (fluorescence intensity in the absence of Triton X-100), B (fluorescence intensity in the presence of Triton X-100) The amount of encapsulation was calculated from the measured values ​​of each sample diluted with HEPES buffer B, prepared by preparing a calibration curve from values ​​measured by the Ribogreen Assay method using siRNA or mRNA solutions of known concentrations.

[0170] [Preparation of Lipid Particles] <Encapsulated Substance: mRNA> [Example 1] Amphipathic Compound Modification Rate: 0.1 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 18.2:63.6:18.1:0.1, with a molar ratio of SM-102 to nucleic acid of 5000 (N / P ratio 2.6)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The obtained lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min: 1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The obtained dispersion was concentrated by centrifugation at 3,000 × G for 10 minutes using a centrifugal filter unit (NMWL: 30 kDa, Amicon (registered trademark) Ultra, Merck Co., Ltd.), resuspended in PBS, washed again by centrifugation at 3,000 × G for 20 minutes, and stored as a lipid particle suspension at 4 ° C until use. The particle size of the lipid particles was measured using a Zetasizer Ultra (Malvern Panalytical).

[0171] [Example 2] Amphiphilic compound modification rate 0.5 mol% (phospholipid:sterol = approximately 1:3.5 (molar ratio)) Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 18.1:63.3:18.0:0.5, and the molar ratio of SM-102 to nucleic acid was 5000 (N / P ratio 2.6)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0172] [Example 3] Amphiphilic compound modification rate 1.0 mol% (phospholipid:sterol = approximately 1:3.5 (molar ratio)) Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), polymer of Production Example 4, respectively, in a molar ratio of 15.3:53.4:30.3:1.0, and the molar ratio of SM-102 to nucleic acid was 10,000 (N / P ratio 5.2)) was dissolved in 99.5 (v / v)% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0173] [Example 4] Amphiphilic compound modification rate 5.0 mol% (phospholipid:sterol = approximately 1:3.5 (molar ratio)) Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), polymer of Production Example 4, respectively, in a molar ratio of 14.7:51.2:29.1:5.0, and the molar ratio of SM-102 to nucleic acid was 10,000 (N / P ratio 5.2) mixed) was dissolved in 99.5 (v / v)% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0174] Example 5 Amphipathic Compound Modification Rate: 10.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 13.9:48.5:27.6:10.0, with a molar ratio of SM-102 to nucleic acid of 10,000 (N / P ratio 5.2)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0175] Example 6 Amphipathic Compound Modification Rate: 20.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 10.6:37.1:42.2:20.0, with a molar ratio of SM-102 to nucleic acid of 10,000 (N / P ratio 5.2)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0176] Example 7 Amphipathic Compound Modification Rate: 1.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 11.7:40.8:46.5:1.0, with a molar ratio of SM-102 to nucleic acid of 20,000 (N / P ratio of 10.4)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of approximately 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0177] Example 8 Amphipathic Compound Modification Rate: 5.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 11.2:39.2:44.6:5.0, with a molar ratio of SM-102 to nucleic acid of 20,000 (N / P ratio of 10.4)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of approximately 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0178] Example 9 Amphipathic Compound Modification Rate: 10.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 10.6:37.1:42.2:10.0, with a molar ratio of SM-102 to nucleic acid of 20,000 (N / P ratio of 10.4)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0179] Example 10 Amphipathic Compound Modification Rate: 20.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 9.4:33.0:37.6:20.0, with a molar ratio of SM-102 to nucleic acid of 20,000 (N / P ratio of 10.4)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0180] [Example 11] Amphiphilic compound modification rate 1.0 mol% (phospholipid: sterol = approximately 1:3.5 (molar ratio)) Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), polymer of Production Example 4, respectively, in a molar ratio of 8.0:27.8:63.2:1.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8) mixed) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0181] [Example 12] Amphiphilic compound modification rate 5.0 mol% (phospholipid: sterol = approximately 1:3.5 (molar ratio)) Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), polymer of Production Example 4, respectively, in a molar ratio of 7.6:26.7:60.7:5.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8) mixed) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0182] Example 13 Amphipathic Compound Modification Rate: 10.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 7.2:25.3:57.5:10.0, with a molar ratio of SM-102 to nucleic acid of 40,000 (N / P ratio of 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0183] Example 14 Amphipathic Compound Modification Rate: 20.0 mol% (Phosphatidylcholine:Sterol = Approximately 1:3.5 (Mole Ratio)) A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 6.4:22.5:51.1:20.0, with a molar ratio of SM-102 to nucleic acid of 40,000 (N / P ratio of 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0184] The above Examples 1 to 14 are summarized as follows.

[0185]

[0186] [Comparative Example 1] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 18.2:63.6:18.1:0.1, and mixed so that the molar ratio of SM-102 to nucleic acid was 5000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0187] [Comparative Example 2] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 18.1:63.3:18.0:0.5, and mixed so that the molar ratio of SM-102 to nucleic acid was 5000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0188] [Comparative Example 3] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 15.3: 53.4: 30.3: 1.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 10,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0189] [Comparative Example 4] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 14.7: 51.2: 29.1: 5.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 10,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0190] [Comparative Example 5] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 13.9: 48.5: 27.6: 10.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 10,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.11 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0191] [Comparative Example 6] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 12.4: 43.1: 24.5: 20.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 10,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0192] [Comparative Example 7] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 11.7: 40.8: 46.5: 1.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 20,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of approximately 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0193] [Comparative Example 8] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 11.2: 39.2: 44.6: 5.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 20,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of approximately 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0194] [Comparative Example 9] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 10.6: 37.1: 42.2: 10.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 20,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0195] [Comparative Example 10] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 9.5: 33.0: 37.5: 20.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 20,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0196] [Comparative Example 11] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 8.0: 27.8: 63.2: 1.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 40,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0197] [Comparative Example 12] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 7.6: 26.7: 60.7: 5.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 40,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0198] [Comparative Example 13] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 7.2: 25.3: 57.5: 10.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 40,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0199] [Comparative Example 14] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 6.4: 22.5: 51.1: 20.0, and mixed so that the molar ratio of SM-102 to nucleic acid was 40,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0200] [Comparative Example 15] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.) in a molar ratio of 11.8:41.3:46.9, and a molar ratio of SM-102 to nucleic acid of 20,000) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.23 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of approximately 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use. The particle diameter of the particles in the resulting dispersion was 424 nm.

[0201] [Comparative Example 16] Amphiphilic compound modification rate 40.0 mol% lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), polymer of Production Example 4, respectively in a molar ratio of 7.4:25.8:26.8:40.0, and the molar ratio of SM-102 to nucleic acid was 20,000 (N / P ratio 10.4) mixed) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.23 mg / mL was obtained. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 1.8 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and no particle formation was observed.

[0202] <Comparison of lipid particle diameters> The particle diameter measurement results for the lipid particles produced in Examples 1 to 14 and Comparative Examples 1 to 14 are shown in Tables 2 and 3. For the polymer-modified lipid particles of Production Example 4, particles with a size of 60 to 240 nm were obtained regardless of the polymer modification rate or the molar ratio of the cationic lipid (ionized lipid), and most lipid particles were particles with a size of 150 nm or less. On the other hand, for lipid particles modified with commercially available PEG lipid, particles with a size exceeding 240 nm were obtained depending on the polymer modification rate and the molar ratio of the cationic lipid.

[0203]

[0204]

[0205] <Storage Stability Test of mRNA-Encapsulating Lipid Particles> The lipid particles prepared in Examples 7, 8, 9, 10, and Comparative Example 15 were suspended in PBS and stored at 4°C, and the change in particle size after three days of storage was measured. The particle size change was compared with that on the start of storage (day 0) using the particle size change rate calculated using the following formula: Particle size change rate (%) = (B ÷ A) × 100 A (particle size on the start of storage (day 0)), B (particle size on day 3 of storage) The results are shown in Table 4.

[0206] The lipid particles (unmodified) of Comparative Example 15 showed a large decrease in particle size, whereas the polymer-modified lipid particles of Examples 7, 8, 9 and 10 showed only a slight change in particle size.

[0207]

[0208] <Cellular introduction test of mRNA-encapsulated lipid particles using cultured cells> Human breast adenocarcinoma-derived cells (MCF-7, DS Pharma Biomedical) were cultured using DMEM medium (Nacalai Tesque) supplemented with fetal bovine serum (FBS) (DS Pharma Biomedical) at a final concentration of 10%. ３ cell / cm ２ The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cells were 100 mm thick and incubated at 37°C, 5% CO ２ MCF-7 cells were cultured under the following conditions. MCF-7 cells were cultured in a 100 mm cell culture dish until they reached 70% confluence, and then treated with a 0.25% trypsin / 50 mM EDTA solution. The serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an MCF-7 cell suspension. The number of cells in the MCF-7 cell suspension was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries). The cell suspension was then diluted to a concentration of 3.2 x 10 cells per well. ３ The cells were seeded in a 96-well plate (Thermo Fisher Science) at 37°C, 5% CO ２The cells were cultured for 24 hours under these conditions. After 24 hours, 50 μL of a diluted solution prepared with Opti-MEM (Thermo Fisher Science) of the lipid particle suspensions prepared in Example 8, Comparative Example 8, and Comparative Example 15 to achieve a nucleic acid encapsulation level of 1 μg / mL was added to each well. Two days after the addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well, and the cells were stirred at 600 rpm for 3 minutes on a plate shaker. The luminescence intensity was then measured using an SH-9000 plate reader (Corona Electric Co., Ltd.). The mRNA expression efficiency was compared with that of the non-polymer-modified lipid particle-added group using the relative luminescence intensity calculated using the following formula: Relative luminescence intensity (%) = (B ÷ A) × 100 A (luminescence intensity in the non-polymer-modified lipid particle-added group), B (luminescence intensity in the polymer-modified lipid particle-added group). The results are shown in Figure 1.

[0209] Compared to the lipid particles of Comparative Example 15 (unmodified), the gene expression level decreased in the lipid particles of Comparative Example 8 (PEG phospholipid modified), whereas the expression level increased in the lipid particles of Example 8.

[0210] While the modifying substance contributes to particle stabilization, unmodified lipid nanoparticles have higher intracellular migration, and typically, modification reduces the expression efficiency of the encapsulated substance (e.g., nucleic acid), as in Comparative Example 8. However, unexpectedly, according to this embodiment, the expression efficiency of the encapsulated substance (e.g., nucleic acid) is higher than that of unmodified lipid nanoparticles, as in Example 8. This is thought to be because the lipid nanoparticles of this embodiment have higher intracellular penetration (translocation) than unmodified or PEG-modified lipid nanoparticles, and also because the expression efficiency of the encapsulated substance (e.g., nucleic acid) after intracellular migration is higher.

[0211] <Encapsulated substance: siRNA> [Example 15] Amphiphilic compound modification rate 5.0 mol% A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Corporation), and the polymer from Production Example 4 mixed in a molar ratio of 11.0:38.4:45.7:5.0, with a molar ratio of stearyltrimethylammonium chloride to nucleic acid of 250 (N / P ratio 0.13)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Corporation) to obtain a lipid solution of approximately 0.17 mg / mL. Luciferase siRNA was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 5.7 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0212] [Example 16] Amphiphilic compound modification rate 10.0 mol% Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Corporation), and the polymer from Production Example 4 mixed in a molar ratio of 10.4:36.3:43.3:10.0, with a molar ratio of stearyltrimethylammonium chloride to nucleic acid of 250 (N / P ratio 0.13)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Corporation) to obtain a lipid solution of approximately 0.17 mg / mL. Luciferase siRNA was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 5.7 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0213] [Example 17] Amphiphilic compound modification rate 20.0 mol% Lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Corporation), and the polymer from Production Example 4 mixed in a molar ratio of 9.3:32.3:38.5:20.0, with a molar ratio of stearyltrimethylammonium chloride to nucleic acid of 250 (N / P ratio 0.13)) was dissolved in 99.5% ethanol (Fujifilm Wako Pure Chemical Corporation) to obtain a lipid solution of approximately 0.17 mg / mL. Luciferase siRNA was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 5.7 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0214] [Comparative Example 16] Amphiphilic compound modification rate 10.0 mol% lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), PEG-phospholipid (NOF Corporation, SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 10.4:36.3:43.3:10.0, and the molar ratio of stearyltrimethylammonium chloride to nucleic acid was 250 (N / P ratio 0.13) mixed) was dissolved in 99.5% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and a lipid solution of approximately 0.17 mg / mL was obtained. Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 5.7 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0215] <Cellular introduction test of siRNA-encapsulated lipid particles using cultured cells> Luciferase-expressing human breast adenocarcinoma-derived cells (MCF-7 / Luc, Cosmo Bio Co., Ltd.) were cultured using DMEM medium (Nacalai Tesque) supplemented with a final concentration of 10% fetal bovine serum (FBS) (DS Pharma Biomedical Co., Ltd.). ３ cell / cm ２ The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cells were 100 mm thick and incubated at 37°C, 5% CO ２ MCF-7 cells were cultured under the following conditions. MCF-7 cells were cultured in a 100 mm cell culture dish until they reached 70% confluence, and then treated with a 0.25% trypsin / 50 mM EDTA solution. The serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an MCF-7 cell suspension. The number of cells in the MCF-7 cell suspension was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries). The cell suspension was then diluted to a concentration of 3.2 x 10 cells per well. ３The cells were seeded in a 96-well plate (Thermo Fisher Science) at 37°C, 5% CO ２ The cells were cultured for 24 hours under these conditions. After 24 hours, 50 μL of a dilution solution prepared with Opti-MEM (Thermo Fisher Science) of the lipid particle suspensions prepared in Examples 15, 16, 17, and Comparative Example 16 so that the nucleic acid encapsulation amount was 80 pmol / mL, and Opti-MEM alone as a control was added to each well. One day after the addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well, and the plates were stirred at 600 rpm for 3 minutes on a plate shaker, after which the luminescence intensity was measured using an SH-9000 plate reader (Corona Electric Co., Ltd.). The efficiency of mRNA expression suppression was compared based on the relative luminescence intensity calculated using the following formula: Relative light output (%) = (B ÷ A) × 100 A (light output in the group with Opti-MEM alone added), B (light output in the group with nucleic acid-encapsulated lipid particles added) A comparison of the gene expression suppression efficiencies of the lipid particles produced in Comparative Example 16, Example 15, Example 16, and Example 17 showed that modifying the lipid nanoparticles with the amphiphilic compound of this embodiment had a significant effect of suppressing gene expression compared to lipid nanoparticles modified with PEG phospholipid, as shown in Figure 2. Therefore, it can be seen that according to this embodiment, siRNA is efficiently taken up into cells, just like mRNA, and functional expression is significantly improved.

[0216] [Example 18] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), and the polymer from Production Example 4 mixed in a molar ratio of 21.5:75.0:3.5) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.17 mg / mL. Luciferase siRNA was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 4.0 μg / mL nucleic acid solution. The obtained lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0217] [Example 19] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids)), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 10.3:36.1:50.1:3.5, and the molar ratio of stearyltrimethylammonium chloride to nucleic acid was 150 (N / P ratio = 0.08)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.17 mg / mL. Luciferase siRNA was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 4.0 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0218] [Example 20] A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 10.3:36.1:50.1:3.5, with a molar ratio of stearyltrimethylammonium chloride to nucleic acid of 250 (N / P ratio = 0.13)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.17 mg / mL. Luciferase siRNA was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 4.0 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0219] <Effect of cationic lipid on siRNA encapsulation efficiency> The siRNA encapsulation efficiency of the lipid particles produced in Examples 18, 19, and 20 was compared, and as a result, it was found that the encapsulation efficiency could be increased by maintaining a constant molar ratio of cationic lipid to nucleic acid (particularly, by setting the N / P ratio to 0.03 or more), as shown in Figure 3.

[0220] Example 21 Amphiphilic Compound Modification Rate: 0.01 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 28.4 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 19.9:51.7:0.01, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0221] Example 22 Amphiphilic Compound Modification Rate: 0.1 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 28.4 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 19.8:51.7:0.1, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0222] Example 23 Amphiphilic Compound Modification Rate: 0.45 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 28.2 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 19.8:51.5:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0223] Example 24 Amphiphilic compound modification rate: 1.5 mol% (phospholipid:sterol = approximately 1:0.7 (molar ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 27.9 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 19.6:51.0:1.5, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0224] Example 25 Amphiphilic Compound Modification Rate: 5.0 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 27.0 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 18.9:49.1:5.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0225] Example 26 Amphiphilic Compound Modification Rate: 10.0 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 25.5 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 17.9:46.6:10.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0226] Example 27 Amphiphilic Compound Modification Rate: 20.0 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 22.7 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 15.9:41.4:20.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0227] Example 28 Amphiphilic Compound Modification Rate: 30.0 mol% (Phospholipid:Sterol = Approximately 1:0.7 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 19.9 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 13.9:36.2:30.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.27 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0228] Example 29 Amphiphilic Compound Modification Rate: 0.45 mol% (Phospholipid:Sterol = Approximately 1:0.1 (Mole Ratio)) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 73.2 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 7.3:19.0:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.79 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0229] [Example 30] Phospholipid: sterol = approximately 1:0.2 (molar ratio) (modification rate of amphipathic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), polymer of Production Example 4, each in a molar ratio of 57.9 (hydrogenated soybean-derived phosphatidylcholine: dioleoylphosphatidylethanolamine = 1:1 molar ratio): 11.6:30.1:0.45, and mixed so that the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.49 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0230] [Example 31] Phospholipid:sterol = approximately 1:0.5 (molar ratio) (modification rate of amphipathic compound: 0.45 mol%) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 35.5 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 17.8:46.2:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.30 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0231] [Example 32] A lipid mixture (phospholipid:sterol = approximately 1:1 (molar ratio) (modification rate of amphipathic compound: 0.45 mol%) (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 21.6 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 21.6:56.3:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.24 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0232] [Example 33] Phospholipid:sterol = approximately 1:4 (molar ratio) (modification rate of amphipathic compound: 0.45 mol%) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 6.5 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 25.8:67.3:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.19 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0233] [Example 34] Phospholipid:sterol = approximately 1:10 (molar ratio) (modification rate of amphipathic compound: 0.45 mol%) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 2.7 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 26.9:70.0:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.18 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0234] [Example 35] Phospholipid:sterol = approximately 1:20 (molar ratio) (modification rate of amphipathic compound 0.45 mol%) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 1.4 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 27.3:70.9:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.18 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0235] [Example 36] Phospholipid:sterol = approximately 1:40 (molar ratio) (modification rate of amphipathic compound 0.45 mol%) A lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.)), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 0.7 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 27.4:71.4:0.45, and the molar ratio of SM-102 to nucleic acid was 40,000 (N / P ratio 20.8)) was dissolved in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.18 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C until use.

[0236] [Example 37] Cationic lipid molar ratio 12.0 mol% (phospholipid:sterol = approximately 1:0.7 (molar ratio), modification rate of amphiphilic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids, dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 51.6 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 36.1:11.8:0.45, and the molar ratio of SM-102 to nucleic acid was 5000 (N / P ratio 2.6)) in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.14 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0237] [Example 38] Cationic lipid molar ratio 21.0 mol% (phospholipid:sterol = approximately 1:0.7 (molar ratio), modification rate of amphiphilic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids, dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 46.2 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 32.3:21.0:0.45, and the molar ratio of SM-102 to nucleic acid was 10,000 (N / P ratio 5.2)) in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.16 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0238] [Example 39] Cationic lipid molar ratio 35.0% (phospholipid:sterol = approximately 1:0.7 (molar ratio), modification rate of amphiphilic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids, dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 38.1 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 26.7:34.7:0.45, and the molar ratio of SM-102 to nucleic acid was 20,000 (N / P ratio 10.4)) in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.19 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0239] [Example 40] Cationic lipid molar ratio 61.0% (phospholipid:sterol = approximately 1:0.7 (molar ratio), modification rate of amphiphilic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids, dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 22.5 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 15.7:61.4:0.45, and the molar ratio of SM-102 to nucleic acid was 60,000 (N / P ratio 31.2)) in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.34 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0240] [Example 41] Cationic lipid molar ratio 68.0% (phospholipid:sterol = approximately 1:0.7 (molar ratio), modification rate of amphiphilic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids, dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 18.6 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 13.0:67.9:0.45, and the molar ratio of SM-102 to nucleic acid was 80,000 (N / P ratio 41.6)) in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.41 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a 3.5 μg / mL nucleic acid solution. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0241] [Example 42] Cationic lipid molar ratio 72.0% (phospholipid:sterol = approximately 1:0.7 (molar ratio), modification rate of amphiphilic compound 0.45 mol%) Lipid mixture (phospholipid (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids, dioleoylphosphatidylethanolamine (Fujifilm Wako Pure Chemical Industries, Ltd.), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer from Production Example 4 were mixed in a molar ratio of 15.9 (hydrogenated soybean-derived phosphatidylcholine:dioleoylphosphatidylethanolamine = 1:1 molar ratio): 11.1:72.5:0.45, and the molar ratio of SM-102 to nucleic acid was 100,000 (N / P ratio 52.1)) in 99.5% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of approximately 0.49 mg / mL. Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 3.5 μg / mL. The resulting lipid solution and nucleic acid solution were mixed at room temperature using a NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL / min:1.33 mL / min to obtain a dispersion containing lipid particles encapsulating nucleic acids. The resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.

[0242] The above Examples 21 to 42 are summarized as follows.

[0243]

[0244] <Comparative Test of Nucleic Acid Recovery Rate> The nucleic acid recovery rate was calculated using the following formula.

[0245] Nucleic acid recovery rate [%] = (amount of nucleic acid (μg) recovered by destroying the prepared lipid nanoparticles / total amount of nucleic acid (μg) used to prepare the lipid nanoparticles) × 100 Here, the "amount of nucleic acid recovered by destroying the prepared lipid nanoparticles" was calculated from the product of the nucleic acid concentration calculated in the same manner as the encapsulation amount described in the encapsulation efficiency column above and the amount of lipid nanoparticle suspension recovered.

[0246] The nucleic acid recovery rates of Comparative Example 15 and Examples 21 to 42 are shown in Figure 4. The nucleic acid recovery rates of the LNPs prepared in Examples 21 to 42 all showed higher values ​​than those of Comparative Example 15. Focusing on the polymer modification rate, extremely high recovery rates were observed in Examples 23 to 28, which had high modification rates, while surprisingly, a significant improvement in recovery rate was observed in Examples 21 and 22, which had low modification rates, compared to Comparative Example 15. Focusing on the phospholipid sterol ratio, a significant improvement in recovery rate was observed in all ranges examined in Examples 29 to 36, compared to Comparative Example 15.

[0247] <Gene Expression Test Using Lipid Nanoparticles with Different Polymer Modification Rates> Human breast adenocarcinoma-derived cells (MCF-7, DS Pharma Biomedical) were cultured in DMEM medium (Nacalai Tesque) supplemented with fetal bovine serum (FBS) (DS Pharma Biomedical) at a final concentration of 10%. ３ cell / cm ２ The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cells were 100 mm thick and incubated at 37°C, 5% CO ２ MCF-7 cells were cultured under the following conditions. MCF-7 cells were cultured in a 100 mm cell culture dish until they reached 70% confluence, and then treated with a 0.25% trypsin / 50 mM EDTA solution. The serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an MCF-7 cell suspension. The number of cells in the MCF-7 cell suspension was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries). The cell suspension was diluted to a concentration of 3.2 x 10 cells per well. ３ The cells were seeded in a 96-well plate (Thermo Fisher Science) at 37°C, 5% CO ２The cells were cultured for 24 hours under these conditions. After 24 hours, the medium was replaced with serum-free DMEM medium minus FBS, and 50 μL of a diluted solution of the lipid particle suspensions prepared in Examples 21 to 28 and Comparative Example 15 in serum-free DMEM medium was added to each well so that the nucleic acid encapsulation amount was 200 ng / mL. Two days after addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well, and the plates were stirred at 600 rpm for 3 minutes on a plate shaker, after which the luminescence level was measured using an SH-9000 plate reader (Corona Electric Co., Ltd.). The mRNA expression efficiency was compared between test sections using the relative luminescence level calculated using the following formula: Relative luminescence intensity (%)=(B÷A)×100 A (luminescence intensity in the area where lipid particles of Comparative Example 15 were added), B (luminescence intensity in the area where lipid particles of each polymer modification rate were added) The results are shown in FIG.

[0248] Compared with the lipid particles of Comparative Example 15, the lipid particles of Examples 21 to 28 showed the same or increased gene expression levels. In particular, Examples 21 to 25, which had low polymer modification rates, showed high gene expression levels, and the luminescence intensity of the lipid particles produced in Example 21 was approximately 170 times that of the lipid particles produced in Example 8. It is generally known that a decrease in the polymer modification rate of lipid particles leads to aggregation of the particles and an increase in particle size. However, unexpectedly, despite the extremely low modification rate of 0.01% for the lipid particles of Example 21, the particle size maintained a good size of 200 nm or less. Conversely, a high polymer modification rate is thought to reduce particle stability and make it difficult to maintain particle shape. However, lipid particles that maintained an appropriate particle size were confirmed even when the polymer modification rate was 30%.

[0249] <Gene expression test using lipid nanoparticles under different pH conditions> Powdered DMEM medium (D2902, Merck) was dissolved in 1 L of ultrapure water sterilized by high-pressure steam, sodium bicarbonate (Wako Pure Chemical Industries) was added, the pH was adjusted to 6.7, and the mixture was sterilized by filtration to prepare a low-pH DMEM medium (pH 6.7).

[0250] Human breast adenocarcinoma-derived cells (MCF-7, DS Pharma Biomedical) were cultured in DMEM medium (Nacalai Tesque) supplemented with fetal bovine serum (FBS) (DS Pharma Biomedical) at a final concentration of 10%. ３ cell / cm ２ The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cells were 100 mm thick and incubated at 37°C, 5% CO ２ MCF-7 cells were cultured under the following conditions. MCF-7 cells were cultured in a 100 mm cell culture dish until they reached 70% confluence, and then treated with a 0.25% trypsin / 50 mM EDTA solution. The serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an MCF-7 cell suspension. The number of cells in the MCF-7 cell suspension was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries). The cell suspension was then diluted to a concentration of 3.2 x 10 cells per well. ３ The cells were seeded in a 96-well plate (Thermo Fisher Science) at 37°C, 5% CO ２ The plates were cultured for 24 hours under these conditions. After 24 hours, the DMEM medium was removed from the well plate and replaced with low-pH DMEM medium. A dilution of the lipid particle suspensions prepared in Examples 21 to 28 and Comparative Example 8 in serum-free DMEM medium was added to each well at a concentration of 200 ng / mL. 50 μL of the diluted solution was added per well. Two days after the addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well. After stirring at 600 rpm for 3 minutes on a plate shaker, the luminescence intensity was measured using an SH-9000 plate reader (Corona Electric Co., Ltd.). The mRNA expression efficiency was compared between test sections using the relative luminescence intensity calculated using the following formula: Relative luminescence intensity (%)=(B÷A)×100 A (luminescence intensity in the area where lipid particles of Comparative Example 15 were added), B (luminescence intensity in the area where lipid particles of each polymer modification rate were added) The results are shown in FIG.

[0251] Even when the medium pH was low, the lipid particles of Examples 21 to 28 were confirmed to have equivalent or more pronounced gene expression levels compared to the lipid particles of Comparative Example 15. It is generally known that the extracellular pH around tumor tissue is lower (pH 6.2 to 6.8) compared to the extracellular pH of normal tissue (7.4), and being able to maintain gene expression ability even in a low pH environment is effective from a therapeutic perspective.

[0252] <Gene Expression Test Using Lipid Nanoparticles with Different Phospholipid-Sterol Ratios> Human breast adenocarcinoma-derived cells (MCF-7, DS Pharma Biomedical) were cultured in DMEM medium (Nacalai Tesque) supplemented with fetal bovine serum (FBS) (DS Pharma Biomedical) at a final concentration of 10%. ３ cell / cm ２ The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cells were 100 mm thick and incubated at 37°C, 5% CO ２ MCF-7 cells were cultured under the following conditions. MCF-7 cells were cultured in a 100 mm cell culture dish until they reached 70% confluence, and then treated with a 0.25% trypsin / 50 mM EDTA solution. The serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an MCF-7 cell suspension. The number of cells in the MCF-7 cell suspension was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries). The cell suspension was then diluted to a concentration of 3.2 x 10 cells per well. ３ The cells were seeded in a 96-well plate (Thermo Fisher Science) at 37°C, 5% CO ２The cells were cultured for 24 hours under these conditions. After 24 hours, the medium was replaced with serum-free DMEM medium minus FBS, and 50 μL of a diluted solution prepared with serum-free DMEM medium to give a nucleic acid encapsulation amount of 200 ng / mL of the lipid particle suspensions prepared in Examples 24, 29 to 36, and Comparative Example 15 was added to each well. Two days after the addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well, and the plates were stirred at 600 rpm for 3 minutes on a plate shaker, after which the luminescence level was measured using a plate reader SH-9000 (Corona Electric Co., Ltd.). The mRNA expression efficiency was compared between test sections using the relative luminescence level calculated using the following formula: Relative luminescence intensity (%) = (B ÷ A) × 100 A (luminescence intensity in the area where lipid particles of Comparative Example 15 were added), B (luminescence intensity in the area where lipid particles of each phospholipid sterol ratio were added) The results are shown in Figure 7.

[0253] Compared with the lipid particles of Comparative Example 15, the gene expression level was significantly increased in the lipid particles of Examples 24 and 29 to 36. Furthermore, for all of the lipid particles, the particle diameter was in the range of 30 to 200 nm, and good particles were obtained.

[0254] <Gene Expression Test Using Lipid Nanoparticles with Different Cationic Ratios> Human breast adenocarcinoma-derived cells (MCF-7, DS Pharma Biomedical) were cultured in DMEM medium (Nacalai Tesque) supplemented with fetal bovine serum (FBS) (DS Pharma Biomedical) at a final concentration of 10%. ３ cell / cm ２ The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cells were 100 mm thick and incubated at 37°C, 5% CO ２ MCF-7 cells were cultured under the following conditions. MCF-7 cells were cultured in a 100 mm cell culture dish until they reached 70% confluence, and then treated with a 0.25% trypsin / 50 mM EDTA solution. The serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an MCF-7 cell suspension. The number of cells in the MCF-7 cell suspension was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries). The cell suspension was then diluted to a concentration of 3.2 x 10 cells per well.３ The cells were seeded in a 96-well plate (Thermo Fisher Science) at 37°C, 5% CO ２ The cells were cultured for 24 hours under these conditions. After 24 hours, the DMEM medium was replaced with serum-free DMEM medium minus FBS, and 50 μL of a diluted solution of the lipid particle suspensions prepared in Examples 24, 37 to 42, and Comparative Example 15 in serum-free DMEM medium was added to each well so that the nucleic acid encapsulation amount was 200 ng / mL. Two days after addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well, and the plates were stirred at 600 rpm for 3 minutes on a plate shaker, after which the luminescence level was measured using an SH-9000 plate reader (Corona Electric Co., Ltd.). The mRNA expression efficiency was compared between test sections using the relative luminescence level calculated using the following formula: Relative luminescence intensity (%)=(B÷A)×100 A (luminescence intensity in the area where lipid particles of Comparative Example 15 were added), B (luminescence intensity in the area where lipid particles of each cationic ratio were added) The results are shown in FIG.

[0255] Compared with the lipid particles of Comparative Example 15, the gene expression level was significantly increased in the lipid particles of Examples 24 and 37 to 42. Furthermore, for all of the lipid particles, the particle diameter was in the range of 30 to 200 nm, and good particles were obtained.

[0256] Gene Expression Tests in Different Cell Types Gene expression tests were performed using human lung cancer-derived cells (A549, DS Pharma Biomedical) and human pancreatic cancer-derived cells (S2-013, obtained from the Medical Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University). Except for the difference in cell type, the cells were cultured in the same manner as in the test using MCF-7 cells. Cells were seeded into a 96-well plate (Thermo Fisher Science Inc.). A diluted solution of the lipid particle suspensions prepared in Example 24 and Comparative Example 15 in serum-free DMEM medium was added to each well at a concentration of 200 ng / mL of nucleic acid. 50 μL per well was added. Two days after addition, 50 μL of ONE-Glo™ EX Luciferase Assay System (Promega Corporation) was added per well. The plates were shaken at 600 rpm for 3 minutes, and then the luminescence was measured using an SH-9000 plate reader (Corona Electric Co., Ltd.). The mRNA expression efficiency was compared between test sections using the relative luminescence intensity calculated using the following formula: Relative luminescence intensity (%) = (B ÷ A) × 100 A (luminescence intensity in the lipid particle-added group of Comparative Example 15), B (luminescence intensity in the lipid particle-added group of each cell type) The results are shown in Figure 9.

[0257] In all the cells used, the amount of gene expression was significantly increased in the lipid particles of Example 24 compared to the lipid particles of Comparative Example 15.

[0258] This application is based on Japanese Patent Application No. 2023-121431, filed on July 26, 2023, the disclosure of which is incorporated by reference in its entirety.

Claims

1. A lipid nanoparticle comprising an amphipathic compound, the amphipathic compound having (i) a portion (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms.

2. The lipid nanoparticle of claim 1, which contains a nucleic acid.

3. The structural unit (A) is represented by the following chemical formula (1): In formula (1), R １ represents a hydrogen atom or a methyl group, and X is -C(=O)-O-, -C(=O)-NH-, -O-, or -CH ２ O- or -CH ２ CH ２ The lipid nanoparticle according to claim 1 or 2, which is a structural unit represented by:

4. The lipid nanoparticle described in claim 3, wherein the proportion of the structural unit (A) in the portion (I) is 80 to 100 mass %.

5. The lipid nanoparticle according to claim 1 or 2, wherein the modification rate (%) of the amphipathic compound calculated by the following formula is 0.01% or more and less than 40%. Modification rate (%) = {content (mol) of the amphipathic compound / total amount (mol) of lipid contained in the lipid nanoparticle} x 100 (%) 6. The lipid nanoparticle of claim 1 or 2, comprising a cationic lipid.

7. The lipid nanoparticle according to claim 6, wherein the molar ratio of the cationic lipid to the total lipid present in the lipid nanoparticle is 0.5 to 75 mol %.

8. The lipid nanoparticle of claim 1 or 2, comprising a phospholipid.

9. The lipid nanoparticle according to claim 8, further comprising a sterol, wherein the molar ratio of the phospholipid to the sterol is phospholipid:sterol=1:0.1 to 1:

40.

10. The lipid nanoparticle according to claim 1 or 2, having a particle diameter of 10 to 300 nm.

11. A pharmaceutical comprising the lipid nanoparticles described in claim 1 or 2.

12. A method for producing lipid nanoparticles comprising a phospholipid, a sterol, a cationic lipid, and an amphipathic compound, wherein the amphipathic compound has (i) a moiety (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms, the method comprising mixing the phospholipid, the sterol, the cationic lipid, and the amphipathic compound with a solvent, and contacting the resulting mixture with an encapsulation substance to obtain lipid nanoparticles encapsulating the encapsulation substance.

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