Method for producing nucleic acid encapsulated lipid nanoparticles

Abstract

The present invention provides a method for producing nucleic acid-encapsulated lipid nanoparticles, the method comprising the following steps (a) and (b): a step (a) for preparing a suspension of nucleic acid-encapsulated lipid nanoparticles by mixing an alcoholic solution containing an ionic lipid having a tertiary amino group, a sterol, and a PEG lipid with a citric acid buffer solution having a pH of 3-6.5 in which a nucleic acid is dispersed; and a step (b) in which a suspension of nucleic acid-encapsulated lipid nanoparticles is subjected to ultrafiltration concentration and diluted with a Tris buffer solution having a pH of 5.2-9.0, thereby replacing the dispersion medium of the suspension with the Tris buffer solution.

Description

Technical Field

[0001] This invention relates to a method for manufacturing nucleic acid-encapsulated lipid nanoparticles, a method for introducing nucleic acids into cells, and a method for manufacturing pharmaceutical compositions. Background Technology

[0002] To practically implement nucleic acid therapy using oligonucleotides such as siRNA and gene therapy using mRNA, pDNA, etc., efficient and safe nucleic acid delivery vectors are needed. Viral vectors are a type of nucleic acid delivery vector with good expression efficiency, but the development of non-viral nucleic acid delivery vectors that can be used more safely is underway.

[0003] Cationic liposomes using cationic lipids with a quaternary ammonium moiety are positively charged and can form complexes (liposome complexes) with negatively charged nucleic acids through electrostatic interactions, thus enabling the delivery of nucleic acids into cells (see, for example, Patent Documents 1 and 2).

[0004] However, liposome complexes produced by such methods are difficult to control in terms of particle size, and the cytotoxicity of positively charged cationic lipids becomes a problem.

[0005] Therefore, a lipid nanoparticle (or LNP) using ionic lipids having a tertiary amino group that is positively charged under acidic conditions and uncharged near neutral conditions has been developed and has now become the most versatile nonviral nucleic acid delivery carrier (see, for example, non-patent literature 1).

[0006] As examples of lipid nanoparticles that utilize ionic lipids having tertiary amino groups within the molecule, there are also examples of ionic lipids endowed with decomposable groups (see, for example, Patent Document 3).

[0007] Various non-viral vectors have been developed in this way, but since nucleic acids are usually unstable compounds, there are still challenges in the preservation stability of nucleic acid-encapsulated lipid nanoparticles. Existing technical documents Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2008-5801 Patent Document 2: Japanese Patent Application Publication No. 2001-2565 Patent Document 3: International Publication No. 2013 / 073480 Patent Document 4: International Publication No. 2019 / 188867 Non-patent literature

[0009] Non-patent literature 1: Gene Therapy (1999) 6.271-281 Summary of the Invention The problem the invention aims to solve

[0010] As a known method for manufacturing nucleic acid-encapsulated lipid nanoparticles, one method involves mixing an acidic buffer solution containing dispersed nucleic acids with a lipid solution dissolved in alcohol, and then replacing the dispersion medium of the resulting suspension with other buffer solutions. For example, while Patent Document 4 describes an example of using a pH 3.0 malate buffer solution as the acidic buffer solution and replacing the dispersion medium of the resulting suspension with pH 7.4 PBS to prepare nucleic acid-encapsulated lipid nanoparticles, it does not mention any impact of the buffer solutions on the performance of the nucleic acid-encapsulated lipid nanoparticles.

[0011] That said, as methods to improve the stability of nucleic acid-encapsulated lipid nanoparticles, attempts have been made to add cryoprotectants to the nucleic acid-encapsulated lipid nanoparticles for cryopreservation, or to freeze-dry the nucleic acid-encapsulated lipid nanoparticles and then add water to reconstruct the suspension of the nucleic acid-encapsulated lipid nanoparticles before use. These methods are useful in improving the stability of nucleic acid-encapsulated lipid nanoparticles, but there is room for improvement in terms of simplicity.

[0012] In view of the above-mentioned problems, the present invention aims to provide a method for manufacturing nucleic acid-encapsulated lipid nanoparticles that improves the storage stability of suspensions of nucleic acid-encapsulated lipid nanoparticles, which has not been achieved in the prior art. Problem-solving methods

[0013] Through in-depth efforts addressing the aforementioned issues, the inventors discovered that the buffer solution used in the preparation process of lipid nanoparticles affects the nucleic acid delivery efficiency of the lipid nanoparticles and the storage stability of their suspensions. Specifically, by replacing the dispersion medium of the suspension prepared using citrate buffer as the acidic buffer with Tris buffer, the nucleic acid delivery efficiency of the nucleic acid-encapsulated lipid nanoparticles and the storage stability of their suspensions were improved, thus completing this invention. The invention based on this discovery is as follows.

[0014] [1] A method for manufacturing nucleic acid-encapsulated lipid nanoparticles, comprising the following steps (a) and (b): Step (a) involves mixing an alcoholic solution containing ionic lipids with tertiary amino groups, sterols, and PEG lipids with a citrate buffer solution at pH 3–6.5 containing dispersed nucleic acids to prepare a suspension of nucleic acid-encapsulated lipid nanoparticles; and... Step (b) involves ultrafiltration concentration of a suspension of nucleic acid-encapsulated lipid nanoparticles and dilution with a Tris buffer solution at pH 5.2–9.0, replacing the dispersion medium of the suspension with the Tris buffer solution. [2] According to the method described in [1], the total concentration of citric acid and its salt in the citric acid buffer used in step (a) is 10 to 100 mM, and the total concentration of tris(hydroxymethyl)aminomethane and its salt in the Tris buffer used in step (b) is 10 to 200 mM. [3] According to the method described in [1] or [2], the alcohol solution used in step (a) further contains phospholipids. [4] According to any one of [1] to [3], the ionic lipid is a compound represented by formula (1).

[0015] Equation (1): [Chemistry 1]

[0016] (In formula (1),) R 1a and R 1b Each independently represents an alkylene group having 1 to 6 carbon atoms. X a and X b Each can be independently represented as a noncyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. R 2a and R 2b Each can independently represent an alkylene group having 1 to 8 carbon atoms or an oxadialkylene group having 2 to 8 carbon atoms. Y a and Y b Each can independently represent an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z a and Z b Each can independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms. na and nb each independently represent 0 or 1. R 3a and R 3b Each of these groups independently represents a residue derived from the reaction of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from the reaction of a sterol derivative with a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring having 3 to 40 carbon atoms, or a group represented by formula (3c). Equation (3c): R 3c -O-CO-(CH2) a -*(3c) (In equation (3c), * indicates the bonding location. R 3c This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms, and a represents an integer from 2 to 10. [5] In any one of the methods described in [1] to [4], the molar ratio of the total amino group of the ionic lipid to the phosphate group of the nucleic acid used in step (a) is 7 or more (total amino group of the ionic lipid / phosphate group of the nucleic acid). [6] A method for introducing the nucleic acid into a cell, comprising a step of contacting the cell with a nucleic acid-encapsulated lipid nanoparticle manufactured by any one of the methods described in [1] to [5] in vitro. [7] A method for introducing the nucleic acid into the target cells of the organism, comprising the step of administering nucleic acid-encapsulated lipid nanoparticles manufactured by any one of the methods described in [1] to [5] to the organism. [8] A method for manufacturing a pharmaceutical composition, comprising any one of the methods described in [1] to [5]. The effects of the invention

[0017] In the manufacturing method of this invention, compared with the prior art, the nucleic acid delivery efficiency is higher immediately after manufacturing, enabling the production of nucleic acid-encapsulated lipid nanoparticles. Furthermore, the suspension of nucleic acid-encapsulated lipid nanoparticles prepared by the manufacturing method of this invention exhibits excellent storage stability. Compared with the prior art, the nucleic acid delivery efficiency of the nucleic acid-encapsulated lipid nanoparticles prepared by the manufacturing method of this invention is higher, thus facilitating gene delivery in cells and organisms. Due to the high storage stability of its suspension, it is particularly useful as a pharmaceutical composition. Attached Figure Description

[0018] [ Figure 1 [Graphs showing the results of nucleic acid delivery efficiency of nucleic acid-encapsulated lipid nanoparticles prepared in Examples 1 and Comparative Examples 1-6] [ Figure 2 The graph shows the time-varying nucleic acid delivery efficiency of the nucleic acid-encapsulated lipid nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 6. [ Figure 3 [Graph showing the results of nucleic acid delivery efficiency of nucleic acid-encapsulated lipid nanoparticles prepared using citrate buffers with different pH values ​​in Example 2.] [ Figure 4 [Graph showing the nucleic acid delivery efficiency of nucleic acid encapsulated lipid nanoparticles prepared under different molar ratios of total amino groups in ionic lipids to phosphate groups in nucleic acids (total amino groups in ionic lipids / phosphate groups in nucleic acids) in Example 3.] [ Figure 5 The graph shows the results of nucleic acid delivery efficiency of the nucleic acid-encapsulated lipid nanoparticles prepared in Example 4 and Comparative Example 7. [ Figure 6 The graph shows the results of nucleic acid delivery efficiency of the nucleic acid-encapsulated lipid nanoparticles prepared in Example 5 and Comparative Example 8. [ Figure 7 [Graphs showing the time-varying efficiency of nucleic acid delivery in vivo for nucleic acid-encapsulated lipid nanoparticles prepared in Examples 1, 1, and 6.] [ Figure 8 The graph shows the results of nucleic acid delivery efficiency of the nucleic acid-encapsulated lipid nanoparticles prepared in Example 6 and Comparative Example 9. Detailed Implementation

[0019] The following describes embodiments of the present invention, but the present invention is not limited to these.

[0020] The method for manufacturing nucleic acid-encapsulated lipid nanoparticles of the present invention includes the following steps (a) and (b): Step (a) involves preparing a suspension of lipid nanoparticles by mixing an alcoholic solution containing amino-containing ionic lipids, sterols, and PEG lipids with a citrate buffer solution of pH 3–6.5 in which nucleic acids are dispersed; and Step (b) involves concentrating the suspension of lipid nanoparticles by ultrafiltration and diluting it with Tris buffer solution at pH 5.2–9.0, thereby replacing the dispersion medium of the suspension with the Tris buffer solution.

[0021] According to the present invention, a suspension of nucleic acid-encapsulated lipid nanoparticles with excellent preservation stability can be manufactured. In other words, the method of the present invention is a method for manufacturing a suspension of nucleic acid-encapsulated lipid nanoparticles with excellent preservation stability.

[0022] In this specification, "nucleic acid-encapsulated lipid nanoparticles" refers to lipid nanoparticles that contain nucleic acids. In this specification, "lipid nanoparticles" refers to membrane-structured particles with hydrophilic groups of amphiphilic lipids arranged facing the aqueous phase side of the interface. In this specification, "amphiphilic lipids" refers to lipids that possess both a hydrophilic group exhibiting hydrophilicity and a hydrophobic group exhibiting hydrophobicity. Examples of amphiphilic lipids include ionic lipids, phospholipids, and PEG lipids. Examples of lipids include ionic lipids, sterols, PEG lipids, and phospholipids.

[0023] Process (a) Ionic lipids with tertiary amino groups The alcohol solution used in step (a) contains an ionic lipid having a tertiary amino group (hereinafter referred to simply as "ionic lipid"). Only one type of ionic lipid may be used, or two or more may be used in combination. The ionic lipids usable in this invention are those composed of a tertiary amino group and a hydrophobic group, as long as they can form lipid nanoparticles.

[0024] Examples of ionic lipids include: 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dilinoleoyloxy-3-dimethylaminopropane (DLinDMA), 2-[2,2-bis[(9Z,12Z)-octadec-9,12-dienyl]-1,3-dioxolane-4-yl]-N,N-dimethylethylamine (DLin-KC2-DMA), (6Z,9Z,28Z,31... Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octate (Lipid5), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octate (Lipid8), and compounds represented by the following formula (1),

[0025] Equation (1): [Chemistry 2]

[0026] (In formula (1),) R 1a and R 1b Each can independently represent an alkylene group having 1 to 6 carbon atoms. X a and X b Each can be independently represented as a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. R 2a and R 2b Each can independently represent an alkylene group having 1 to 8 carbon atoms or an oxadialkylene group having 2 to 8 carbon atoms. Y a and Y b Each can independently represent an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z a and Z bEach can independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms. na and nb each independently represent 0 or 1. R 3a and R 3b Each of these groups independently represents a residue derived from the reaction of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from the reaction of a sterol derivative with a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring having 3 to 40 carbon atoms, or a group represented by formula (3c). Equation (3c): R 3c -O-CO-(CH2) a -*(3c) (In equation (3c), * indicates the bonding location. R 3c This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms, and 'a' represents an integer from 2 to 10. (There are cases described below as "ionic lipids (1)").

[0027] The ionic lipid used in this invention is preferably an ionic lipid (1). Only one type of ionic lipid (1) may be used, or two or more types may be used in combination. The groups in the ionic lipid (1) will be described in turn below.

[0028] R 1a and R 1b Each alkylene group, independently representing 1 to 6 carbon atoms, can be linear or branched, but is preferably linear. The number of carbon atoms in this alkylene group is preferably 1 to 4, more preferably 1 to 2. Examples of alkylene groups with 1 to 6 carbon atoms include: methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. 1a and R 1b Preferably, each of the following is independently methylene, ethylene, trimethylene, isopropylene, or tetramethylene, with ethylene being the most preferred.

[0029] R 1a With R 1b They can be the same or different, but R is preferred. 1a To be with R 1b Same group.

[0030] X a and X bEach of the above can be independently represented as a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, preferably a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0031] The alkyl group with 1 to 6 carbon atoms and one tertiary amino group in the noncyclic alkyl tertiary amino group can be linear, branched, or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of alkyl groups with 1 to 6 carbon atoms include: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, cyclohexyl, etc., preferably methyl, ethyl, propyl, or isopropyl, with methyl being the most preferred.

[0032] Preferred specific structures of noncyclic alkyl tertiary amino groups having 1 to 6 carbon atoms and 1 tertiary amino group are shown in X. 1 As shown.

[0033] [Chemistry 3]

[0034] X 1 R 5 The alkyl group having 1 to 6 carbon atoms can be linear, branched, or cyclic. The preferred carbon number of the alkyl group is 1 to 3. Specific examples of alkyl groups having 1 to 6 carbon atoms include: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, cyclohexyl, etc., with methyl, ethyl, propyl, or isopropyl being preferred, and methyl being the most preferred.

[0035] The cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups preferably has 4 to 5 carbon atoms. Specifically, the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups includes aziridinidinediyl, aziridinediyl, pyrrolidinediyl, piperidinediyl, imidazolediyl, and piperazinediyl, with pyrrolidinediyl, piperidinediyl, and piperazinediyl being preferred, and piperidinediyl being the most preferred.

[0036] Preferred specific structures of cyclic alkylene tertiary amino groups having 2 to 5 carbon atoms and 1 tertiary amino group are shown in X. 2 As shown.

[0037] [Chemistry 4]

[0038] X 2 p is 1 or 2. When p is 1, X 2 It is pyrrolidine dimethyl; when p is 2, X 2 It is a piperidine dimethyl group. Preferably, p is 2.

[0039] Preferred specific structures of cyclic alkylene tertiary amino groups having 2 to 5 carbon atoms and 2 tertiary amino groups are shown in X. 3 As shown.

[0040] [Chemistry 5]

[0041] X 3 w is 1 or 2. When w is 1, X 3 For imidazole dimethyl; when w is 2, X 3 It is piperazine dimethyl.

[0042] X a With X b They can be the same or different, but X is preferred. a To be with X b Same group.

[0043] R 2a and R 2b Each of the terms independently represents an alkylene group having 1 to 8 carbon atoms or an oxadialkylene group having 2 to 8 carbon atoms, preferably an alkylene group having 1 to 8 carbon atoms.

[0044] The alkylene group having 1 to 8 carbon atoms can be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 6 or less, and most preferably 4 or less. Examples of alkylene groups having 1 to 8 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, and octamethylene, with methylene, ethylene, trimethylene, and tetramethylene being preferred, and ethylene being most preferred.

[0045] In this specification, "oxadiene with 2 to 8 carbon atoms" refers to an alkylene group (alkylene-O-alkylene, in other words, "alkyloxyalkylene") separated by an ether bond, wherein the total number of carbon atoms in the two alkylene groups is 8 or less. The two alkylene groups may be the same or different, but are preferably the same. Specific examples of oxadiene with 2 to 8 carbon atoms include: oxadiethylene, oxadiethylene, oxadi(trimethylene) (i.e., trimethyleneoxytrimethylene), and oxadi(tetramethylene) (i.e., tetramethyleneoxytetramethylene). Oxadiethylene, oxadiethylene, and oxadi(trimethylene) are preferred, with oxadiethylene being the most preferred.

[0046] R 2a With R 2b They can be the same or different, but R is preferred. 2a To be with R 2b Same group.

[0047] Y a and Y b Each bond is independently an ester bond, amide bond, carbamate bond, ether bond, or urea bond, preferably an ester bond, amide bond, or carbamate bond, more preferably an ester bond or amide bond, and most preferably an ester bond. a and Y b The bonding orientation is not restricted, Y a and Y b In the case of an ester bond, it is preferred to have a -Z bond. a -CO-OR 2a -and-Z b -CO-OR 2b - structure.

[0048] Y a With Y b They can be the same or different, but Y is preferred. a To be with Y b Same group.

[0049] Z a and Z b Each of these terms independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms. The aromatic compound preferably contains 6 to 12 carbon atoms, and most preferably 6 to 7. Furthermore, the aromatic compound preferably contains only one aromatic ring.

[0050] As for the types of aromatic rings contained in aromatic compounds with 3 to 16 carbon atoms, examples of aromatic hydrocarbon rings include: benzene rings, naphthalene rings, and anthracene rings; examples of aromatic heterocycles include: imidazole rings, pyrazole rings, oxazole rings, isoxazole rings, thiazole rings, isothiazole rings, triazine rings, pyrrole rings, furan rings, thiophene rings, pyrimidine rings, pyrazine rings, pyridine rings, purine rings, pteridine rings, benzimidazole rings, indole rings, benzofuran rings, quinazoline rings, phthalazine rings, quinoline rings, isoquinoline rings, coumarin rings, chromone rings, and benzodiazepine rings. Rings, phenoxazine rings, phenothiazine rings, acridine rings, etc., are preferred, with benzene rings, naphthalene rings, and anthracene rings being the most preferred.

[0051] The aromatic ring may have substituents, such as: acyl group with 2-4 carbon atoms, alkoxycarbonyl group with 2-4 carbon atoms, alkylcarbamoyl group with 2-4 carbon atoms, acyloxy group with 2-4 carbon atoms, acylamino group with 2-4 carbon atoms, alkoxycarbonylamino group with 2-4 carbon atoms, fluorine atom, chlorine atom, bromine atom, iodine atom, alkylthio group with 1-4 carbon atoms, alkylsulfonyl group with 1-4 carbon atoms, arylsulfonyl group with 6-10 carbon atoms, nitro group, trifluoromethyl group, cyano group, alkyl group with 1-4 carbon atoms, ureo group, carbonyl group, etc. Preferred examples include alkoxy groups with 1 to 4 atoms, aryl groups with 6 to 10 carbon atoms, and aryloxy groups with 6 to 10 carbon atoms, such as acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylsulfanyl, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, ureo, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, and phenoxy.

[0052] As Z a and Z b A preferred specific structure can be exemplified by Z. 1 .

[0053] [Chemistry 6]

[0054] In the formula, s represents an integer from 0 to 3, t represents an integer from 0 to 3, u represents an integer from 0 to 4, and u represents R. 4 Each substituent is represented independently. In this specification, "s = 0" means Z 1 (CH2) s It does not exist; "t is 0" means Z 1 (CH2) t It does not exist; "u is 0" means Z 1 (R) 4 ) u It does not exist.

[0055] Z 1 The s in the formula is preferably an integer from 0 to 1, and more preferably 0. Z 1 In this context, t is preferably an integer from 0 to 2, and more preferably 1. Z 1 The u in the figure is preferably an integer from 0 to 2, and more preferably an integer from 0 to 1.

[0056] Z 1 R in 4 Substituents in aromatic compounds with 3 to 16 carbon atoms that do not hinder the reaction in the synthesis of this ionic lipid are called substituents for the aromatic ring (benzene ring). Examples of such substituents include: acyl groups with 2 to 4 carbon atoms, alkoxy carbonyl groups with 2 to 4 carbon atoms, alkyl carbamoyl groups with 2 to 4 carbon atoms, acyloxy groups with 2 to 4 carbon atoms, acylamino groups with 2 to 4 carbon atoms, alkoxy carbonylamino groups with 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkyl thiols with 1 to 4 carbon atoms, alkyl sulfonyl groups with 1 to 4 carbon atoms, aryl sulfonyl groups with 6 to 10 carbon atoms, nitro groups, trifluoromethyl groups, cyano groups, alkyl groups with 1 to 4 carbon atoms, and urea groups. Preferred examples include alkoxy groups with 1 to 4 carbon atoms, aryl groups with 6 to 10 carbon atoms, and aryloxy groups with 6 to 10 carbon atoms, such as acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylthio, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, ureo, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, and phenoxy. Multiple R groups are present. 4 At that time, each R 4 They can be the same or different.

[0057] Z a With Z b They can be the same or different, but Z is preferred. a To be with Z b Same group.

[0058] na and nb are each independently 0 or 1. In this specification, "na is 0" means that (Z) in equation (1) a -Y a ) na It does not exist; "nb is 0" refers to (Z) in equation (1). b -Y b ) nb It does not exist.

[0059] na and nb can be the same or different, but it is preferred that na be the same as nb.

[0060] R 3a and R 3b Each of these groups independently represents a residue derived from the reaction of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from the reaction of a sterol derivative with a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring having 3 to 40 carbon atoms, or a group represented by formula (3c). Equation (3c): R 3c -O-CO-(CH2) a -*(3c) (In equation (3c), * indicates the bonding location. R 3c Aliphatic hydrocarbon groups representing 2 to 20 carbon atoms, and 'a' represents an integer from 2 to 10.

[0061] R 3a and R 3b Each of the residues is preferably derived from the reaction product of a fat-soluble vitamin with hydroxyl groups and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group with 12 to 22 carbon atoms, or a group represented by the above formula (3c). More preferably, it is derived from the reaction product of a fat-soluble vitamin with hydroxyl groups and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group with 12 to 22 carbon atoms. Most preferably, it is an aliphatic hydrocarbon group with 12 to 22 carbon atoms.

[0062] Residues derived from the reaction product of a hydroxyl-containing fat-soluble vitamin with succinic anhydride or glutaric anhydride indicate a group in which the hydroxyl group of the fat-soluble vitamin is replaced with a structure of *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2- (where * indicates the bonding position with the fat-soluble vitamin). Residues derived from the reaction product of a hydroxyl-containing sterol derivative with succinic anhydride or glutaric anhydride indicate a group in which the hydroxyl group of the sterol derivative is replaced with a structure of *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2- (where * indicates the bonding position with the sterol derivative).

[0063] Examples of fat-soluble vitamins containing hydroxyl groups include retinol, ergosterol, 7-dehydrocholesterol, calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. Tocopherol is preferred among fat-soluble vitamins containing hydroxyl groups.

[0064] Examples of sterol derivatives containing hydroxyl groups include cholesterol, cholesterol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, with cholesterol or cholesterol being preferred.

[0065] The aliphatic hydrocarbon group with 1 to 40 carbon atoms can be straight-chain or branched. The aliphatic hydrocarbon group can be saturated or unsaturated. When it is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds it contains is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, preferably carbon-carbon double bonds. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes alkyl, alkenyl, and alkynyl groups, preferably alkyl or alkenyl groups. Examples of aliphatic hydrocarbon groups with 1 to 40 carbon atoms include: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, triadecyl, tetradecyl, ... Alkenyl, pentadecenyl, hexadecenyl, heptadecanyl, octadecenyl, nonadecenyl, eicosenecanyl, icosenecanyl, icosenecanyl, dodecanediyl, tridecadecanyl, tetradecadecanyl, pentadecadecanyl, hexadecadecanyl, heptadecanyl, octadecadecanyl, nonadecadecanyl, eicosenecanyl, icosenecanyl, octadectrienyl, eicoseneteratenyl, eicosenepentenyl, eicosenehexatenyl, isostearyl, 1-hexylheptyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, etc. The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably tridecyl, pentadecyl, heptadecanyl, nonadecanyl, heptadecanyl-enyl, heptadecanyl-dienyl, or 1-hexylnonyl, and particularly preferably tridecyl, heptadecanyl, heptadecanyl-enyl, or heptadecanyl-dienyl.

[0066] In one embodiment of the present invention, R 3a and R 3b The aliphatic hydrocarbon group representing 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) is derived from fatty acids. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). As a specific example of an aliphatic hydrocarbon group, when linoleic acid is used as the fatty acid, it is a heptadecanediol, and when oleic acid is used as the fatty acid, it is a heptadecanedenyl.

[0067] R 3a and R 3bThe alkyl group having 3 to 40 carbon atoms in a cyclopropane ring refers to an alkyl chain having at least one cyclopropane ring with 3 to 40 carbon atoms. The 3 to 40 carbon atoms in the alkyl group do not include the carbon atoms of the cyclopropane ring. Preferably, the alkyl group has one cyclopropane ring. 3a and R 3b The alkyl group having 3 to 40 carbon atoms in the cyclopropane ring is preferably the group represented by formula (4):

[0068] [Chemistry 7]

[0069] (in equation (4),) * indicates the bonding location, and b and c are each an independent integer, and the sum of b and c is 2 to 39. Preferably, b is an integer from 1 to 20, and c is an integer from 1 to 19. More preferably, b is an integer from 2 to 18, even more preferably, an integer from 3 to 17, and particularly preferably, an integer from 4 to 12. More preferably, c is an integer from 3 to 15, even more preferably, an integer from 3 to 11, and particularly preferably, an integer from 3 to 9. Examples of the groups represented by formula (4) include 7-(2-octylcyclopropyl)heptyl.

[0070] In equation (3c), R 3cThe aliphatic hydrocarbon group representing 2 to 20 carbon atoms can be linear or branched. This aliphatic hydrocarbon group can be saturated or unsaturated. When it is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds it contains is typically 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, preferably carbon-carbon double bonds. The number of carbon atoms in this aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, further preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes alkyl, alkenyl, alkynyl, etc., preferably alkyl or alkenyl, and more preferably alkyl. Examples of aliphatic hydrocarbon groups with 2 to 20 carbon atoms include: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecylenyl, tridecylenyl, tetradecylenyl, pentadecylenyl, hexadecylenyl, heptadecanyl, and octadecyl. Alkenyl, 19-carbon-alkenyl, 10-carbon-alkenyl, 11-carbon-alkenyl, 12-carbon-alkenyl, 12-carbon-dienyl, 13-carbon-dienyl, 14-carbon-dienyl, 15-carbon-dienyl, 16-carbon-dienyl, 17-carbon-dienyl, 18-carbon-dienyl, 19-carbon-dienyl, 10-carbon-dienyl, 10-carbon-trienyl, 10-carbon-tetraenyl, 10-carbon-pentaenyl, isostearyl, 1-hexylheptyl, 1-ethylnonyl, 1-butylnonyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 3-octylundecyl, etc. The aliphatic hydrocarbon groups with 2 to 20 carbon atoms are preferably tridecyl, pentadecyl, heptadecanyl, nonadecanyl, heptadecanyl, heptadecanyl, and 1-hexylnonyl, and particularly preferably tridecyl, heptadecanyl, heptadecanyl, and heptadecanyldienyl.

[0071] In formula (3c), a is preferably an integer from 3 to 9, more preferably an integer from 3 to 7, even more preferably an integer from 5 to 7, and most preferably 5 or 7.

[0072] R 3a With R 3b They can be the same or different, but R is preferred. 3a To be with R 3b Same group.

[0073] In one embodiment of the present invention, R 1a With R 1b Same, X a With X b Same, R 2a With R 2b Same, Y a With Y bSame, Z a With Z b Same, R 3a With R 3b same.

[0074] As a preferred example of ionic lipid (1), the following ionic lipids can be cited. [Ionic lipids (1-1a)] An ionic lipid (1): R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently a noncyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinediyl) having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups; R 2a and R 2b Each is independently an alkylene group having 1 to 8 carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is an ester bond or an amide bond independently; Z a and Z b Each is independently a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-) derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms; na and nb are each independently 0 or 1; R 3a and R 3b Each of these residues is independently derived from a reaction product of a fat-soluble vitamin (e.g., tocopherol) with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl), or a group represented by formula (3c): R 3c -O-CO-(CH2) a -*(3c) (In equation (3c), * indicates the bonding location. R 3c This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms, and 'a' represents an integer from 2 to 10.

[0075] [Ionic lipids (1-1b)] An ionic lipid (1): R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently a noncyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinediyl) having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups; R 2a and R 2b Each is independently an alkylene group having 1 to 8 carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is an ester bond or an amide bond independently; Z a and Z b Each is independently a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-) derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms; na and nb are each independently 0 or 1; R 3a and R 3b Each is independently derived from the residues of a fat-soluble vitamin with a hydroxyl group (e.g., tocopherol) reacted with succinic anhydride or glutaric anhydride, or from an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl).

[0076] [Ionic lipids (1-2a)] An ionic lipid (1): R 1a and R 1b Each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently a noncyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 3 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinediyl) having 2 to 5 carbon atoms and 1 tertiary amino group; R 2a and R 2b Each is independently an alkylene group having 6 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y bEach is an ester bond or an amide bond independently; Z a and Z b Each is an independent divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and possibly heteroatoms (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-). na and nb are each independently 0 or 1; R 3a and R 3b Each of these groups is independently a residue derived from the reaction of a fat-soluble vitamin (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group with 13 to 19 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl) or a group represented by formula (3c): R 3c -O-CO-(CH2) a -*(3c) (In equation (3c), * indicates the bonding location. R 3c Aliphatic hydrocarbon groups representing 8 to 20 carbon atoms, and 'a' represents an integer from 3 to 9.

[0077] [Ionic lipids (1-2b)] An ionic lipid (1): R 1a and R 1b Each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently a noncyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 3 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinediyl) having 2 to 5 carbon atoms and 1 tertiary amino group; R 2a and R 2b Each is independently an alkylene group having 6 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is an ester bond or an amide bond independently; Z a and Z b Each is independently a divalent group (e.g. -C6H4-CH2-, -CH2-C6H4-CH2-) of an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and possibly heteroatoms; na and nb are each independently 0 or 1; R 3a and R 3b Each is independently derived from the residue of a fat-soluble vitamin (e.g., tocopherol) with hydroxyl groups and succinic anhydride, or from an aliphatic hydrocarbon group with 13 to 19 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl).

[0078] [Ionic lipids (1-3a)] An ionic lipid (1): R 1a and R 1b Each is independently an alkylene group having 1 to 2 carbon atoms (i.e., methylene or ethylene); X a and X b Each independently as X 1 or X 2 :

[0079] [Chemistry 8]

[0080] (where R is in the formula) 5 It is an alkyl group having 1 to 3 carbon atoms (e.g., methyl).

[0081] [Chemistry 9]

[0082] (In the formula, p is 1 or 2.) R 2a and R 2b Each is independently an alkylene group having 4 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is an ester bond or an amide bond independently; Z a and Z b Each independently as Z 1 :

[0083] [Chemistry 10]

[0084] (In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (preferably 0), and u represents R.) 4 Each substituent can be represented independently. na and nb are each independently 0 or 1; R 3a and R 3bEach of these groups is independently a residue derived from the reaction of a fat-soluble vitamin (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group with 13 to 17 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl) or a group represented by formula (3c): R 3c -O-CO-(CH2) a -* (3c) (In equation (3c), * indicates the bonding location. R 3c The aliphatic hydrocarbon group representing 13 to 19 carbon atoms (preferably an aliphatic hydrocarbon group representing 3 to 17 carbon atoms), and 'a' represents an integer from 5 to 7 (preferably 5 or 7).

[0085] [Ionic lipids (1-3b)] An ionic lipid (1): R 1a and R 1b Each is independently an alkylene group having 1 to 2 carbon atoms (i.e., methylene or ethylene); X a and X b Each independently as X 1 or X 2 :

[0086] [Chemistry 11]

[0087] (where R is in the formula) 5 It is an alkyl group having 1 to 3 carbon atoms (e.g., methyl).

[0088] [Chemistry 12]

[0089] (In the formula, p is 1 or 2.) R 2a and R 2b Each is independently an alkylene group having 4 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is an ester bond or an amide bond independently; Z a and Z b Each independently as Z 1 :

[0090] [Chemistry 13]

[0091] (In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (preferably 0), and u represents R.) 4 Each substituent can be represented independently. na and nb are each independently 0 or 1; R 3a and R 3b Each is independently derived from the residue of a fat-soluble vitamin (e.g., tocopherol) with hydroxyl groups and succinic anhydride, or from an aliphatic hydrocarbon group with 13 to 17 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl).

[0092] Specific examples of ionic lipids (1) include SS-OP, SS-EC, O-Ph-P3C1, O-Ph-P4C1, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, B-2, B-2-5, TS-P4C2, L-P4C2, and O-P4C2, as described in Tables 1-1 to 1-3 below.

[0093] [Table 1-1]

[0094] [Table 1-2]

[0095] [Table 1-3]

[0096] Furthermore, specific examples of ionic lipids (1) include Lipid1 to Lipid20 as described in WO2021 / 195529A2.

[0097] The ionic lipid (1) is preferably at least one selected from the group consisting of SS-OP, SS-EC, O-Ph-P3C1, O-Ph-P4C1, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, B-2, B-2-5, TS-P4C2, L-P4C2, O-P4C2 and compound (5), more preferably at least one selected from the group consisting of SS-OP, SS-EC and compound (5), and even more preferably SS-OP and / or SS-EC.

[0098] Ionic lipids (1) can be manufactured by known methods (e.g., the methods described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, WO2021 / 195529A2).

[0099] From the viewpoint of lipid solubility, the concentration of ionic lipids in the alcohol solution is preferably 0.1 to 40 mM, more preferably 1 to 20 mM.

[0100] Sterols The alcohol solution used in step (a) contains sterols. Only one type of sterol may be used, or two or more may be used in combination. Sterols are components that regulate the fluidity of the lipid membrane of lipid nanoparticles. Examples of sterols include: cholesterol, lanosterol, phytosterol, zymosterol, dihydrozymostenol, sterol, stigmasterol, dihydrolanosterol, and 7-dehydrocholesterol. The sterol is preferably at least one selected from the group consisting of cholesterol, lanosterol, and phytosterols, and more preferably cholesterol.

[0101] From the viewpoint of lipid solubility, the concentration of sterols in the alcohol solution is preferably 0.1 to 40 mM, more preferably 0.5 to 20 mM.

[0102] From the viewpoint of the stability of nucleic acid encapsulated lipid nanoparticles and the efficiency of nucleic acid delivery, the molar ratio of sterol to ionic lipid (sterol / ionic lipid) used in step (a) is preferably 0.01 to 1.0, more preferably 0.1 to 0.9.

[0103] PEG lipids The alcohol solution used in step (a) contains PEG lipids. In this specification, "PEG lipid" refers to a lipid having polyethylene glycol (PEG) chains. One type of PEG lipid may be used, or two or more may be used in combination. PEG lipids coat the surface of lipid nanoparticles with hydrophilic PEG chains, serving as a stabilizer to inhibit particle aggregation or to inhibit the interaction between biological components and particles when administered to a living organism.

[0104] The PEG chains can have any molecular weight. In some embodiments, the PEG chains have a number average molecular weight of 200 to 10,000, and can be straight or branched. The number average molecular weight can be determined by MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry.

[0105] Examples of PEG lipids include PEG-phospholipids, PEG-ceramides, PEG-diacylglycerols, and PEG-cholesterols. PEG lipids are preferably diacylglycerols (PEG) with a number-average molecular weight of 1,000 to 10,000 for the PEG chain; more preferably, dimyristoylglycerols (PEG) with a number-average molecular weight of 1,000 to 10,000 (particularly 1,2-dimyristoyl-rac-glycerol-3-methylpolyoxyethylene, i.e., compounds in which a methoxy polyethylene glycol chain is added to 1,2-dimyristoyl-rac-glycerol) and / or distearate glycerols (PEG) with a number-average molecular weight of 1,000 to 10,000 for the PEG chain; and even more preferably, dimyristoylglycerols (PEG) with a number-average molecular weight of 1,000 to 10,000 for the PEG chain (particularly 1,2-dimyristoyl-rac-glycerol-3-methylpolyoxyethylene).

[0106] From the viewpoint of lipid solubility, the concentration of PEG lipid in the alcohol solution is preferably 0.01 to 10 mM, more preferably 0.05 to 5 mM.

[0107] From the perspective of the stability of nucleic acid encapsulated lipid nanoparticles and the efficiency of nucleic acid delivery, the molar ratio of PEG lipid to ionic lipid (PEG lipid / ionic lipid) used in step (a) is preferably 0.001 to 0.5, more preferably 0.01 to 0.05.

[0108] Phospholipids The alcohol solution used in step (a) may further contain phospholipids. Only one type of phospholipid may be used, or two or more types may be used in combination.

[0109] Examples of phospholipids include: 1,2-diacyl-sn-glycerol-3-phosphocholine (PC), 1,2-diacyl-sn-glycerol-3-phosphatidylethanolamine (PE), 1,2-diacyl-sn-glycerol-3-phosphatidylserine (PS), 1,2-diacyl-sn-glycerol-3-phosphatidylglycerol (PG), 1,2-diacyl-sn-glycerol-3-phosphatidylglycerol (PA), or hemolytic forms of these.

[0110] Specific examples of phospholipids include: 1,2-Didecanoyl-sn-glycerol-3-phosphate choline (DDPC) 1,2-Dilauroyl-sn-glycerol-3-phosphocholine (DLPC) 1,2-Dimyristico-sn-glycerol-3-phosphocholine (DMPC) 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC) 1,2-Distearyl-sn-glycerol-3-phosphocholine (DSPC) 1,2-Dioleoyl-sn-glycerol-3-phosphate choline (DOPC) 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (DLoPC) 1,2-Disorhoyl-sn-glycerol-3-phosphorylcholine (DEPC) 1-Myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine (MPPC) 1-Myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine (MSPC) 1-Palmitoyl-2-myristoyl-sn-glycerol-3-phosphocholine (PMPC) 1-Palmitoyl-2-stearoyl-sn-glycerol-3-phosphocholine (PSPC) 1-Palmyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) 1-Stearyl-2-oleoyl-sn-glycerol-3-phosphocholine (SOPC).

[0111] Phospholipids in which PC is appropriately replaced with PE, PS, PG, or PA can be used. In other words, the following can be used: (i) Phospholipids in which “sn-glycerol-3-phosphocholine” in the above-mentioned specific examples of phospholipids is replaced with “sn-glycerol-3-phosphatidylethanolamine” (e.g., 1,2-dicepanoyl-sn-glycerol-3-phosphatidylethanolamine); (ii) Phospholipids in which “sn-glycerol-3-phosphocholine” in the above-mentioned specific examples of phospholipids is replaced with “sn-glycerol-3-phosphatidylserine” (e.g., 1,2-decanoyl-sn-glycerol-3-phosphatidylserine); (iii) Phospholipids in which the specific example of the above phospholipids is replaced with "sn-glycerol-3-phosphocholine" (e.g., 1,2-dicepanoyl-sn-glycerol-3-phosphatidylglycerol) or (iv) Phospholipids in which “sn-glycerol-3-phosphocholine” in the above-mentioned specific examples of phospholipids is replaced with “sn-glycerol-3-phosphatidic acid” (e.g., 1,2-dicacyl-sn-glycerol-3-phosphatidic acid).

[0112] The phospholipids used in this invention are preferably PC and / or PE, more preferably at least one selected from the group consisting of DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), and POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine), further preferably DOPC and / or DOPE, and particularly preferably DOPC or DOPE.

[0113] When phospholipids are used in step (a), from the viewpoint of lipid solubility, the concentration of phospholipids in the alcohol solution is preferably 0.1 to 25 mM, more preferably 0.1 to 20 mM.

[0114] When using phospholipids in step (a), from the viewpoint of the stability of nucleic acid encapsulated lipid nanoparticles and the efficiency of nucleic acid delivery, the molar ratio of phospholipids to ionic lipids (phospholipids / ionic lipids) is preferably 0.01 to 1.0, more preferably 0.1 to 0.9.

[0115] alcohol Examples of alcohols used in the alcohol solution in step (a) include ethanol and tert-butanol. Only one type of alcohol may be used, or two or more may be used in combination. From the viewpoint of lipid solubility and the manufacture of nucleic acid-encapsulated lipid nanoparticles, ethanol and / or tert-butanol are preferred, with ethanol being more preferred.

[0116] Citrate buffer From the viewpoint of the particle size of nucleic acid encapsulated lipid nanoparticles and the nucleic acid encapsulation rate, the pH of the citrate buffer used in step (a) is 3 to 6.5, preferably 3 to 6, and more preferably 4 to 6.

[0117] The citrate buffer includes citric acid and its salts (specifically, trisodium citrate). From the viewpoint of nucleic acid stability, the total concentration of citric acid and its salts in the citrate buffer used in step (a) is preferably 10 to 100 mM, more preferably 10 to 80 mM.

[0118] Nucleic acid Examples of nucleic acids used in this invention include, but are not limited to, DNA, RNA, chimeric RNA nucleic acids, and DNA / RNA hybrids. Furthermore, nucleic acids can be of any type with 1 to 3 strands, preferably single-stranded or double-stranded. Nucleic acids can also be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers with a non-nucleotide backbone (e.g., commercially available peptide nucleic acids (PNAs)), or other oligomers with specific bonds (wherein the oligomer contains nucleotides configured to allow base pairing and base adhesion as found in DNA and RNA). Furthermore, nucleic acids can also be, for example, nucleic acids with known modifications, nucleic acids with labels known in the field, capped nucleic acids, methylated nucleic acids, nucleic acids in which one or more natural nucleotides are replaced by analogs, nucleic acids with intramolecularly modified nucleotides, nucleic acids with non-charged bonds (e.g., methanesulfonates, triphosphates, aminophosphates, carbamates, etc.), nucleic acids with charged bonds or sulfur-containing bonds (e.g., thiophosphates, dithiophosphates, etc.), nucleic acids with side chains such as proteins (e.g., nucleases, nuclease / inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (e.g., monosaccharides, etc.), nucleic acids with intercalated compounds (e.g., acridine, psoralen, etc.), nucleic acids containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), nucleic acids containing alkylating agents, and nucleic acids with modified bonds (e.g., α-terminal epimers, etc.).

[0119] The type of DNA used in this invention is not particularly limited and can be appropriately selected according to the intended use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligonucleotides, etc. Plasmid DNA, cDNA, and antisense DNA are preferred, and plasmid DNA is more preferred. Circular DNA such as plasmid DNA can also be digested by appropriate restriction enzymes and used as linear DNA.

[0120] The type of RNA used in this invention is not particularly limited and can be appropriately selected according to the purpose of use. Examples include: siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., with siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon being preferred.

[0121] The nucleic acids used in this invention are preferably purified according to methods commonly used by those skilled in the art.

[0122] Regarding the nucleic acid in the citrate buffer used in step (a), the preferred concentration is determined relative to the total lipid concentration in the alcohol. From this viewpoint, the nucleic acid concentration in the citrate buffer is preferably 0.5 to 400 μg / mL, more preferably 1 to 200 μg / mL.

[0123] When the molar ratio of total amino groups to phosphate groups of nucleic acids in the ionic lipids used in step (a) is low (total amino groups of ionic lipids / phosphate groups of nucleic acids (in this specification, this may be referred to as "N / P ratio"), nucleic acid encapsulation efficiency and gene delivery efficiency decrease. Therefore, an N / P ratio of 7 or higher is preferred. On the other hand, a high N / P ratio may exhibit toxicity originating from the ionic lipids. Therefore, an N / P ratio of 7 to 130 is more preferred, and 17 to 70 is even more preferred.

[0124] In step (a) of the present invention, a suspension of nucleic acid-encapsulated lipid nanoparticles is prepared by mixing an ionic lipid having a tertiary amino group, an alcoholic solution containing sterols and PEG lipids (hereinafter referred to as "alcoholic solution of lipids"), and a citrate buffer solution with a pH of 3 to 6.5 in which nucleic acids are dispersed (hereinafter referred to as "buffer solution containing nucleic acids"). Furthermore, in the buffer solution containing nucleic acids, the nucleic acids may be dissolved in the buffer solution or may not be dissolved. That is, the buffer solution containing nucleic acids may be a nucleic acid solution in which nucleic acids are dispersed in a dissolved state, or it may be a dispersion in which the nucleic acids are dispersed without being dissolved.

[0125] The mixing in step (a) is preferably performed using a device containing microchannels or a vortex, and more preferably using a device containing microchannels. Examples of devices containing microchannels include: the NanoAssemblr (registered trademark) ultra-high-speed nanomedicine fabrication device (Precision NanoSystems), the fully automated library synthesis system ALiS (Particle Works), and the ANP System (Particle Works).

[0126] When mixing a buffer solution containing nucleic acid with an alcoholic solution containing lipids, from the viewpoint of the particle size of nucleic acid-encapsulated lipid nanoparticles and the nucleic acid encapsulation efficiency, the volume ratio (buffer solution containing nucleic acid: alcoholic solution containing lipids) is preferably 1:1 to 20:1, more preferably 2:1 to 12:1. When mixing the buffer solution containing nucleic acid and the alcoholic solution containing lipids using a device containing microfluidics, from the viewpoint of the particle size of nucleic acid-encapsulated lipid nanoparticles, the total flow rate of the buffer solution containing nucleic acid and the alcoholic solution containing lipids is preferably 1 to 20 mL / min, more preferably 10 to 15 mL / min. Here, "total flow rate of the buffer solution containing nucleic acid and the alcoholic solution containing lipids" refers to the sum of the flow rates of the buffer solution containing nucleic acid and the alcoholic solution containing lipids. Furthermore, for example, when the volume ratio of the buffer solution containing nucleic acid to the alcoholic solution containing lipids is 3:1 and the total flow rate is 4 mL / min, the flow rate of the buffer solution containing nucleic acid is 3 mL / min and the flow rate of the alcoholic solution containing lipids is 1 mL / min.

[0127] From the viewpoint of ensuring that the lipid state during the formation of nucleic acid-encapsulated lipid nanoparticles is constant and that nucleic acid-encapsulated lipid nanoparticles can be prepared with high reproducibility, the temperature for mixing the buffer solution containing nucleic acid and the alcohol solution containing lipids is preferably 0–50°C, and more preferably 10–30°C.

[0128] When using a device containing microchannels, the cross-sectional shape of the microchannels can be any shape, such as circular or rectangular. When using a device containing square microchannels with a rectangular cross-sectional shape, from the viewpoint of the particle size of nucleic acid encapsulated lipid nanoparticles and the nucleic acid encapsulation efficiency, the longitudinal and transverse lengths of the inner diameter of the square microchannels are each preferably 50 to 700 μm, more preferably 70 to 500 μm.

[0129] The nucleic acid concentration in the suspension of nucleic acid-encapsulated lipid nanoparticles obtained in step (a) is preferably 0.01 to 100 μg / mL, more preferably 0.05 to 50 μg / mL.

[0130] Process (b) In step (b) of the present invention, the dispersion medium of the suspension is replaced with the Tris buffer by ultrafiltration concentration of the suspension of nucleic acid-encapsulated lipid nanoparticles and dilution with Tris buffer at pH 5.2 to 9.0.

[0131] The dilution and concentration in step (b) may each be performed only once or multiple times. Preferably, the dilution and concentration are performed multiple times.

[0132] The dilution and concentration in step (b) can be performed in any order. In other words, (i) the suspension of nucleic acid-encapsulated lipid nanoparticles can be diluted by adding the Tris buffer and then concentrated by ultrafiltration; (ii) the suspension of nucleic acid-encapsulated lipid nanoparticles can be concentrated by ultrafiltration and then diluted by adding the Tris buffer to the resulting concentrate. Wherein, after concentration, the dilution is performed, and the dispersion medium of the suspension of nucleic acid-encapsulated lipid nanoparticles is replaced with the Tris buffer to produce a suspension of nucleic acid-encapsulated lipid nanoparticles with good preservation stability (i.e., the Tris buffer containing nucleic acid-encapsulated lipid nanoparticles).

[0133] The ultrafiltration in step (b) is preferably centrifugal ultrafiltration. Here, "centrifugal ultrafiltration" refers to ultrafiltration that utilizes centrifugal force.

[0134] Between steps (a) and (b), step (c) can be performed, in which the dispersion medium of the suspension of nucleic acid-encapsulated lipid nanoparticles prepared in step (a) is replaced with a dispersion medium other than the Tris buffer (e.g., PBS) by ultrafiltration concentration and dilution. In this case, the dispersion medium of the suspension of nucleic acid-encapsulated lipid nanoparticles prepared in step (c) is replaced with the Tris buffer in step (b). Similar to step (b), the dilution and concentration in step (c) can each be performed only once or multiple times. Furthermore, similar to step (b), the dilution and concentration in step (c) can be performed in any order. For ease of operation, step (c) is preferably omitted. In other words, in step (b), it is preferable to replace the dispersion medium of the suspension of nucleic acid-encapsulated lipid nanoparticles prepared in step (a) with the Tris buffer.

[0135] Tris buffer From the viewpoint of particle size and nucleic acid encapsulation efficiency, the pH of the Tris buffer used in step (b) is 5.2–9.0, more preferably 5.5–8.5, and even more preferably 6.0–8.0. The Tris buffer may contain sodium chloride. When sodium chloride is present, its concentration in the Tris buffer is preferably 10–154 mM, more preferably 30–154 mM.

[0136] Examples of Tris buffers include: Tris-buffered saline (TBS), Tris-hydrochloric acid buffer, TE buffer, TAE buffer, and TBE buffer. Tris-buffered saline (TBS) contains tris(hydroxymethyl)aminomethane and its hydrochloride salt, as well as sodium chloride. Tris-hydrochloric acid buffer contains tris(hydroxymethyl)aminomethane and its hydrochloride salt. TE buffer contains tris(hydroxymethyl)aminomethane and its hydrochloride salt, as well as ethylenediaminetetraacetic acid (EDTA). TAE buffer contains tris(hydroxymethyl)aminomethane and its acetate salt, as well as EDTA. TBE buffer contains tris(hydroxymethyl)aminomethane and its borate, as well as EDTA. Tris-buffered saline (TBS) is preferred.

[0137] From the viewpoint of nucleic acid encapsulation efficiency, the total concentration of tris(hydroxymethyl)aminomethane and its salt in the Tris buffer used in step (b) is preferably 10 to 200 mM, more preferably 10 to 100 mM.

[0138] When using Tris-buffered saline (TBS) in step (b), from the viewpoint of nucleic acid stability, the concentration of sodium chloride in the TBS is preferably 140–160 mM.

[0139] The nucleic acid concentration in the suspension of nucleic acid-encapsulated lipid nanoparticles obtained in step (b) is preferably 0.01 to 100 μg / mL, more preferably 0.05 to 50 μg / mL.

[0140] Nucleic acid encapsulated lipid nanoparticles The nucleic acid encapsulation efficiency of the nucleic acid-encapsulated lipid nanoparticles manufactured by the method of the present invention is preferably 45% or higher. The nucleic acid encapsulation efficiency can be determined using Ribogreen (registered trademark) assay.

[0141] The particle size of the nucleic acid-encapsulated lipid nanoparticles manufactured by the method of the present invention is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm, and even more preferably 50 nm to 120 nm. The particle size can be determined, for example, using a particle size distribution measuring device such as the Zetasizer Nano (Malvern). In this specification, "particle size" refers to the average particle size (number average) determined by dynamic light scattering.

[0142] The surface potential (ZETA potential) of the nucleic acid-encapsulated lipid nanoparticles manufactured by the method of the present invention is not particularly limited, but is preferably -15 to +15 mV, more preferably -10 to +10 mV. In existing gene delivery methods, particles with positively charged surface potentials are mainly used. While this is useful for promoting electrostatic interactions between negatively charged cell surfaces and heparan sulfate, thus promoting cell uptake, a positive surface potential may inhibit the release of nucleic acids from the carrier due to interactions with the delivered nucleic acid, and inhibit protein synthesis due to interactions between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface potential to the aforementioned range. The surface potential can be measured, for example, using a ZETA potential measuring device such as the Zetasizer Nano. The surface potential of the lipid nanoparticles can be adjusted by the composition of the constituent components of the lipid nanoparticles.

[0143] Nucleic acid-encapsulated lipid nanoparticles manufactured using the method of the present invention can, for example, be administered into a living organism for the purpose of preventing and / or treating diseases. Therefore, the nucleic acid used in the present invention is preferably a nucleic acid that has preventive and / or therapeutic activity against a specific disease (a nucleic acid for prevention / treatment). Examples of such nucleic acids include, for instance, nucleic acids used in so-called gene therapy.

[0144] Preservation of nucleic acid-encapsulated lipid nanoparticles The storage temperature of the nucleic acid encapsulated lipid nanoparticles manufactured by the method of the present invention is preferably 0℃~50℃, more preferably 0℃~30℃, further preferably 0℃~20℃, particularly preferably 0℃~10℃, and most preferably 0℃~5℃.

[0145] Nucleic acid-encapsulated lipid nanoparticles manufactured using the method of the present invention are preferably cold-stored. In other words, the preferred method of the present invention is a method for manufacturing nucleic acid-encapsulated lipid nanoparticles for cold storage. Herein, "nucleic acid-encapsulated lipid nanoparticles for cold storage" refers to nucleic acid-encapsulated lipid nanoparticles stored at 0°C to 20°C. The storage temperature for nucleic acid-encapsulated lipid nanoparticles for cold storage is preferably 0°C to 10°C, more preferably 0°C to 5°C.

[0146] The storage stability of nucleic acid-encapsulated lipid nanoparticles can be evaluated by measuring the particle size, polydispersity index (PdI), encapsulation efficiency of nucleic acids (e.g., mRNA), and nucleic acid delivery efficiency in vitro or in vivo before and after storage. In evaluating the nucleic acid delivery efficiency in vitro or in vivo, when stored at a temperature of 0°C to 50°C for 7 days, a reduction in nucleic acid delivery efficiency before and after storage is preferably less than 30%; when stored at a temperature of 0°C to 5°C for 30 days, a reduction in nucleic acid delivery efficiency before and after storage is more preferably less than 20%.

[0147] Methods of introducing nucleic acids into cells This invention also provides: (i) A method for introducing the nucleic acid into the cells, comprising a step of contacting nucleic acid-encapsulated lipid nanoparticles manufactured by the method with cells in vitro; and (ii) A method for introducing the nucleic acid into target cells of the organism, comprising the step of administering nucleic acid-encapsulated lipid nanoparticles manufactured by the above method to the organism.

[0148] The following details the process of bringing nucleic acid-encapsulated lipid nanoparticles into contact with cells in vitro. Several days before contact with nucleic acid-encapsulated lipid nanoparticles, cells are suspended in a suitable culture medium and cultured under appropriate conditions. Cells may or may not be in the proliferative phase upon contact with the nucleic acid-encapsulated lipid nanoparticles.

[0149] The culture medium used for this contact can be a serum-containing medium or a serum-free medium. The serum concentration in the medium is preferably 30% by weight or less, more preferably 20% by weight or less. When the culture medium contains excess serum or other proteins, there is a possibility that the contact between the nucleic acid-encapsulated lipid nanoparticles and the cells may be hindered.

[0150] There is no specific limit to the cell density at this contact point; it can be appropriately set considering factors such as cell type, but it is typically 1×10⁻⁶. 4 ~1×10 7 The range of cells / mL.

[0151] A suspension of nucleic acid-encapsulated lipid nanoparticles is added to cells as described above. The amount of this suspension added is not particularly limited and can be appropriately set considering factors such as the number of cells. As long as the target nucleic acid can be introduced into the cells, the concentration of the nucleic acid-encapsulated lipid nanoparticles in contact with the cells is not particularly limited. The lipid concentration is typically 1–300 nmol / mL, preferably 10–200 nmol / mL; the nucleic acid concentration is typically 0.01–100 μg / mL, preferably 0.05–10 μg / mL.

[0152] After adding the above suspension to the cells, the cells are cultured. The temperature, humidity, CO2 concentration, etc., during culture can be appropriately set depending on the cell type. When the cells are derived from mammals, the typical temperature is approximately 37°C, humidity is approximately 95%, and CO2 concentration is approximately 5%. Furthermore, the culture time can also be appropriately set considering the cell type and other conditions, typically 0.1–96 hours, preferably 0.2–72 hours, and more preferably 0.5–48 hours. If the culture time is too short, nucleic acids cannot be sufficiently introduced into the cells; if the culture time is too long, the cells may become weak.

[0153] Through the above culture, nucleic acids are introduced into the cells, but it is preferable to replace the culture medium with fresh culture medium or add fresh culture medium to the culture medium for further culture. When the cells are derived from mammalian cells, the fresh culture medium preferably contains serum or nutrient factors.

[0154] Furthermore, as described above, by using nucleic acid-encapsulated lipid nanoparticles, nucleic acids can be introduced into target cells of an organism not only in vitro but also in vivo. That is, by administering nucleic acid-encapsulated lipid nanoparticles into an organism, the nanoparticles reach and contact the target cells, and the nucleic acids within the lipid nanoparticles are introduced into the cells within the organism. There are no particular limitations on the objects to which these nucleic acid-encapsulated lipid nanoparticles can be administered; examples include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cattle), birds (e.g., chickens, ostriches), amphibians (e.g., frogs), and fish (e.g., zebrafish, medaka), invertebrates such as insects (e.g., silkworms, moths, fruit flies), and plants. Humans or other mammals are preferred as the recipients of these nucleic acid-encapsulated lipid nanoparticles.

[0155] There are no particular limitations on the types of target cells. By using nucleic acid-encapsulated lipid nanoparticles, nucleic acids can be introduced into cells in various tissues (such as liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0156] For methods of administering nucleic acid-encapsulated lipid nanoparticles to target organisms (e.g., vertebrates, invertebrates, etc.), there are no particular limitations on the method of introducing the compound into the lipid nanoparticles into the cells, as long as the lipid nanoparticles reach and come into contact with the target cells. Well-known administration methods (e.g., oral administration, non-oral administration (e.g., intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected based on the type and location of the target cells. As long as the amount of nucleic acid-encapsulated lipid nanoparticles administered is within the range that allows for the introduction of nucleic acid into the cells, there are no particular limitations on the dosage. The dosage can be appropriately selected based on the type of target organism, administration method, type of compound introduced, type and location of the target cells.

[0157] Method for manufacturing pharmaceutical compositions The nucleic acid-encapsulated lipid nanoparticles manufactured using the method of the present invention can be used as drug delivery systems for selectively delivering nucleic acids and the like into specific cells. For example, they are useful for DNA vaccines based on the introduction of antigen genes into dendritic cells, gene therapy drugs for tumors, and nucleic acid drugs that inhibit the expression of target genes using RNA interference. Therefore, the present invention also provides a method for manufacturing a pharmaceutical composition comprising the above-described method.

[0158] Nucleic acid delivery agent When using nucleic acid-encapsulated lipid nanoparticles manufactured by the method of the present invention as nucleic acid delivery agents, the nucleic acid-encapsulated lipid nanoparticles can be formulated using conventional methods.

[0159] When this nucleic acid delivery agent is provided as a research reagent, it can be provided directly as nucleic acid-encapsulated lipid nanoparticles, or as a sterile solution or suspension of nucleic acid-encapsulated lipid nanoparticles in, for example, water or other physiologically acceptable liquids (e.g., water-soluble solvents (e.g., malate buffer), organic solvents (e.g., ethanol, methanol, DMSO, tert-butanol), or mixtures of water-soluble and organic solvents). The nucleic acid delivery agent may appropriately contain physiologically acceptable additives known to the public (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0160] Furthermore, when this nucleic acid delivery agent is provided as a pharmaceutical product, it can be used directly by encapsulating lipid nanoparticles with nucleic acids, or used with pharmaceutically acceptable and well-known additives (such as carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.), by mixing in the unit dosage form required by generally recognized formulation implementation, and manufactured as an oral formulation (e.g., tablets, capsules, etc.) or a non-oral formulation (e.g., injections, sprays, etc.), preferably manufactured as a non-oral formulation (more preferably an injection).

[0161] In addition to being used for adults, nucleic acid delivery agents can also be used as pediatric preparations. Example

[0162] The present invention will be described in more detail below with examples, but the present invention is not limited to the following examples.

[0163] In the following examples, the ionic lipids represented by formula (1) are shown by the names described in the table above. Nucleic acid-encapsulated lipid nanoparticles may be simply referred to as "nucleic acid-encapsulated nanoparticles." Furthermore, the meanings of the abbreviations used in the following examples are each as described below. Cholesterol DMG-PEG2000: 1,2-Dimyristoyl-rac-glycerol-3-methylpolyoxyethylene (N-average molecular weight of PEG chain: 2000) DOPC: 1,2-Dioleoyl-sn-glycerol-3-phosphocholine DOPE: 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine FBS: Fetal bovine serum LNP: Lipid nanoparticles MES: 2-Morpholinoethanesulfonic acid N / P ratio: The molar ratio of total amino groups in ionic lipids to phosphate groups in nucleic acids (total amino groups in ionic lipids / phosphate groups in nucleic acids). PBS: Phosphate-buffered saline TBS: Tris-buffered saline Tris: Tris(hydroxymethyl)aminomethane

[0164] Example 1 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0165] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 5.0) and 450 μL of lipid-containing ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid-containing ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0166] After adding 1000 μL of TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.4) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0167] The concentrate obtained was diluted to 4 mL using the TBS described above, and then ultrafiltered again under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate it to approximately 200 μL. After repeating this dilution and concentration process twice more, 0.2 mL of the above TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0168] Comparative Example 1 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0169] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in malate buffer (total concentration of malate and its salts: 20 mM, pH: 3.0) and 450 μL of lipid ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0170] After adding 1000 μL of MES buffer (total concentration of MES and its salts: 20 mM, pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0171] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 200 μL. This dilution and concentration process was repeated twice more. Then, 0.2 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0172] Comparative Example 2 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0173] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in malate buffer (total concentration of malate and its salts: 20 mM, pH: 5.0) and 450 μL of lipid ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0174] After adding 1000 μL of MES buffer (total concentration of MES and its salts: 20 mM, pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0175] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 200 μL. This dilution and concentration process was repeated twice more. Then, 0.2 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0176] Comparative Example 3 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0177] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in malate buffer (total concentration of malate and its salts: 20 mM, pH: 3.0) and 450 μL of lipid ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), at a flow rate (volume ratio) of 3:1 for the mRNA-containing buffer to the lipid ethanol solution, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C, the mRNA-containing buffer and the lipid ethanol solution were mixed to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 200, nucleic acid concentration in the suspension: 3.7 μg / mL).

[0178] After adding 1000 μL of MES buffer (total concentration of MES and its salts: 20 mM, pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0179] The resulting concentrate was diluted to 4 mL using TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.4), and then ultrafiltered again under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate it to approximately 200 μL. This dilution and concentration process was repeated twice more. Then, 0.2 mL of the aforementioned TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0180] Comparative Example 4 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0181] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 3.0) and 450 μL of lipid-ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid-ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0182] After adding 1000 μL of MES buffer (total concentration of MES and its salts: 20 mM, pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0183] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 200 μL. This dilution and concentration process was repeated twice more. Then, 0.2 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0184] Comparative Example 5 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0185] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 5.0) and 450 μL of lipid-containing ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid-containing ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0186] After adding 1000 μL of MES buffer (20 mM, pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0187] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 200 μL. This dilution and concentration process was repeated twice more. Then, 0.2 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0188] Comparative Example 6 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0189] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (20 mM, pH: 5.0) and 450 μL of lipid ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0190] After adding 1000 μL of PBS (pH: 7.4) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0191] The resulting concentrate was diluted to 4 mL with PBS and then ultrafiltered again under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate it to approximately 500 μL. After performing this process more than twice, nucleic acid-encapsulated nanoparticles were obtained (nucleic acid concentration in suspension: 10 μg / mL).

[0192] Determination of particle size, etc. The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the nucleic acid encapsulated nanoparticles prepared in Examples 1 and Comparative Examples 1-6 were analyzed. Particle size and PdI were determined using dynamic light scattering with a Zetasizer (registered trademark). The mRNA encapsulation efficiency was determined using a Ribogreen (registered trademark) assay. The results are shown in Table 2. Furthermore, particle size is expressed as Z-Ave (Z-mean) and number Mean (hereinafter the same). Additionally, PdI can vary depending on the type of average particle size; therefore, these PdI values ​​are shown to the right of Z-Ave and number Mean in Table 2 (the same applies below).

[0193] [Table 2]

[0194] As shown in Table 2, in Example 1 using citrate buffer and TBS, the mRNA was encapsulated in LNPs, and no difference in mRNA encapsulation rate was found in Comparative Examples 1-6 with other conditions.

[0195] Evaluation of nucleic acid import efficiency The nucleic acid-encapsulated nanoparticles prepared in Examples 1 and Comparative Examples 1-6 were applied to HeLa cells to evaluate nucleic acid delivery efficiency. Specifically, in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin), at a concentration of 3 × 10⁻⁶... 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 600 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to achieve a nucleic acid concentration of 0.03 μg / 30 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence intensity was measured every hour. Results are as follows: Figure 1 As shown. Additionally... Figure 1 The vertical axis represents luciferase activity (RLU), which is the total cumulative luminescence over 24 hours. Furthermore, Figure 1 The "3.00.E+07" in the text refers to "3.00 × 10 7 Other entries that are identical to "3.00.E+07" also have the same meaning as "3.00.E+07".

[0196] like Figure 1 As shown, compared to the nucleic acid encapsulated nanoparticles of Comparative Examples 1-6 which used other combinations, the nucleic acid encapsulated nanoparticles of Example 1, which used a combination of citrate buffer and TBS, showed improved nucleic acid delivery efficiency.

[0197] Evaluation of preservation stability Determination of particle size, etc. The suspensions of nucleic acid-encapsulated nanoparticles prepared in Examples 1, 1, and 6 were stored at 4°C for 2 months. The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the stored nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were determined using dynamic light scattering assay with a Zetasizer (registered trademark). The mRNA encapsulation efficiency was determined using a Ribogreen (registered trademark) assay. The results are shown in Table 3.

[0198] [Table 3]

[0199] Evaluation of changes in nucleic acid delivery efficiency over time The suspensions of nucleic acid-encapsulated nanoparticles prepared in Examples 1, 1, and 6 were stored at 4°C. The nucleic acid-encapsulated nanoparticles were applied to HeLa cells on the start of storage (day 0), day 21, day 28, and day 57 to evaluate nucleic acid delivery efficiency. Specifically, the nanoparticles were indwelling at 3 × 10⁻⁶ cells / day in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin). 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 600 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to a nucleic acid concentration of 0.03 μg / 30 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in a Kronos incubator-type photometer, and the cumulative luminescence intensity was measured every hour. Luciferase activity (RLU) was calculated as the total cumulative luminescence intensity over 24 hours, and the relative RLU (%) was calculated using the following formula. Relative RLU (%) = 100 × RLU on day x of storage / RLU on day 0 of storage (storage start date) (In the above formula, x represents 0, 21, 28, or 57.) The result is as follows: Figure 2 As shown.

[0200] like Figure 2 As shown, compared to the nucleic acid encapsulated nanoparticles of Comparative Examples 1 or 6 using other combinations, the nucleic acid encapsulated nanoparticles of Example 1 using a combination of citrate buffer and TBS showed a suppressed time-dependent decrease in nucleic acid delivery efficiency.

[0201] Example 2 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 1.8mM DOPC: 0.3mM Chol: 1.3mM DMG-PEG2000: 0.05mM

[0202] 1200 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 50 mM, pH: 3, 4, 5, or 6) and 450 μL of lipid-ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by PrecisionNanoSystems), the mRNA-containing buffer and lipid-ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 68, nucleic acid concentration in suspension: 3.7 μg / mL).

[0203] After adding 1000 μL of TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.4) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0204] The concentrate obtained was diluted to 4 mL using the TBS described above, and then ultrafiltered again under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate it to approximately 200 μL. After repeating this dilution and concentration process twice more, 0.2 mL of the above TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0205] Determination of particle size, etc. The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of nucleic acid encapsulated nanoparticles prepared using citrate buffers with different pH values ​​in Example 2 were analyzed. Particle size and PdI were determined using dynamic light scattering assay with Zetasizer (registered trademark). mRNA encapsulation efficiency was determined using Ribogreen (registered trademark) assay. The results are shown in Table 4.

[0206] [Table 4]

[0207] As shown in Table 4, regardless of the pH of the citrate buffer, the mRNA was encapsulated in LNPs, and no difference in encapsulation efficiency was found.

[0208] Evaluation of nucleic acid import efficiency In Example 2, nucleic acid-encapsulated nanoparticles prepared using citrate buffers with different pH values ​​were applied to HeLa cells to evaluate nucleic acid delivery efficiency. Specifically, in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin), at a concentration of 3 × 10⁻⁶... 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 600 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to achieve a nucleic acid concentration of 0.03 μg / 60 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence intensity was measured every hour. Results are as follows: Figure 3 As shown. Additionally... Figure 3 The vertical axis represents luciferase activity (RLU), which is the total cumulative luminescence over 24 hours. Furthermore, Figure 3 The "6.0.E+07" in the text refers to "6.0×10". 7 Other entries that are identical to "6.0.E+07" also have the same meaning as "6.0.E+07".

[0209] like Figure 3 As shown, compared with nucleic acid encapsulated nanoparticles prepared using citrate buffer at pH 4, nucleic acid encapsulated nanoparticles prepared using citrate buffer at pH 4 exhibit improved nucleic acid delivery efficiency.

[0210] Example 3 SS-OP was used as the ionic lipid. The lipid composition, in molar ratio, was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 3.5mM DOPC: 0.5mM Chol: 2.7mM DMG-PEG2000: 0.1mM

[0211] Measure 1200 μL of a buffer containing mRNA dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 5.0) (mRNA concentrations: (1) 147 μg / mL, (2) 122 μg / mL, (3) 50 μg / mL, (4) 12.5 μg / mL, (5) 6.3 μg / mL, (6) 3.1 μg / mL) and 450 μL of an ethanol solution of lipids into separate syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), a suspension of nucleic acid-encapsulated nanoparticles was prepared with a flow rate (volume ratio) of 3:1 for a buffer solution containing mRNA and an ethanol solution containing lipids, a total flow rate of 4 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C. The suspensions contained N / P ratios of (1) 5.7, (2) 7, (3) 17, (4) 68, (5) 136, and (6) 272, respectively, and the nucleic acid concentrations in the suspensions of (1) 20.8 μg / mL, (2) 19.2 μg / mL, (3) 7.7 μg / mL, (4) 3.0 μg / mL, (5) 1.6 μg / mL, and (6) 0.9 μg / mL.

[0212] After adding 1000 μL of TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.4) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 500 μL.

[0213] The concentrate obtained was diluted to 4 mL using the above-mentioned TBS and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate to about 150-1000 μL. After the dilution and concentration were repeated twice, 0.1-3.5 mL of the above-mentioned TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: (1)-(6) 10 μg / mL).

[0214] Determination of particle size, etc. In Example 3, the particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of nucleic acid encapsulated nanoparticles prepared under different N / P ratio conditions were analyzed. Particle size and PdI were determined using dynamic light scattering assay with Zetasizer (registered trademark). The mRNA encapsulation efficiency was determined using Ribogreen (registered trademark) assay. The results are shown in Table 5.

[0215] [Table 5]

[0216] Evaluation of nucleic acid import efficiency In Example 3, nucleic acid-encapsulated nanoparticles prepared under different N / P ratio conditions were applied to HeLa cells to evaluate nucleic acid delivery efficiency. Specifically, in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin), at a concentration of 3 × 10⁻⁶... 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 0.03 μg / 60 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to a nucleic acid concentration of 0.03 μg / 60 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence intensity was measured every hour. Results are as follows: Figure 4 As shown. Additionally... Figure 4 The vertical axis represents luciferase activity (RLU), which is the total amount of light emitted over 24 hours.

[0217] Example 4 SS-EC was used as the ionic lipid. The lipid composition, in molar ratio, was SS-EC:DOPE:Chol:DMG-PEG2000 = 60:30:10:3. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-EC: 4.0mM DOPE: 2.0mM Chol: 0.7mM DMG-PEG2000: 0.2mM

[0218] 1300 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 5.0) and 600 μL of lipid-ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), a suspension of nucleic acid-encapsulated nanoparticles was prepared at a flow rate (volume ratio) of 3:1 for the mRNA-containing buffer to the lipid-ethanol solution, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C. The suspension contained 15 μg / mL of nucleic acid.

[0219] After adding 1000 μL of TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.4) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 1000 μL.

[0220] The concentrate obtained was diluted to 4 mL using the aforementioned TBS and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 750 μL. After repeating this dilution and concentration process twice more, 0.75 mL of the aforementioned TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0221] Comparative Example 7 SS-EC was used as the ionic lipid. The lipid composition, in molar ratio, was SS-EC:DOPE:Chol:DMG-PEG2000 = 60:30:10:3. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-EC: 4.0mM DOPE: 2.0mM Chol: 0.7mM DMG-PEG2000: 0.2mM

[0222] 1300 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in malate buffer (pH: 3.0) and 600 μL of lipid-ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid-ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 45, nucleic acid concentration in suspension: 15 μg / mL).

[0223] After adding 1000 μL of MES buffer (pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 1000 μL.

[0224] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 750 μL. This dilution and concentration process was repeated twice more. Then, 0.75 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0225] Determination of particle size, etc. The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the nucleic acid encapsulated nanoparticles prepared in Example 4 and Comparative Example 7 were analyzed. Particle size and PdI were determined using dynamic light scattering assay with a Zetasizer (registered trademark). The mRNA encapsulation efficiency was determined using a Ribogreen (registered trademark) assay. The results are shown in Table 6.

[0226] [Table 6]

[0227] As shown in Table 6, in Example 4, where SS-EC was used as an ionic lipid, mRNA was encapsulated in LNPs. No difference in mRNA encapsulation rate was found in Example 4 compared with other conditions in Example 7.

[0228] Evaluation of changes in nucleic acid delivery efficiency over time The suspensions of nucleic acid-encapsulated nanoparticles prepared in Example 4 and Comparative Example 7 were stored at 4°C. The nucleic acid-encapsulated nanoparticles were applied to HeLa cells on the start of storage (day 0), day 7, and day 14 to evaluate nucleic acid delivery efficiency. Specifically, the nanoparticles were in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin) at a concentration of 3 × 10⁻⁶. 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 600 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to achieve a nucleic acid concentration of 0.03 μg / 30 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence intensity was measured every hour. Results are as follows: Figure 5 As shown. Additionally... Figure 5 The vertical axis represents luciferase activity (RLU), which is the total cumulative luminescence over 24 hours. Furthermore, Figure 5The three bar charts for Example 4 and Comparative Example 7, from left to right, show the results from the start date of storage (day 0), day 7 of storage, or day 14 of storage. Additionally, Figure 5 The "8.00.E+06" in the text refers to "8.00 × 10 6 Other records identical to "8.00.E+06" also have the same meaning as "8.00.E+06".

[0229] like Figure 5 As shown, compared to the nucleic acid encapsulated nanoparticles of Comparative Example 7 which used other combinations, the nucleic acid encapsulated nanoparticles of Example 4, which used a combination of citrate buffer and TBS, showed improved nucleic acid delivery efficiency and further suppressed the time-dependent decline in nucleic acid delivery efficiency.

[0230] Example 5 SS-OP and SS-EC were used as ionic lipids. The lipid composition, in molar ratio, was SS-OP:SS-EC:DOPE:Chol:DMG-PEG2000 = 32.5:20:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 2.2mM SS-EC: 1.3mM DOPE: 0.5mM Chol: 2.7mM DMG-PEG2000: 0.1mM

[0231] 1300 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 5.0) and 600 μL of lipid-containing ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid-containing ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 34, nucleic acid concentration in suspension: 20 μg / mL).

[0232] After adding 1000 μL of TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.4) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 1000 μL.

[0233] The concentrate obtained was diluted to 4 mL using the aforementioned TBS and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 750 μL. After repeating this dilution and concentration process twice more, 0.75 mL of the aforementioned TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0234] Comparative Example 8 SS-OP and SS-EC were used as ionic lipids. The lipid composition, in molar ratio, was SS-OP:SS-EC:DOPE:Chol:DMG-PEG2000 = 32.5:20:7.5:40:1.5. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. SS-OP: 2.2mM SS-EC: 1.3mM DOPE: 0.5mM Chol: 2.7mM DMG-PEG2000: 0.1mM

[0235] 1300 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in malate buffer (total concentration of malate and its salts: 20 mM, pH: 3.0) and 600 μL of lipid ethanol solution were separately measured into syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 34, nucleic acid concentration in suspension: 20 μg / mL).

[0236] After adding 1000 μL of MES buffer (pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 1000 μL.

[0237] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 750 μL. This dilution and concentration process was repeated twice more. Then, 0.75 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0238] Determination of particle size, etc. The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the nucleic acid encapsulated nanoparticles prepared in Example 5 and Comparative Example 8 were analyzed. Particle size and PdI were determined using dynamic light scattering assay with a Zetasizer (registered trademark). The mRNA encapsulation efficiency was determined using a Ribogreen (registered trademark) assay. The results are shown in Table 7.

[0239] [Table 7]

[0240] As shown in Table 7, in Example 5, where SS-OP and SS-EC were used as ionic lipids, mRNA was encapsulated in LNPs. No difference in mRNA encapsulation rate was found in Example 5 compared with other conditions in Example 8.

[0241] Evaluation of nucleic acid import efficiency The nucleic acid-encapsulated nanoparticles prepared in Examples 5 and 8 were applied to HeLa cells to evaluate nucleic acid delivery efficiency. Specifically, in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin), at a concentration of 3 × 10⁻⁶... 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 600 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to achieve a nucleic acid concentration of 0.4 μg / 200 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence intensity was measured every hour. Results are as follows: Figure 6 As shown. Additionally... Figure 6The vertical axis represents luciferase activity (RLU), which is the total cumulative luminescence over 24 hours. Furthermore, Figure 6 The "4.40.E+07" in the text refers to "4.40 × 10 7 Other records identical to "4.40.E+07" also have the same meaning as "4.40.E+07".

[0242] like Figure 6 As shown, compared to the nucleic acid encapsulated nanoparticles of Comparative Example 8 which used other combinations, the nucleic acid encapsulated nanoparticles of Example 5, which used a combination of citrate buffer and TBS, showed improved nucleic acid delivery efficiency.

[0243] Evaluation of changes in nucleic acid delivery efficiency over time in vivo The nucleic acid-encapsulated nanoparticles prepared in Examples 1, 1, and 6 were stored at 4°C. On day 1 and day 28 of storage, 200 μL of the nanoparticles (0.1 μg of mRNA per mouse) was administered intravenously to each 6-week-old female C57BL / 6J mouse via the tail vein. Four hours after administration, the liver was harvested, and the expression level of luciferase protein was evaluated using an IVIS imaging system. Results are as follows. Figure 7 As shown. Additionally... Figure 7 The vertical axis represents luciferase activity (RLU), which is the total cumulative luminescence over 24 hours. Furthermore, Figure 7 The "1.4E+10" in the text refers to "1.4 × 10⁻¹⁰". 10 Other records that are identical to "1.4E+10" also have the same meaning as "1.4E+10".

[0244] like Figure 7 As shown, among the nucleic acid encapsulated nanoparticles of Example 1, Comparative Example 1, and Comparative Example 6, only the nucleic acid encapsulated nanoparticles of Example 1, which used a combination of citrate buffer and TBS, had the same nucleic acid delivery efficiency on day 28 of storage as on day 1 of storage.

[0245] Example 6 Compound (5) was used as an ionic lipid. The lipid composition was in the molar ratio of compound (5): DOPC:Chol:DMG-PEG2000 = 49:7.5:42.5:1. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. Compound (5): 5.0 mM DOPC: 5.0mM Chol: 10mM DMG-PEG2000: 0.5mM

[0246] 1300 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in citrate buffer (total concentration of citrate and its salts: 20 mM, pH: 5.0) and 600 μL of lipid-ethanol solution were separately measured in syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), a suspension of nucleic acid-encapsulated nanoparticles was prepared at a flow rate (volume ratio) of 3:1 for the mRNA-containing buffer to the lipid-ethanol solution, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C. The suspension contained 3.7 μg / mL of nucleic acid.

[0247] After adding 1000 μL of TBS (total concentration of Tris and its salts: 50 mM, NaCl concentration: 150 mM, pH: 7.6) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 1000 μL.

[0248] The concentrate obtained was diluted to 4 mL using the aforementioned TBS and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 200 μL. After repeating this dilution and concentration process twice more, 0.20 mL of the aforementioned TBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0249] Comparative Example 9 Compound (5) was used as an ionic lipid. The lipid composition was in the molar ratio of compound (5): DOPC:Chol:DMG-PEG2000 = 49:7.5:42.5:1. Furthermore, the concentrations of each lipid in the ethanol solution of the lipids used are as described below. Compound (5): 5.0 mM DOPC: 5.0mM Chol: 10mM DMG-PEG2000: 0.5mM

[0250] 1300 μL of mRNA-containing buffer (mRNA concentration: 5 μg / mL) dispersed in malate buffer (total concentration of malate and its salts: 20 mM, pH: 3.0) and 600 μL of lipid-ethanol solution were separately measured in syringes. Using the NanoAssmblr (registered trademark) ultra-high-speed nanomedicine fabrication device (manufactured by Precision NanoSystems), the mRNA-containing buffer and lipid-ethanol solution were mixed at a flow rate (volume ratio) of 3:1, a total flow rate of 12 mL / min, a microchannel inner diameter of 130 × 300 μm (square), and a syringe holder temperature of 25 °C to prepare a suspension of nucleic acid-encapsulated nanoparticles (N / P ratio: 34, nucleic acid concentration in suspension: 3.7 μg / mL).

[0251] After adding 1000 μL of MES buffer (pH: 6.5) to the recovered suspension, the resulting mixture was transferred to an Amicon Ultra 4 and ultrafiltered under centrifugation conditions (25 °C, 1000 g, 3 min) to concentrate to approximately 1000 μL.

[0252] The resulting concentrate was diluted to 4 mL with PBS (pH: 7.4) and then ultrafiltered again under centrifugation conditions (25°C, 1000 g, 3 min) to concentrate it to approximately 750 μL. This dilution and concentration process was repeated twice more. Then, 0.75 mL of the aforementioned PBS was added to the concentrate to obtain a suspension of nucleic acid-encapsulated nanoparticles (nucleic acid concentration in the suspension: 10 μg / mL).

[0253] Determination of particle size, etc. The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the nucleic acid encapsulated nanoparticles prepared in Example 6 and Comparative Example 9 were analyzed. Particle size and PdI were determined using dynamic light scattering assay with a Zetasizer (registered trademark). The mRNA encapsulation efficiency was determined using a Ribogreen (registered trademark) assay. The results are shown in Table 8.

[0254] [Table 8]

[0255] As shown in Table 8, in Example 6 where compound (5) was used as an ionic lipid, mRNA was encapsulated in LNPs, and no difference in mRNA encapsulation rate was found in Example 6 compared with other conditions in Example 9.

[0256] Evaluation of nucleic acid import efficiency The nucleic acid-encapsulated nanoparticles prepared in Example 6 and Comparative Example 9 were applied to HeLa cells to evaluate nucleic acid delivery efficiency. Specifically, in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (a solution supplemented with 10 v / v % FBS and 1 v / v % penicillin-streptomycin), at a concentration of 3 × 10⁻⁶... 4 HeLa cells were seeded in 24-well, flat-bottomed, transparent white culture plates at a concentration of 600 μL. The obtained nucleic acid encapsulated particles were added to the culture medium to achieve a nucleic acid concentration of 0.4 μg / 200 μL. Luciferin was added to the culture medium to a concentration of 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using luminescence intensity. HeLa cells cultured in the medium containing nucleic acid encapsulated nanoparticles and luciferin were placed in an incubator-type photometer (Kronos), and the cumulative luminescence intensity was measured every hour. Results are as follows: Figure 8 As shown. Additionally... Figure 8 The vertical axis represents luciferase activity (RLU), which is the total cumulative luminescence over 24 hours. Furthermore, Figure 8 The "1.40E+08" in the text refers to "1.40×10". 8 Other records identical to "1.40E+08" also have the same meaning as "1.40E+08".

[0257] like Figure 8 As shown, compared to the nucleic acid encapsulated nanoparticles of Comparative Example 9 which used other combinations, the nucleic acid encapsulated nanoparticles of Example 6 which used a combination of citrate buffer and TBS showed improved nucleic acid delivery efficiency. Industrial availability

[0258] According to the method of the present invention, a suspension of nucleic acid-encapsulated lipid nanoparticles with excellent preservation stability can be prepared. The nucleic acid-encapsulated lipid nanoparticles prepared according to the method of the present invention are useful for nucleic acid drugs, gene therapy, and biochemical experiments.

[0259] This application is based on Japanese Patent Application No. 2023-053865, the entire contents of which are contained in this specification.

Claims

1. A method for manufacturing nucleic acid-encapsulated lipid nanoparticles, comprising the following steps (a) and (b): Step (a) involves mixing an alcoholic solution containing ionic lipids with tertiary amino groups, sterols, and PEG lipids with a citrate buffer solution at pH 3–6.5 containing dispersed nucleic acids to prepare a suspension of nucleic acid-encapsulated lipid nanoparticles; and... Step (b) involves ultrafiltration concentration of a suspension of nucleic acid-encapsulated lipid nanoparticles and dilution with a Tris buffer solution at pH 5.2–9.0, replacing the dispersion medium of the suspension with the Tris buffer solution.

2. According to the method of claim 1, the total concentration of citric acid and its salts in the citrate buffer used in step (a) is 10-100 mM, and the total concentration of tris(hydroxymethyl)aminomethane and its salts in the Tris buffer used in step (b) is 10-200 mM.

3. The method according to claim 1, wherein the alcohol solution used in step (a) further contains phospholipids.

4. The method according to claim 1, wherein the ionic lipid is a compound represented by formula (1): In equation (1), R 1a and R 1b Each can independently represent an alkylene group having 1 to 6 carbon atoms. X a and X b Each can be independently represented as a noncyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. R 2a and R 2b Each can independently represent an alkylene group having 1 to 8 carbon atoms or an oxadialkylene group having 2 to 8 carbon atoms. Y a and Y b Each can independently represent an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z a and Z b Each of these groups independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and with or without heteroatoms. na and nb each independently represent 0 or 1. R 3a and R 3b Each of the following groups independently represents a residue derived from the reaction of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from the reaction of a sterol derivative with a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring having 3 to 40 carbon atoms, or a group represented by formula (3c): R 3c -O-CO-(CH2) a -*(3c) In equation (3c), * indicates the bonding location. R 3c This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms, and a represents an integer from 2 to 10.

5. The method according to claim 1, wherein the molar ratio of the total amino groups of the ionic lipids to the phosphate groups of the nucleic acids used in step (a), i.e., the ratio of the total amino groups of the ionic lipids to the phosphate groups of the nucleic acids, is 7 or more.

6. A method for introducing nucleic acids into cells, comprising a step of contacting said cells in vitro with nucleic acid-encapsulated lipid nanoparticles manufactured by any one of claims 1 to 5.

7. A method for delivering nucleic acids into target cells of an organism, comprising the step of administering nucleic acid-encapsulated lipid nanoparticles manufactured by any one of claims 1 to 5 into the organism.

8. A method for manufacturing a pharmaceutical composition, comprising the method according to any one of claims 1 to 5.

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