Frozen composition containing nucleic acid-encapsulated lipid nanoparticles

Abstract

The present invention provides a frozen composition containing nucleic acid-encapsulated lipid nanoparticles, wherein the nucleic acid-encapsulated lipid nanoparticles contain an ionic lipid represented by formula (1), and the molar ratio of amino groups of the ionic lipid represented by formula (1) to nucleic acid phosphate groups is 7-250 inclusive (the definitions of the symbols in formula (1) are as described in the description).

Description

Frozen compositions containing nucleic acid-encapsulated lipid nanoparticles

[0001] The present invention relates to frozen compositions comprising nucleic acid-encapsulated lipid nanoparticles.

[0002] In order to put into practical use nucleic acid therapy using oligonucleic acids such as siRNA and gene therapy using mRNA, pDNA, etc., an effective and safe nucleic acid delivery carrier is required (see Non-Patent Document 1). Viral vectors are nucleic acid delivery carriers with high expression efficiency, but development of non-viral nucleic acid delivery carriers that can be used more safely is underway.

[0003] Lipid nanoparticles are one type of nucleic acid delivery carrier. Important parameters to consider when using lipid nanoparticles as nucleic acid delivery carriers include lipid nanoparticle formation, storage stability of lipid nanoparticles, components of lipid nanoparticles, physical properties of lipid nanoparticles, nucleic acid encapsulation efficiency, and toxicity of lipid nanoparticles.

[0004] Ionic lipids are known to be significantly involved in the toxicity of nucleic acid-encapsulated lipid nanoparticles, with the higher the ionic lipid content (amino group content), the greater the toxicity. The molar ratio of amino groups in the ionic lipid to phosphate groups in the nucleic acid (sometimes referred to as the "N / P ratio" herein) is used as an indicator of the ionic lipid content in nucleic acid-encapsulated lipid nanoparticles. The N / P ratio is typically 3 to 6 (see Non-Patent Document 2). However, a higher ionic lipid content in nucleic acid-encapsulated lipid nanoparticles is desirable to increase nucleic acid delivery efficiency (see Non-Patent Document 3).

[0005] International Publication No. 2017 / 218704

[0006] Advanced Drug Delivery Reviews 154-155 (2020) 37-63International Journal of Pharmaceutics 601 (2021) 120586ACS Biomater Sci Eng. 2015 Sep 14; 1(9): 834-844

[0007] From the viewpoint of toxicity, a low N / P ratio is preferable, but from the viewpoint of nucleic acid delivery efficiency, a high N / P ratio is preferable, which is a contradictory relationship. Various companies are working on developments to resolve this dilemma, but considering use as a pharmaceutical, low toxicity is a priority, so progress is being made on nucleic acid-encapsulated lipid nanoparticles with a low N / P ratio.

[0008] As mentioned above, nucleic acid-encapsulated lipid nanoparticles with a high N / P ratio have increased toxicity due to the increased amount of amino groups. However, from the viewpoint of enhancing gene expression activity, a high N / P ratio is desirable. Therefore, it can be said that producing nucleic acid-encapsulated lipid nanoparticles with a high N / P ratio and reduced toxicity is the best way to solve the above problems.

[0009] Furthermore, one method for increasing the stability of nucleic acid-encapsulated lipid nanoparticle formulations has been to freeze-dry the nucleic acid-encapsulated lipid nanoparticle formulation and then hydrate it at the time of use to reconstitute the nucleic acid-encapsulated lipid nanoparticle formulation (see, for example, Patent Document 1). In this regard, when freeze-drying a nucleic acid-encapsulated lipid nanoparticle formulation, it is required that the physical properties of the nucleic acid-encapsulated lipid nanoparticles do not change and that the gene delivery efficiency does not decrease even after the freeze-drying and hydration process after storage.

[0010] Another way to improve the stability of nucleic acid-encapsulated lipid nanoparticle formulations is to freeze and store the nucleic acid-encapsulated lipid nanoparticles. When freezing nucleic acid-encapsulated lipid nanoparticle formulations, it is required that the physical properties of the nucleic acid-encapsulated lipid nanoparticles do not change and that gene expression activity does not decrease even after thawing processes after freezing and storage. Furthermore, since the physical properties of nucleic acid-encapsulated lipid nanoparticles vary greatly depending on the ratio of ionic lipids and other components that make up the nucleic acid-encapsulated lipid nanoparticles, it is necessary to consider the freezing conditions for each ionic lipid.

[0011] In view of the above circumstances, the present invention aims to provide a frozen composition containing nucleic acid-encapsulated lipid nanoparticles that exhibit low toxicity and high gene expression activity after freezing and thawing, despite having a high N / P ratio.

[0012] The toxicity of nucleic acid-encapsulated lipid nanoparticles increases with an increase in amino groups. Therefore, the N / P ratio of typical nucleic acid-encapsulated lipid nanoparticles is low, about 5. However, a high N / P ratio is desirable for enhancing gene expression activity. In light of this situation, the present inventors have made extensive efforts and found that by using an ionic lipid represented by the following formula (1), a frozen composition containing nucleic acid-encapsulated lipid nanoparticles can be obtained that exhibits low toxicity and high gene expression activity after freezing and thawing, despite having a high N / P ratio. The present invention based on this finding is as follows.

[0013] [1] A frozen composition comprising nucleic acid-encapsulated lipid nanoparticles, wherein the nucleic acid-encapsulated lipid nanoparticles are represented by the formula (1):

[0014]

[0015] (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms; Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond; Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom; na and nb each independently represent 0 or 1; R ３ａ and R ３ｂare each independently: (i) a monovalent group having 10 to 50 carbon atoms and having one carbonyl group and at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond (excluding monovalent groups containing a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group), (ii) a monovalent group having 10 to 50 carbon atoms and having at least two carbonyl groups (excluding monovalent groups containing a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group), (iii) a group represented by formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 10 carbon atoms; １ represents a carbamate bond, a carbonate bond, or an amide bond, and R ５ represents an alkyl group having 1 to 25 carbon atoms, and R ５ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, (iv) a monovalent group represented by formula (3): *-R ６ -CO-O-R ７ (3) (In formula (3), * represents a bonding position, R ６ represents an alkylene group having 1 to 10 carbon atoms, and R ７ represents an alkyl group having 1 to 25 carbon atoms substituted with at least one halogen atom, (v) a monovalent group represented by formula (4):

[0016]

[0017] (In formula (4), * represents a bonding position, R ８ and R ９ each independently represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms; R １０ ~R １２ are each independently a hydrogen atom, a benzyl group, or *-Si(R １３ ) (R １４ ) (R １５) group (where * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group. ) represents a monovalent group represented by formula (5):

[0018]

[0019] (In formula (5), * represents a bonding position, X ２ is a nitrogen atom or a group of formula (6):

[0020]

[0021] (In formula (6), * represents R １６ and ** represents the bonding position with R １７ or R １８ represents a bonding position with ２ is a nitrogen atom, R １６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, ２ is a trivalent group represented by formula (6), R １６ represents an alkylene group having 1 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, ２ is a nitrogen atom, R １７ and R １８ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; and R １７ and R １８ may each independently be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and ２ is a trivalent group represented by formula (6), R １７ and R １８ each independently represents an alkyl group having 1 to 10 carbon atoms, and R １７ and R１８ may each independently be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, (vii) a monovalent group represented by formula (7):

[0022]

[0023] (In formula (7), * represents a bonding position, and R １９ represents a hydrogen atom, a benzyl group, *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group, or *-CO-R ２０ group (where * represents a bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms. (viii) a monovalent group represented by formula (8):

[0024]

[0025] (In formula (8), * represents a bonding position, and R ２１ and R ２２ are each independently a hydrogen atom, a benzyl group, or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group. (ix) represents a monovalent group represented by formula (9):

[0026]

[0027] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom, a benzyl group, or *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R １３ ~R １５each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group; (x) a monovalent group represented by formula (10):

[0028]

[0029] (In formula (10), * represents a bonding position. R ２４ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２５ represents an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, or an alkynyl group having 2 to 30 carbon atoms, (xi) a monovalent group represented by formula (11):

[0030]

[0031] (In formula (11), * represents a bonding position, and R ２６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２７ and R ２８ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms.), or (xii) a monovalent group represented by formula (12):

[0032]

[0033] (In formula (12), * represents a bonding position, R ２９ represents an alkylene group having 1 to 10 carbon atoms, and R ３０ and R ３１ each independently represents an alkyl group having 1 to 10 carbon atoms, (xiii) a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond, or (xiv) R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃrepresents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8; ３ａ and R ３ｂ may be the same as or different from each other.) A frozen composition comprising an ionic lipid represented by formula (1):

[0034] [2] R ３ａ and R ３ｂ are each independently: (i) a monovalent group having 10 to 50 carbon atoms and having one carbonyl group and at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond (excluding monovalent groups containing a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group); (ii) a monovalent group having 10 to 50 carbon atoms and having at least two carbonyl groups (excluding monovalent groups containing a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group); (iii) a monovalent group represented by the formula (2); (vii) a monovalent group represented by the formula (7); (ix) a monovalent group represented by the formula (9); (xiii) a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond, or (xiv) R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8.

[0035] [3] The frozen composition according to [1] or [2], wherein the nucleic acid is RNA or DNA. [4] The frozen composition according to [1] or [2], wherein the nucleic acid is mRNA.

[0036] [5] The frozen composition according to any one of [1] to [4], wherein the particle size of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition is 10 nm or more and 200 nm or less. [6] The frozen composition according to any one of [1] to [5], wherein the PdI of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition is 0.01 or more and 0.30 or less. [7] The frozen composition according to any one of [1] to [6], wherein the nucleic acid encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition is 75% or more and 100% or less.

[0037] [8] A method for producing a frozen composition containing nucleic acid-encapsulated lipid nanoparticles, wherein the nucleic acid-encapsulated lipid nanoparticles contain an ionic lipid represented by formula (1), and the molar ratio of amino groups in the ionic lipid represented by formula (1) to phosphate groups in the nucleic acid is 7 or more and 250 or less, and the method comprises the following steps a), b), c), and d), or the following steps a), b'), and d), or the following steps a), b'), c), and d): a) mixing an alcohol solution containing the ionic lipid represented by formula (1), a sterol, and a PEG lipid with a nucleic acid solution containing an acidic buffer solution having a pH of 1.0 to 6.5 as a solvent to prepare a suspension containing nucleic acid-encapsulated lipid nanoparticles, b) exchanging the dispersion medium of the suspension obtained in step a) for a buffer solution having a pH of 4.5 to 8.0 other than the acidic buffer solution used in step a) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles, b') replacing the dispersion medium of the suspension obtained in step a) with a buffer solution having a pH of 4.5 to 8.0 and containing a cryoprotectant, other than the acidic buffer solution used in step a), to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant; c) mixing the suspension obtained in step b) or step b') with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant; d) freezing the suspension obtained in step b') or step c) at -10°C or below.

[0038] [9] The method according to [8], which comprises steps a), b), c), and d), or comprises steps a), b'), and d), and in step c), the suspension obtained in step b) is mixed with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant.

[0039]

[10] The method according to [8] or [9], wherein the ratio of (particle size of nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) / (particle size of nucleic acid-encapsulating lipid nanoparticles in the suspension obtained in step b') or step c) is 0.8 or more and 1.5 or less.

[11] The method according to any one of [8] to

[10] , wherein the PdI of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) is 0.01 or more and 0.25 or less.

[12] The method according to any one of [8] to

[11] , wherein the ratio of (nucleic acid encapsulation rate of nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) / (nucleic acid encapsulation rate of nucleic acid-encapsulating lipid nanoparticles in the suspension obtained in step b') or step c) is 0.8 or more and 1.0 or less.

[0040]

[13] A method for producing a pharmaceutical composition, comprising the method according to any one of [8] to

[12] above.

[0041]

[14] A method for introducing nucleic acid contained in the suspension into cells, the method comprising the step of contacting cells ex vivo with a suspension obtained by thawing the frozen composition according to any one of [1] to [7] above or the frozen composition produced by the method according to any one of [8] to

[12] above, or a suspension obtained by exchanging the dispersion medium of the suspension with another dispersion medium.

[0042]

[15] A method for introducing nucleic acid contained in the suspension into target cells of a living organism, the method comprising the step of administering to a living organism a suspension obtained by thawing the frozen composition according to any one of [1] to [7] above or the frozen composition produced by the method according to any one of [8] to

[12] above, or a suspension obtained by exchanging the dispersion medium of the suspension with another dispersion medium.

[0043] Although the frozen composition containing the nucleic acid-encapsulated lipid nanoparticles of the present invention has a higher N / P ratio than conventional techniques, the physical properties and gene expression activity of the nucleic acid-encapsulated lipid nanoparticles do not change significantly before and after freezing. Furthermore, despite the high N / P ratio, the nucleic acid-encapsulated lipid nanoparticles contained in the frozen composition of the present invention can achieve both high gene expression activity and low toxicity after freezing and thawing. Therefore, the frozen composition containing the nucleic acid-encapsulated lipid nanoparticles of the present invention is advantageous for gene transfer into cells in vitro or in vivo, and is also useful as a pharmaceutical composition.

[0044] 1 shows the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspensions obtained by thawing the frozen compositions of Example 3 (N / P ratio: 60), Example 4 (N / P ratio: 15), and Example 5 (N / P ratio: 8). 2 shows the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspensions obtained by thawing the frozen compositions of Comparative Example 1 (N / P ratio: 62), Comparative Example 2 (N / P ratio: 31), and Comparative Example 3 (N / P ratio: 6). 1 shows the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspensions obtained by thawing the frozen compositions of Example 6 (buffer in step a): acidic malic acid buffer, buffer in step b): MES buffer), Example 7 (buffer in step a): acidic citrate buffer, buffer in step b): MES buffer), Example 8 (buffer in step a): acidic citrate buffer, buffer in step b): Tris / maleic acid buffer), and Example 9 (buffer in step a): acidic citrate buffer, buffer in step b'): Tris / NaCl / sucrose buffer), as well as the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in a suspension of Comparative Example 4 (buffer in step a): acidic citrate buffer, buffer in step b): Tris-buffered saline). This figure shows the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspensions obtained by thawing the frozen compositions of Example 11 (ionic lipid: SS-OP described in Table 2-1), Example 12 (ionic lipid: SS-EC described in Table 2-3), Example 13 (ionic lipid: SS-OP described in Table 2-1 and SS-EC described in Table 2-3), and Example 14 (ionic lipid: Compound 13 described in Table 1-3). This figure shows the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspensions obtained by thawing the frozen compositions of Example 9 (freezing temperature: -80 ° C., storage temperature: -20 ° C.), Example 15 (freezing temperature: -20 ° C. or -80 ° C., storage temperature: -20 ° C.), and Example 16 (freezing temperature: -20 ° C. or -80 ° C., storage temperature: -20 ° C.) after storing for a predetermined period, as well as the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in a suspension of Comparative Example 5 (storage temperature: 4 ° C).Example 17 (ionic lipid: compound 7 listed in Table 1-2), Example 20 (ionic lipid: compound 11 listed in Table 1-2), Example 22 (ionic lipid: compound 23 listed in Table 1-4), Example 24 (ionic lipid: compound 34 listed in Table 1-6), Example 25 (ionic lipid: compound 37 listed in Table 1-6), Example 26 (ionic lipid: compound 58 listed in Table 1-10), Example 27 (ionic lipid: compound 155 listed in Table 1-29), Example 29 (ionic lipid: compound 161 listed in Table 1-31) 1-31), Example 30 (ionic lipid: compound 162 described in Table 1-31), Example 38 (ionic lipid: compound 163 described in Table 1-31), and Example 40 (ionic lipid: compound 196 described in Table 1-38), as well as the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in a suspension of Comparative Example 6 (ionic lipid: D-Lin-MC3-DMA (CAS number: 1224606-06-7)). Example 18 (ionic lipid: compound 9 described in Table 1-2), Example 19 (ionic lipid: compound 10 described in Table 1-2), Example 21 (ionic lipid: compound 12 described in Table 1-2), Example 23 (ionic lipid: compound 33 described in Table 1-6), Example 28 (ionic lipid: compound 159 described in Table 1-31), Example 31 (ionic lipid: compound 163 described in Table 1-31), Example 32 (ionic lipid: compound 164 described in Table 1-32), Example 34 (ionic lipid: compound 166 described in Table 1-32), and Example 36 (ionic lipid: compound 168 described in Table 1-32) The gene expression activity of nucleic acid-encapsulated lipid nanoparticles in a suspension obtained by thawing the frozen composition, as well as Comparative Example 6 (ionic lipid: D-Lin-MC3-DMA (CAS number: 1224606-06-7)) is a figure showing the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in a suspension.Example 18 (ionic lipid: compound 9 listed in Table 1-2, N / P ratio: 16), Example 19 (ionic lipid: compound 10 listed in Table 1-2, N / P ratio: 16), Example 20 (ionic lipid: compound 11 listed in Table 1-2, N / P ratio: 64), Example 21 (ionic lipid: compound 12 listed in Table 1-2, N / P ratio: 16), Example 23 (ionic lipid: compound 33 listed in Table 1-6, N / P ratio: 16), Example 24 (ionic lipid: compound 41 listed in Table 1-6, N / P ratio: 16), Example 25 (ionic lipid: compound 42 listed in Table 1-6, N / P ratio: 16), Example 26 (ionic lipid: compound 43 listed in Table 1-6, N / P ratio: 16), Example 27 (ionic lipid: compound 44 listed in Table 1-6, N / P ratio: 16), Example 28 (ionic lipid: compound 45 listed in Table 1-6, N / P ratio: 16), Example 29 (ionic lipid: compound 46 listed in Table 1-6, N / P ratio: 16), Example 30 (ionic lipid: compound 47 listed in Table 1-6, N / P ratio: 16), Example 31 (ionic lipid: compound 48 listed in Table 1-6, N / P ratio: 16), Example 32 (ionic lipid: compound 49 listed in Table 1-6, N / P ratio: 16), Example 33 (ionic lipid: compound 49 listed in Table 1-6, N / P ratio: 16), Example 34 (ionic lipid: compound 49 listed in Table 1-6, N / P ratio: 16), Example 35 (ionic lipid: compound 46 listed in Table 1-6, N / P ratio: 16), Example 36 (ionic lipid: compound 47 listed in Table 1-6 Example 25 (ionic lipid: compound 37 described in Table 1-7, N / P ratio: 64), Example 28 (ionic lipid: compound 159 described in Table 1-31, N / P ratio: 16), Example 29 (ionic lipid: compound 161 described in Table 1-31, N / P ratio: 16), Example 30 (ionic lipid: compound 162 described in Table 1-31, N / P ratio: 64), Example 31 (ionic lipid: compound 163 described in Table 1-31, N / P ratio: 16) , Example 32 (ionic lipid: compound 164 described in Table 1-32, N / P ratio: 16), Example 33 (ionic lipid: compound 165 described in Table 1-32, N / P ratio: 16), Example 34 (ionic lipid: compound 166 described in Table 1-32, N / P ratio: 16), Example 35 (ionic lipid: compound 167 described in Table 1-32, N / P ratio: 16), Example 36 (ionic lipid: compound 168 described in Table 1-32, N / P ratio: 16), Example 37 (ionic lipid Lipid: Compound 169 described in Table 1-32, N / P ratio: 16), and Example 39 (ionic lipid: Compound 197 described in Table 1-38, N / P ratio: 16) toxicity of nucleic acid-encapsulated lipid nanoparticles in suspension obtained by thawing the frozen composition (fluorescence of dead cells), and Comparative Example 6 (ionic lipid: D-Lin-MC3-DMA (CAS number: 1224606-06-7), N / P ratio: 6) toxicity of nucleic acid-encapsulated lipid nanoparticles in suspension (fluorescence of dead cells). Example 41 (N / P ratio: 16), Example 42 (N / P ratio: 12), and Example 43 (N / P ratio: 8) gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspension obtained in step c) ("Step c" in Figure 9)), and gene expression activity of nucleic acid-encapsulated lipid nanoparticles in suspension obtained by thawing the frozen composition ("After freezing and thawing" in Figure 9).10) in the suspension obtained in step c) of Example 47 (phospholipid: DOPE) gene expression activity of nucleic acid-encapsulated lipid nanoparticles ("step c" in FIG. 10) and the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition ("after freezing and thawing" in FIG. 10). 11) in the suspension obtained in step c) of Example 48 (phospholipid: POPE) gene expression activity of nucleic acid-encapsulated lipid nanoparticles ("step c" in FIG. 11) and the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition ("after freezing and thawing" in FIG. 11). 12) in the suspension obtained in step c) of Example 49 (phospholipid: DSPC) gene expression activity of nucleic acid-encapsulated lipid nanoparticles ("step c" in FIG. 12) and the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition ("after freezing and thawing" in FIG. 12). 13 )) in the suspension obtained in step c) of Example 50 (phospholipid: POPC) gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition ("After freezing and thawing" in FIG. 13). It is a diagram showing the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition of Example 49 (freezing temperature: -80 ° C., storage temperature: -80 ° C.) after storing for a predetermined period. It is a diagram showing the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing after storing for a predetermined period of time. It is a diagram showing the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained in step c) of Example 52 (N / P ratio: 12) ("Step c" in FIG. 15)) and the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition ("After freezing and thawing" in FIG. 15), as well as the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension of Comparative Example 8 (N / P ratio: 12). It is to be noted that FIG. 15 shows the relative value when the gene expression activity (luciferase activity) of Comparative Example 8 is taken as 1. 16A and 16B show the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained in step c) of Example 54 (N / P ratio: 18) ("Step c" in FIG. 16), the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition ("After freezing and thawing" in FIG. 16), and the gene expression activity of nucleic acid-encapsulated lipid nanoparticles in the suspension of Comparative Example 10 (N / P ratio: 18).16 shows relative values ​​with the gene expression activity (luciferase activity) of Comparative Example 10 set at 1.

[0045] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these. Note that the descriptions in this specification can be combined with each other unless it is clear that they cannot be combined.

[0046] The present invention provides a frozen composition comprising nucleic acid-encapsulated lipid nanoparticles, wherein the nucleic acid-encapsulated lipid nanoparticles comprise an ionic lipid represented by formula (1) (hereinafter sometimes abbreviated as "ionic lipid (1)"), and the molar ratio of amino groups in the ionic lipid represented by formula (1) to phosphate groups in the nucleic acid (hereinafter sometimes abbreviated as "N / P ratio") is 7 or more and 250 or less.

[0047] From the viewpoint of nucleic acid encapsulation efficiency, the N / P ratio is preferably 10 or more, more preferably 13 or more. From the viewpoint of toxicity, the N / P ratio is preferably 200 or less, more preferably 150 or less, even more preferably 100 or less, and particularly preferably 75 or less.

[0048] In one embodiment of the present invention, from the viewpoint of nucleic acid encapsulation efficiency and toxicity, the N / P ratio is preferably 7 to 200, more preferably 7 to 150, even more preferably 7 to 100, and most preferably 7 to 75.

[0049] In another embodiment of the present invention, the N / P ratio is preferably 10 to 200, more preferably 13 to 150, from the viewpoint of nucleic acid encapsulation efficiency and toxicity.

[0050] The N / P ratio can be roughly calculated from the following formula: N / P ratio = amount of amino groups in ionic lipid (mol) / (mass of nucleic acid (g) / 330), where "330" is the average molecular weight of deoxynucleosides.

[0051] "Lipid nanoparticles" refer to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are aligned toward the aqueous phase side of the interface. "Amphipathic lipids" refer to lipids having both hydrophilic groups that exhibit hydrophilicity and hydrophobic groups that exhibit hydrophobicity. Examples of amphipathic lipids include ionic lipids, phospholipids, and PEG lipids.

[0052] In the present invention, the term "nucleic acid-encapsulated lipid nanoparticles" refers to lipid nanoparticles in which nucleic acid is encapsulated inside the lipid nanoparticles.

[0053] The nucleic acid-encapsulating lipid nanoparticles in the frozen composition of the present invention contain an ionic lipid (1) as a membrane constituent. The nucleic acid-encapsulating lipid nanoparticles preferably further contain a sterol, a PEG lipid, and a phospholipid.

[0054] In the present invention, the term "total lipids" refers to the total amount of lipids. Examples of lipids include ionic lipids, sterols, PEG lipids, and phospholipids.

[0055] <Ionic lipid (1)> The ionic lipid (1) used in the present invention is a compound represented by the following formula (1): Ionic lipid (1) may be used alone or in combination of two or more types.

[0056]

[0057] First, the alkylene group and the like contained in formula (1) will be described. In this specification, the alkylene group may be either linear or branched. Examples of the alkylene group include a methylene group, an ethylene group, and a trimethylene group (-(CH ２ ) ３ -), propylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), tetramethylene group (-(CH ２ ) ４ -), butylene group (-CH(C ２ H ５ ) CH ２ -, -CH ２ CH(C ２ H ５)-), pentamethylene group (-(CH ２ ) ５ -), hexamethylene group (-(CH ２ ) ６ -), heptamethylene group (-(CH ２ ) ７ -), octamethylene group (-(CH ２ ) ８ -), nonamethylene group (-(CH ２ ) ９ -), decamethylene group (-(CH ２ ) １０ -) (in the above formula, "-" represents a single bond).

[0058] As used herein, the term "alkenediyl group" refers to a divalent group having a structure obtained by removing two hydrogen atoms from an alkene. The alkenediyl group may be linear or branched. The number of olefinic carbon-carbon double bonds in the alkene or alkenediyl group may be one or two or more. Examples of alkenediyl groups include ethenediyl, propenediyl, butenediyl, pentenediyl, hexenediyl, heptenediyl, octenediyl, nonenediyl, and decenediyl. The term "compound name + diyl group (e.g., ethenediyl)" as used herein refers to a divalent group having a structure obtained by removing two hydrogen atoms from the compound.

[0059] As used herein, the term "alkynediyl group" refers to a divalent group having a structure obtained by removing two hydrogen atoms from an alkyne. As used herein, the alkynediyl group may be either linear or branched. Furthermore, as used herein, the number of carbon-carbon triple bonds in either the alkyne or the alkynediyl group may be either one or two or more. Examples of the alkynediyl group include an ethynediyl group, a propynediyl group, a butynediyl group, a pentynediyl group, a hexynediyl group, a heptynediyl group, an octynediyl group, a nonynediyl group, and a decynediyl group.

[0060] In this specification, the term "oxydialkylene group" refers to a divalent group having a structure in which two alkylene groups are bonded via an oxy group (-O-) ("-" in the above formula represents a single bond). The alkylene group in the "oxydialkylene group" is as described above.

[0061] As used herein, an "ester bond" refers to -CO-O- or -O-CO- (where "-" in the formula represents a single bond). As used herein, an "amide bond" refers to -CO-NH- or -NH-CO- (where "-" in the formula represents a single bond).

[0062] As used herein, a "carbamate bond" refers to -O-CO-NH- or -NH-CO-O- (where "-" in the formula represents a single bond). As used herein, an "ether bond" refers to -O- (where "-" in the formula represents a single bond).

[0063] As used herein, a "urea bond" refers to -NH-CO-NH- (where "-" in the formula represents a single bond). As used herein, a "carbonate bond" refers to -O-CO-O- (where "-" in the formula represents a single bond).

[0064] As used herein, the term "residue of a fat-soluble vitamin having a hydroxyl group" refers to a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of the fat-soluble vitamin. Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol.

[0065] As used herein, the term "residue of a sterol derivative having a hydroxyl group" refers to a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of the sterol derivative. Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol.

[0066] In this specification, the alkyl group may be either linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, Examples of such groups include a henicosyl group, a docosyl group, a tricosyl group, a tetracosyl group, a pentacosyl group, a hexacosyl group, a heptacosyl group, an octacosyl group, a nonacosyl group, a triacontyl group, a hentriacontyl group, a dotriacontyl group, a tritriacontyl group, a tetratriacontyl group, a pentatriacontyl group, a hexatriacontyl group, a tetracontyl group, a hentetracontyl group, a dotetracontyl group, a tritetracontyl group, and a tetratetracontyl group.

[0067] In this specification, the alkenyl group may be either linear or branched, and the number of olefinic carbon-carbon double bonds in the alkenyl group may be one or two or more. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, henicosenyl, docosenyl, tricosenyl, tetracosenyl, pentacosenyl, hexacosenyl, heptacosenyl, octacosenyl, nonacosenyl, triacontenyl, hentriacontenyl, and dotriacontenyl groups.

[0068] In this specification, the alkynyl group may be either linear or branched, and the number of carbon-carbon triple bonds in the alkynyl group may be one or two or more. Examples of the alkynyl group include an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tridecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecinyl group, a heptadecynyl group, an octadecynyl group, a nonadecinyl group, an icosinyl group, a henicosinyl group, a docosinyl group, a tricosinyl group, a tetracosinyl group, a pentacosinyl group, a hexacosinyl group, a heptacosinyl group, an octacosinyl group, a nonacosinyl group, a triacontinyl group, a hentriacontinyl group, and a dotriacontinyl group.

[0069] In this specification, examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms.

[0070] In this specification, the term "hydrocarbon ring group having 3 to 12 carbon atoms" refers to a cyclic group whose ring is composed of 3 to 12 carbon atoms. Examples of hydrocarbon ring groups having 3 to 12 carbon atoms include cycloalkyl groups having 3 to 8 carbon atoms, phenyl groups, naphthyl groups, and adamantyl groups. Examples of cycloalkyl groups having 3 to 8 carbon atoms include cyclopropyl groups, cyclobutyl groups, cyclopentyl groups, cyclohexyl groups, cycloheptyl groups, and cyclooctyl groups. The hydrocarbon ring group having 3 to 12 carbon atoms is preferably a non-aromatic hydrocarbon ring group having 3 to 12 carbon atoms, more preferably a cycloalkyl group having 3 to 8 carbon atoms or an adamantyl group, and even more preferably a cyclohexyl group or an adamantyl group.

[0071] In the present specification, the term "3- to 14-membered heterocyclic group" means a heterocyclic group having 3 to 14 ring-constituting atoms. Examples of the 3- to 14-membered heterocyclic group include 5- to 14-membered aromatic heterocyclic groups and 3- to 14-membered non-aromatic heterocyclic groups.

[0072] In the present specification, examples of the 5- to 14-membered aromatic heterocyclic group include the following: (i) 5- to 6-membered monocyclic aromatic heterocyclic groups such as a thienyl group, a furyl group, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a 1,2,4-oxadiazolyl group, a 1,3,4-oxadiazolyl group, a 1,2,4-thiadiazolyl group, a 1,3,4-thiadiazolyl group, a triazolyl group, a tetrazolyl group, and a triazinyl group; (ii) a benzothiophenyl group, a benzofuranyl group, a benzimidazolyl group, a benzoxazolyl group, a benzisoxazolyl group, a benzothiazolyl group, a benzisothiazolyl group, a benzotriazolyl group, an imidazopyridinyl group, a thienopyridinyl group, a furopyridinyl group, a pyrrolopyridinyl group, a pyrazolopyridinyl group, an oxazolopyridinyl group, a thiazolopyridinyl group, an imidazopyrazinyl group, an imidazopyrimidinyl group, a thienopyrimidinyl group, a furopyrimidinyl group, a pyrrolopyrimidinyl group, a pyrazolopyrimidinyl group, an oxazolopyridinyl group, and 8- to 14-membered fused polycyclic aromatic heterocyclic groups such as azolopyrimidinyl group, thiazolopyrimidinyl group, pyrazolotriazinyl group, naphtho[2,3-b]thienyl group, phenoxathiinyl group, indolyl group, isoindolyl group, 1H-indazolyl group, purinyl group, isoquinolyl group, quinolyl group, phthalazinyl group, naphthyridinyl group, quinoxalinyl group, quinazolinyl group, cinnolinyl group, carbazolyl group, β-carbolinyl group, phenanthridinyl group, acridinyl group, phenazinyl group, phenothiazinyl group, and phenoxazinyl group.

[0073] In the present specification, examples of the 3- to 14-membered non-aromatic heterocyclic group include the following: (i) an aziridinyl group, an oxiranyl group, a thiiranyl group, an azetidinyl group, an oxetanyl group, a thietanyl group, a tetrahydrothienyl group, a tetrahydrofuranyl group, a pyrrolinyl group, a pyrrolidinyl group, an imidazolinyl group, an imidazolidinyl group, an oxazolinyl group, an oxazolidinyl group, a pyrazolinyl group, a pyrazolidinyl group, a thiazolinyl group, a thiazolidinyl group, a tetrahydroisothiazolyl group, a tetrahydrooxazolyl group, a tetrahydroisoxazolyl group, a piperidin ... 3- to 8-membered monocyclic non-aromatic heterocyclic groups such as a radinyl group, a tetrahydropyridinyl group, a dihydropyridinyl group, a dihydrothiopyranyl group, a tetrahydropyrimidinyl group, a tetrahydropyridazinyl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a morpholinyl group, a thiomorpholinyl group, an azepanyl group, a diazepanyl group, an azepinyl group, an oxepanyl group, an azocanyl group, a diazocanyl group, or a dithiolanyl group (e.g., a 1,2-dithiolan-3-yl group); (ii) a dihydrobenzofuranyl group, a dihydrobenzimidazolyl group, a dihydrobenzoxazolyl group, a dihydrobenzothiazolyl group, a dihydrobenzisothiazolyl group, a dihydronaphtho[2,3-b]thienyl group, a tetrahydroisoquinolyl group, a tetrahydroquinolyl group, a 4H-quinolidinyl group, an indolinyl group, an isoindolinyl group, a tetrahydrothieno[2,3-c]pyridinyl group, a tetrahydrobenzazepinyl group, a tetrahydroquinoxalinyl group, a tetrahydro and 9- to 14-membered fused polycyclic non-aromatic heterocyclic groups such as a tetrahydrophenanthridinyl group, a hexahydrophenothiazinyl group, a hexahydrophenoxazinyl group, a tetrahydrophthalazinyl group, a tetrahydronaphthyridinyl group, a tetrahydroquinazolinyl group, a tetrahydrocinnolinyl group, a tetrahydrocarbazolyl group, a tetrahydro-β-carbolinyl group, a tetrahydroacridinyl group, a tetrahydrophenazinyl group, a tetrahydrothioxanthenyl group, and an octahydroisoquinolyl group.

[0074] Next, R in formula (1) １ａ , R １ｂ The following R １ａ , R１ｂ The above descriptions and preferred aspects can be combined with each other.

[0075] In formula (1), R １ａ and R １ｂ R each independently represents an alkylene group having 1 to 6 carbon atoms. １ａ is R １ｂ may be the same as or different from, but preferably, R １ａ is R １ｂ is the same group as １ａ and R １ｂ are preferably each independently an alkylene group having 1 to 3 carbon atoms, more preferably both are alkylene groups having 1 to 3 carbon atoms, and even more preferably both are ethylene groups. １ａ and R １ｂ are both alkylene groups having 1 to 3 carbon atoms" means that R １ａ and R １ｂ are the same and are alkylene groups having 1 to 3 carbon atoms. １ａ and R １ｂ are both alkylene groups having 1 to 3 carbon atoms." Other expressions similar to "R １ａ and R １ｂ and each represent an alkylene group having 1 to 3 carbon atoms.

[0076] In formula (1), X ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups.

[0077] In the present specification, the term "acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group" refers to a group represented by the formula (25):

[0078]

[0079] (In formula (25), * represents a bonding position, and R ６０represents an alkyl group having 1 to 6 carbon atoms.) In this specification, "*" and the like indicate a bonding position, as described above, rather than a carbon atom. Therefore, in this specification, "-*" indicates a single bond.

[0080] R in formula (25) ６０ is preferably a methyl group, an ethyl group, a propyl group, or an isopropyl group, and more preferably a methyl group.

[0081] In this specification, the term "cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups" refers to a divalent group in which an alkylene group having 2 to 5 carbon atoms and a tertiary amino group form a cyclic structure, and the cyclic structure contains one or two tertiary amino groups. The number of carbon atoms is preferably 4 or 5.

[0082] Examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups include an aziridinediyl group, an azetidinediyl group, a pyrrolidinediyl group, a piperidinediyl group, an imidazolidinediyl group, and a piperazinediyl group.

[0083] The cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is preferably represented by the formula (26):

[0084]

[0085] (In formula (26), * represents R in formula (1) １ａ or R １ｂ ** represents the bonding position with R in formula (1). ２ａ or R ２ｂ and q represents 1 or 2.

[0086] Hereinafter, the "divalent group represented by formula (26)" may be abbreviated as "group (26)." Groups represented by other formulas may also be abbreviated in the same manner as the "divalent group represented by formula (26)." When q is 1, group (26) is a pyrrolidinediyl group, and when q is 2, group (26) is a piperidinediyl group.

[0087] The cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is preferably represented by the formula (27):

[0088]

[0089] (In formula (27), * represents a bonding position, and r represents 1 or 2.) When r is 1, group (27) is an imidazolidinediyl group, and when r is 2, group (27) is a piperazinediyl group.

[0090] X ａ is X ｂ may be the same as or different from, but preferably, X ａ is X ｂ In formula (1), X ａ and X ｂ are preferably each independently a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups, more preferably each independently a group (26) or a group (27), even more preferably each independently a group (26), and particularly preferably both a group (26) where q is 2 (i.e., a piperidinediyl group).

[0091] In formula (1), R ２ａ and R ２ｂ each independently represents an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms. The oxydialkylene group having 2 to 8 carbon atoms is preferably an oxydimethylene group, an oxydiethylene group, or an oxydipropylene group, and more preferably an oxydiethylene group.

[0092] In formula (1), R ２ａ and R ２ｂ are preferably each independently an alkylene group having 1 to 8 carbon atoms, more preferably each independently an alkylene group having 1 to 4 carbon atoms, and even more preferably both are ethylene groups.

[0093] In formula (1), Y ａ and Y ｂeach independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond. ａ Is Y ｂ may be the same as or different from, but preferably, Y ａ Is Y ｂ It is the same bond as Y. ａ and Y ｂ are preferably each independently an ester bond or an amide bond, more preferably both are ester bonds, and even more preferably both are *-CO-O-** (in the formula, * represents Z in formula (1)). ａ or Z ｂ and ** represents the bonding position of R ２ａ or R ２ｂ represents the bonding position with .

[0094] In formula (1), Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. Hereinafter, the aromatic ring and the aromatic compound will be referred to as "Z ａ and Z ｂ It is sometimes abbreviated as "aromatic ring of

[0095] Z ａ and Z ｂ The aromatic ring in Z may be either an aromatic hydrocarbon ring or an aromatic heterocycle. Examples of aromatic hydrocarbon rings include a benzene ring, a naphthalene ring, and an anthracene ring. Examples of aromatic heterocycles include an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furanthiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring. ａ and Z ｂThe aromatic ring is preferably an aromatic hydrocarbon ring, more preferably a benzene ring.

[0096] Z ａ and Z ｂ The aromatic ring may be substituted with a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, an alkylcarbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a halogen atom (i.e., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom), an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Suitable examples of the substituent include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0097] Z ａ and Z ｂ are preferably each independently represented by formula (13):

[0098]

[0099] (In formula (13), * represents the bonding position with O in formula (1), ** represents the bonding position with Y in formula (1) ａ or Y ｂ represents a bonding position with s, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and ３２ Each of "s" and "t" independently represents a substituent. ２) and "s or t is 0" means that the corresponding methylene group is not present. ３２ "u is 0" means that R ３２ means that does not exist.

[0100] s is preferably 0 or 1, and more preferably 0. t is preferably an integer of 0 to 2, and more preferably 1. u is preferably an integer of 0 to 2, and more preferably 0.

[0101] R ３２is preferably an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a halogen atom (i.e., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom), an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aryloxy group having 6 to 10 carbon atoms; More preferred are an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a halogen atom (i.e., a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, or a tert-butyl group), a ureido group, an alkoxy group having 1 to 4 carbon atoms (e.g., a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, or a tert-butoxy group), a phenyl group, or a phenoxy group; More preferred are halogen atoms (i.e., fluorine atoms, chlorine atoms, bromine atoms, iodine atoms), alkyl groups having 1 to 4 carbon atoms (e.g., methyl groups, ethyl groups, propyl groups, isopropyl groups, tert-butyl groups), or alkoxy groups having 1 to 4 carbon atoms (e.g., methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups), and particularly preferred are halogen atoms (i.e., fluorine atoms, chlorine atoms, bromine atoms, iodine atoms) or alkoxy groups having 1 to 4 carbon atoms (e.g., methoxy groups, ethoxy groups). ３２ If there are multiple R ３２ may be the same or different from each other.

[0102] Z ａ is Z ｂ may be the same as or different from, but preferably, Z ａ is Z ｂ Z in formula (1) is the same group as ａ and Zｂ are preferably each independently a group (13), more preferably each independently a group (13) in which s is 0 or 1, t is an integer of 0 to 2, and u is an integer of 0 to 2, and more preferably both are a group (13) in which s is 0, t is 1, and u is 0.

[0103] In formula (1), na and nb are each independently 0 or 1. Note that "na is 0" means that Y ａ and Z ａ means that there is no nb, and "nb is 0" means that Y ｂ and Z ｂ is not present. na may be the same as or different from nb, but preferably na is the same as nb.

[0104] In formula (1), R ３ａ and R ３ｂ are each independently a monovalent group as listed below: (i) a monovalent group having 10 to 50 carbon atoms and having one carbonyl group and at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond (excluding monovalent groups containing a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group) (hereinafter sometimes abbreviated as "group (i)");

[0105] (ii) a monovalent group having 10 to 50 carbon atoms and having at least two carbonyl groups (excluding monovalent groups containing a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group) (hereinafter sometimes abbreviated as "group (ii)");

[0106] (iii) Formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 10 carbon atoms; １ represents a carbamate bond, a carbonate bond, or an amide bond, and R ５ represents an alkyl group having 1 to 25 carbon atoms, and R ５may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group (hereinafter sometimes abbreviated as "group (iii)");

[0107] (iv) Formula (3): *-R ６ -CO-O-R ７ (3) (In formula (3), * represents a bonding position, R ６ represents an alkylene group having 1 to 10 carbon atoms, and R ７ represents an alkyl group having 1 to 25 carbon atoms substituted with at least one halogen atom (hereinafter sometimes abbreviated as "group (iv)"),

[0108] (v) Formula (4):

[0109]

[0110] (In formula (4), * represents a bonding position, R ８ and R ９ each independently represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms; R １０ ~R １２ are each independently a hydrogen atom, a benzyl group, or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group;

[0111] (vi) Formula (5):

[0112]

[0113] (In formula (5), * represents a bonding position, X ２ is a nitrogen atom or a group of formula (6):

[0114]

[0115] (In formula (6), * represents R １６and ** represents the bonding position with R １７ or R １８ represents a bonding position with ２ is a nitrogen atom, R １６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, ２ is a trivalent group represented by formula (6), R １６ represents an alkylene group having 1 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, ２ is a nitrogen atom, R １７ and R １８ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; and R １７ and R １８ may each independently be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and ２ is a trivalent group represented by formula (6), R １７ and R １８ each independently represents an alkyl group having 1 to 10 carbon atoms, and R １７ and R １８ each independently may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group (hereinafter sometimes abbreviated as "group (vi)");

[0116] (vii) Formula (7):

[0117]

[0118] (In formula (7), * represents a bonding position, and R １９ represents a hydrogen atom, a benzyl group, *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R１３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group, or *-CO-R ２０ group (where * represents a bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms.) represents a monovalent group represented by the formula (hereinafter sometimes abbreviated as "group (vii)"),

[0119] (viii) Formula (8):

[0120]

[0121] (In formula (8), * represents a bonding position, and R ２１ and R ２２ are each independently a hydrogen atom, a benzyl group, or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group;

[0122] (ix) Formula (9):

[0123]

[0124] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom, a benzyl group, or *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group;

[0125] (x) Formula (10):

[0126]

[0127] (In formula (10), * represents a bonding position. R ２４represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２５ represents an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, or an alkynyl group having 2 to 30 carbon atoms.)

[0128] (xi) Formula (11):

[0129]

[0130] (In formula (11), * represents a bonding position, and R ２６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２７ and R ２８ each independently represent an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms (hereinafter sometimes abbreviated as "group (xi)")),

[0131] (xii) Formula (12):

[0132]

[0133] (In formula (12), * represents a bonding position, R ２９ represents an alkylene group having 1 to 10 carbon atoms, and R ３０ and R ３１ each independently represents an alkyl group having 1 to 10 carbon atoms (hereinafter sometimes abbreviated as "group (xii)"),

[0134] (xiii) a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond (hereinafter sometimes abbreviated as "group (xiii)"), or (xiv) R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃrepresents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8 (hereinafter sometimes abbreviated as "group (xiv)").

[0135] R in formula (1) ３ａ and R ３ｂ may be the same or different from each other. ３ａ and R ３ｂ are preferably each independently a group (i), a group (ii), a group (iii), a group (vii), a group (ix), a group (xiii), or a group (xiv), and more preferably a group (i) or a group (xiii).

[0136] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a group (ii), a group (xiii), or a group (xiv), and more preferably each independently a group (xiii).

[0137] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a group (i) or a group (ii). In other words, in another aspect of the present invention, R ３ａ is preferably group (i) or group (ii), and R ３ｂ is preferably any one of groups (i) to (xiv). ３ｂ is more preferably group (i), group (ii), group (xiii), or group (xiv). ３ａ and R ３ｂ may be the same as each other or may be different.

[0138] In another aspect of the present invention, R ３ａ is preferably any one of groups (iii) to (xii), and R ３ｂ is preferably any one of groups (iii) to (xiv). ３ｂ is more preferably group (xiii) or group (xiv), and even more preferably group (xiii).３ａ and R ３ｂ may be the same as each other or may be different.

[0139] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a monovalent group listed below: (i-1) Formula (14): *-R ３３ -CO-X ３ -R ３４ (14) (In formula (14), * represents a bonding position, R ３３ represents an alkylene group having 2 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ３３ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms; R ３４ represents an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms, and R ３４ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms; R ３３ and R ３４ at least one of X has at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond, and ３ represents an oxygen atom, NH, or a sulfur atom.) (hereinafter, may be abbreviated as "group (i-1)"),

[0140] (i-2) Formula (15):

[0141]

[0142] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms; R ３５At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ３５ may be substituted with a substituent selected from the group consisting of an alkoxy group having 1 to 4 carbon atoms, a 3- to 14-membered heterocyclic group, and a hydrocarbon ring group having 3 to 12 carbon atoms (preferably an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ３５ (which may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms) (hereinafter sometimes abbreviated as "group (i-2)"),

[0143] (ii-1) Formula (16): *-R ３６ -CO-X ４ -R ３７ (16) (In formula (16), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, and R ３６ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms; R ３７ represents an alkyl group having 7 to 45 carbon atoms, an alkenyl group having 7 to 45 carbon atoms, or an alkynyl group having 7 to 45 carbon atoms; R ３７ at least one ethylene group or at least one trimethylene group in R is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ３７ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, and X ４ represents an oxygen atom, NH, or a sulfur atom) (hereinafter, may be abbreviated as "group (ii-1)"),

[0144] (ii-2) Formula (17): *-R ３８ -O-R ３９ (17) (In formula (17), * represents a bonding position, and R ３８ represents an alkylene group having 2 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms; R ３８ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ３８ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, and R ３９ represents an alkyl group having 3 to 30 carbon atoms, an alkenyl group having 4 to 30 carbon atoms, or an alkenyl group having 4 to 30 carbon atoms; R ３９ at least two methylene groups of R are replaced with at least two carbonyl groups, and ３９ at least one methylene group may be replaced by at least one ether bond) (hereinafter, may be abbreviated as "group (ii-2)");

[0145] (ii-3) Formula (18):

[0146]

[0147] (In formula (18), * represents a bonding position, R ４０ and R ４１ each independently represents an alkylene group having 3 to 10 carbon atoms, and R ４２ ~R ４４ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; R ４２ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ４３At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ４４ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ４２ ~R ４４ (ii-4) a monovalent group having 50 or less carbon atoms, represented by formula (19):

[0148]

[0149] (In formula (19), * represents a bonding position, R ４５ represents an alkylene group having 5 to 10 carbon atoms, and R ４６ ~R ４８ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; R ４６ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ４７ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ４８ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ４６ ~R ４８may each independently be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms; a monovalent group having 50 or less carbon atoms represented by (hereinafter sometimes abbreviated as "group (ii-4)"), or any of groups (iii) to (xiv).

[0150] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a group (i-2) (i.e., a group (15)), a group (ii-1) (i.e., a group (16)), a group (iii) (i.e., a group (2)), a group (ix) (i.e., a group (9)), a group (xiii), or a group (xiv), and more preferably each independently a group (i-2) (i.e., a group (15)) or a group (xiii).

[0151] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a group (ii-1) (i.e., a group (16)), a group (ix) (i.e., a group (9)), a group (xiii), or a group (xiv), and more preferably each independently a group (xiii).

[0152] The group (i) and the like will be explained in order below. The group (i) is preferably the group (i-1) (i.e., the group (14)) or the group (i-2) (i.e., the group (15)), and more preferably the group (i-2) (i.e., the group (15)).

[0153] R in formula (14) ３３ represents an alkylene group having 2 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, as described above, and R ３３ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0154] In this specification, "R ３３ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms" means that R ３３The alkylene group, the alkenediyl group, and the alkynediyl group may each independently be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms. ３３ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms," and other expressions similar to "R ３３ is optionally substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms."

[0155] R in formula (14) ３３ is preferably an alkylene group having 2 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and more preferably an alkylene group having 2 to 8 carbon atoms or an alkenediyl group having 2 to 8 carbon atoms. In this specification, unless otherwise specified regarding a substituent, "alkylene group," "alkenediyl group," "alkynediyl group," "alkyl group," "alkenyl group," and "alkynyl group" represent unsubstituted groups.

[0156] R in formula (14) ３４ is preferably an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms, and more preferably an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms.

[0157] R in formula (14) ３３ and R ３４ As described above, at least one of X in formula (14) has at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond. ３ is preferably an oxygen atom.

[0158] R in formula (15) ３５ preferably represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ３５may be substituted with a substituent selected from the group consisting of 3- to 14-membered heterocyclic groups and hydrocarbon ring groups having 3 to 12 carbon atoms.

[0159] R in formula (15) ３５ is more preferably an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and the alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms.

[0160] In one embodiment of the present invention, R ３５ is more preferably an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a cyclohexyl group, and the alkyl group may be substituted with an adamantyl group.

[0161] In one embodiment of the present invention, R in formula (15) ３５ is more preferably an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a cyclohexyl group.

[0162] R in formula (15) ３５ is particularly preferably an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms.

[0163] The group (ii) is preferably the group (ii-1) (i.e., the group (16)), the group (ii-2) (i.e., the group (17)), the group (ii-3) (i.e., the group (18)), or the group (ii-4) (i.e., the group (19)), and more preferably the group (ii-1) (i.e., the group (16)).

[0164] X in formula (16) ４ is preferably an oxygen atom or NH, more preferably an oxygen atom.

[0165] In one embodiment of the present invention, R in formula (16) ３６is preferably an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, more preferably an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms, and even more preferably an alkylene group having 2 or 3 carbon atoms or an alkenediyl group having 2 or 3 carbon atoms.

[0166] In one embodiment of the present invention, R in formula (16) ３６ is preferably an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms, more preferably an alkylene group having 2 to 9 carbon atoms, and even more preferably an alkylene group having 2 or 3 carbon atoms.

[0167] R in formula (16) ３７ As described above, R represents an alkyl group having 7 to 45 carbon atoms, an alkenyl group having 7 to 45 carbon atoms, or an alkynyl group having 7 to 45 carbon atoms. ３７ at least one ethylene group or at least one trimethylene group in R is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ３７ may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0168] In this specification, "R ３７ at least one ethylene group or at least one trimethylene group in R is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond" means that R ３７ At least one ethylene group or at least one trimethylene group in the alkyl group of R is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, or ３７At least one ethylene group or at least one trimethylene group in the alkenyl of R is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond, or ３７ means that at least one ethylene group or at least one trimethylene group in the alkynyl group is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond. ３７ and at least one ethylene group or at least one trimethylene group in the alkyl group is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond." ３７ at least one ethylene group or at least one trimethylene group in the alkylene group is replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond."

[0169] R in formula (16) ３７ is preferably (ii-1-1) Formula (20):

[0170]

[0171] (In formula (20), * represents a bonding position, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms; R ４９ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ５０ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ４９ and R５０ (ii-1-2) a monovalent group represented by formula (21):

[0172]

[0173] (In formula (21), * represents a bonding position, and R ５１ and R ５２ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; R ５１ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ５２ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ５１ and R ５２ may each independently be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, or (ii-1-3) a monovalent group represented by formula (22):

[0174]

[0175] (In formula (22), * represents a bonding position, and R ５３ ~R ５５ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; R ５３ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ５４At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ５５ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ５３ ~R ５５ are each independently a monovalent group represented by the formula: which may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0176] In other words, the group (16) is preferably a monovalent group having 50 or less carbon atoms and represented by the following formula (16-20), a monovalent group having 50 or less carbon atoms and represented by the following formula (16-21), or a monovalent group having 50 or less carbon atoms and represented by the following formula (16-22), more preferably a monovalent group having 50 or less carbon atoms and represented by the following formula (16-20) or a monovalent group having 50 or less carbon atoms and represented by the following formula (16-22), and even more preferably a monovalent group having 50 or less carbon atoms and represented by the following formula (16-20) (the definitions of the symbols in the following formulas are as described above).

[0177]

[0178] R in formula (16-20) to formula (16-22) ３６ and X ４ The explanation of R in formula (20) and formula (16-20) is as above. ４９ and R ５０ are preferably each independently an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, more preferably each independently an alkyl group having 1 to 17 carbon atoms or an alkenyl group having 2 to 17 carbon atoms, and even more preferably each independently an alkyl group having 1 to 17 carbon atoms.

[0179] R in formula (21) and formula (16-21) ５１and R ５２ are preferably each independently an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and more preferably each independently an alkyl group having 1 to 10 carbon atoms.

[0180] R in formula (22) and formula (16-22) ５３ ~R ５５ are preferably each independently an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and more preferably an alkyl group having 1 to 10 carbon atoms.

[0181] R in formula (17) ３８ is preferably an alkylene group having 2 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, more preferably an alkylene group having 2 to 10 carbon atoms, and even more preferably an alkylene group having 2 or 3 carbon atoms.

[0182] R in formula (17) ３９ is preferably (ii-2-1) Formula (23):

[0183]

[0184] (In formula (23), * represents a bonding position, Me represents a methyl group, and R ５６ and R ５７ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms; R ５６ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ５７ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ５６ and R ５７may each independently be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms, or (ii-2-2) a monovalent group represented by formula (24):

[0185]

[0186] (In formula (24), * represents a bonding position, and R ５８ and R ５９ each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms; R ５８ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; ５９ At least one ethylene group or at least one trimethylene group in R may be replaced with at least one bond selected from the group consisting of an ester bond, an amide bond, a carbamate bond, and a carbonate bond; and ５８ and R ５９ are each independently a monovalent group represented by the formula: which may be substituted with a substituent selected from the group consisting of a halogen atom, a hydroxyl group, and a hydrocarbon ring group having 3 to 12 carbon atoms.

[0187] In other words, the group (17) is preferably a monovalent group having 50 or less carbon atoms and represented by the following formula (17-23), or a monovalent group having 1 to 50 carbon atoms and represented by the following formula (17-24) (the symbols in the following formulas are defined as above).

[0188]

[0189] R in formula (17-23) and formula (17-24) ３８ The explanation of is as above. Me in formula (17-23) represents a methyl group as described above. R in formula (23) and formula (17-23) ５６ and R ５７are preferably each independently an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and more preferably each independently an alkyl group having 1 to 10 carbon atoms.

[0190] R in formula (24) and formula (17-24) ５８ and R ５９ are preferably each independently an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, more preferably each independently an alkyl group having 1 to 17 carbon atoms, and even more preferably each independently an alkyl group having 1 to 10 carbon atoms.

[0191] R in formula (18) ４０ and R ４１ As described above, each independently represents an alkylene group having 3 to 10 carbon atoms. ４２ ~R ４４ are preferably each independently an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and more preferably an alkyl group having 1 to 10 carbon atoms.

[0192] R in formula (19) ４５ As described above, R in formula (19) is an alkylene group having 5 to 10 carbon atoms. ４６ ~R ４８ are preferably each independently an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and more preferably each independently an alkyl group having 1 to 10 carbon atoms.

[0193] R in formula (2) ４ is preferably an alkylene group having 1 to 8 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, and even more preferably an alkylene group having 1 to 4 carbon atoms.

[0194] X in formula (2) １ is preferably *-NH-CO-O-** (where * represents R in formula (2)) ４and ** represents the bonding position of R ５ represents a bonding position with R in formula (2), or *-O-CO-O-* (where * represents a bonding position), more preferably *-NH-CO-O-** (where * represents a bonding position with R in formula (2)). ４ and ** represents the bonding position of R ５ represents the bonding position with R in formula (2). ５ is preferably an alkyl group having 1 to 25 carbon atoms.

[0195] R in formula (3) ６ As described above, R in formula (3) is an alkylene group having 1 to 10 carbon atoms. ７ is preferably an alkyl group having 1 to 25 carbon atoms substituted with at least one fluorine atom.

[0196] R in formula (4) ８ and R ９ Preferably, each independently represents an alkylene group having 1 to 10 carbon atoms. １０ ~R １２ are preferably each independently a hydrogen atom, a benzyl group, or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and each independently represent a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and each independently represent a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３represents a tert-butyl group, and R １４ and R １５ Both represent methyl groups.

[0197] X in formula (5) ２ As described above, X in formula (5) is a nitrogen atom or group (6). ２ is a nitrogen atom, R in formula (5) １６ is preferably an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and more preferably an alkylene group having 1 to 10 carbon atoms. ２ is group (6), R in formula (5) １６ is preferably an alkylene group having 1 to 10 carbon atoms.

[0198] X in formula (5) ２ is a nitrogen atom or group (6), R in formula (5) １７ and R １８ are preferably each independently an alkyl group having 1 to 10 carbon atoms, and R １７ and R １８ may each independently be substituted with a hydroxyl group.

[0199] R in formula (7) １９ is preferably a hydrogen atom, a benzyl group, *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ represent a methyl group or a phenyl group.) or *-CO-R ２０ group (where * represents a bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms, and more preferably a hydrogen atom, *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５represent a methyl group or a phenyl group.) or *-CO-R ２０ group (where * represents a bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms, and more preferably a hydrogen atom, *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ Both represent a methyl group. ２０ group (where * represents a bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms.

[0200] R in formula (8) ２１ and R ２２ are preferably each independently a hydrogen atom, a benzyl group, or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and each independently represent a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and each independently represent a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ Both represent methyl groups.

[0201] R in formula (9) ２３is preferably a hydrogen atom, a benzyl group, or *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and *—Si(R) are each a methyl group or a phenyl group, and more preferably a hydrogen atom or *—Si(R) １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and *—Si(R) are each preferably a hydrogen atom or a phenyl group. １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ Both represent methyl groups.

[0202] R in formula (10) ２４ is preferably an alkylene group having 1 to 10 carbon atoms. ２５ is preferably an alkyl group having 1 to 30 carbon atoms.

[0203] R in formula (11) ２６ is preferably an alkylene group having 1 to 10 carbon atoms. ２７ and R ２８ are preferably each independently an alkyl group having 2 to 10 carbon atoms or an alkenyl group having 2 to 10 carbon atoms, more preferably each independently an alkenyl group having 2 to 10 carbon atoms.

[0204] R in formula (12) ２９ As described above, R in formula (12) is an alkylene group having 1 to 10 carbon atoms. ３０ and R ３１ are each independently an alkyl group having 1 to 10 carbon atoms.

[0205] R in formula (1) ３ａ and R ３ｂ The group (xiii), which is one of the options above, is preferably a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and one ethylene group in the alkyl group may be replaced by one ester bond. In other words, the group (xiii) is preferably an alkyl group having 1 to 30 carbon atoms, in which one ethylene group may be replaced by one ester bond, or an alkenyl group having 2 to 20 carbon atoms. The group (xiii) is more preferably an alkenyl group having 2 to 20 carbon atoms.

[0206] The group (xiii) is more preferably a monovalent group derived from oleic acid. In this specification, the term "monovalent group derived from oleic acid" refers to a monovalent group having a structure obtained by removing a carboxy group from oleic acid (i.e., a (Z)-8-heptadecenyl group).

[0207] R in formula (1) ３ａ and R ３ｂ The group (xiv) is one of the options for R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8. p is preferably 2 or 3. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol. The sterol derivative having a hydroxyl group is preferably cholesterol or cholestanol, more preferably cholesterol. R ３ｃ is preferably a residue of a fat-soluble vitamin having a hydroxyl group, more preferably a residue of tocopherol.

[0208] In another aspect of the present invention, R ３ａ and R ３ｂare preferably each independently a group (i-2) (i.e., a group (15)), a group (16-20), a group (16-22), a group (iii) (i.e., a group (2)), a group (ix) (i.e., a group (9)), a group (xiii), or a group (xiv), and more preferably each independently a group (i-2) (i.e., a group (15)) or a group (xiii).

[0209] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a group (16-20), a group (xiii), or a group (xiv), and more preferably each independently a group (xiii).

[0210] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently represented by formula (15):

[0211]

[0212] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ３５ may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms), a monovalent group having 50 or less carbon atoms and represented by formula (16-20):

[0213]

[0214] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom, NH, or sulfur atom, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms.) A monovalent group having 50 or less carbon atoms, represented by formula (2): *-R４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 8 carbon atoms; １ represents *-NH-CO-O-** (where * represents R in formula (2)) ４ and ** represents the bonding position of R ５ represents the bonding position with R), or *-O-CO-O-* (where * represents the bonding position), and ５ represents an alkyl group having 1 to 25 carbon atoms, a monovalent group represented by formula (9):

[0215]

[0216] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom, a benzyl group, or *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and R both represent a methyl group or a phenyl group. ) A monovalent group represented by the formula: A monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond, or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8.

[0217] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently represented by formula (15):

[0218]

[0219] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and the alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms.), a monovalent group having 50 or less carbon atoms, represented by formula (16-20):

[0220]

[0221] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom or NH, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms or an alkenyl group having 2 to 17 carbon atoms, a monovalent group having 50 or less carbon atoms, represented by formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 6 carbon atoms; １ represents *-NH-CO-O-** (where * represents R in formula (2)) ４ and ** represents the bonding position of R ５ represents the bonding position with ５ represents an alkyl group having 1 to 25 carbon atoms, a monovalent group represented by formula (9):

[0222]

[0223] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５and R both represent a methyl group or a phenyl group. ) A monovalent group represented by the formula: a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond, or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group, and p represents 2 or 3.

[0224] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently represented by formula (15):

[0225]

[0226] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a cyclohexyl group.), a monovalent group having 50 or less carbon atoms, represented by formula (16-20):

[0227]

[0228] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 or 3 carbon atoms or an alkenediyl group having 2 or 3 carbon atoms; X ４ represents an oxygen atom, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms, represented by formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 4 carbon atoms; X １ represents *-NH-CO-O-** (where * represents R in formula (2)) ４and ** represents the bonding position of R ５ represents the bonding position with ５ represents an alkyl group having 1 to 25 carbon atoms, a monovalent group represented by formula (9):

[0229]

[0230] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and R both represent a methyl group. ) represents a monovalent group represented by the formula: ), an alkenyl group having 2 to 20 carbon atoms, or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group, and p represents 2 or 3.

[0231] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently represented by formula (16-20):

[0232]

[0233] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom, NH, or sulfur atom, and R ４９ and R ５０each independently represent an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms represented by the following formula: a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond; or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8.

[0234] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently represented by formula (16-20):

[0235]

[0236] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom or NH, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms or an alkenyl group having 2 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms represented by the following formula: a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond; or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group, and p represents 2 or 3.

[0237] In another aspect of the present invention, R ３ａ and R ３ｂare preferably each independently represented by formula (16-20):

[0238]

[0239] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 or 3 carbon atoms or an alkenediyl group having 2 or 3 carbon atoms; X ４ represents an oxygen atom, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms represented by the formula: ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group, and p represents 2 or 3.

[0240] In another aspect of the present invention, R ３ａ and R ３ｂ are preferably each independently a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, wherein one ethylene group in the alkyl group may be replaced by one ester bond; more preferably are each independently a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, wherein one ethylene group in the alkyl group may be replaced by one ester bond; and even more preferably are each independently an alkenyl group having 2 to 20 carbon atoms.

[0241] R ３ａ is R ３ｂ may be the same as or different from, but preferably, R ３ａ is R ３ｂ is the same group as

[0242] In one embodiment of the present invention, R １ａ is R １ｂ is the same as X ａ is X ｂ is the same as R２ａ is R ２ｂ is the same as Y ａ Is Y ｂ is identical to Z ａ is Z ｂ is the same as R ３ａ is R ３ｂ is the same as

[0243] Suitable examples of the ionic lipid represented by formula (1) (sometimes abbreviated as "ionic lipid (1)" in this specification) include the following ionic lipids.

[0244] [Ionic lipid (1-1a)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 8 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently represented by the formula (15):

[0245]

[0246] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ３５ may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms), a monovalent group having 50 or less carbon atoms and represented by formula (16-20):

[0247]

[0248] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom, NH, or sulfur atom, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, a monovalent group having 50 or less carbon atoms, represented by formula (16-22):

[0249]

[0250] (In formula (16-22), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom, NH, or sulfur atom, and R ５３ ~R ５５ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms.) A monovalent group having 50 or less carbon atoms, represented by formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 8 carbon atoms;１ represents *-NH-CO-O-** (where * represents R in formula (2)) ４ and ** represents the bonding position of R ５ represents the bonding position with R), or *-O-CO-O-* (where * represents the bonding position), and ５ represents an alkyl group having 1 to 25 carbon atoms, a monovalent group represented by formula (9):

[0251]

[0252] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom, a benzyl group, or *—Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and R both represent a methyl group or a phenyl group. ) A monovalent group represented by the formula: A monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond, or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8; ionic lipid (1).

[0253] [Ionic lipid (1-2a)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently represented by the formula (15):

[0254]

[0255] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and the alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms.), a monovalent group having 50 or less carbon atoms, represented by formula (16-20):

[0256]

[0257] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms (preferably an alkylene group having 2 to 9 carbon atoms); X ４ represents an oxygen atom or NH, and R ４９ and R ５０each independently represents an alkyl group having 1 to 17 carbon atoms or an alkenyl group having 2 to 17 carbon atoms, a monovalent group having 50 or less carbon atoms, represented by formula (16-22):

[0258]

[0259] (In formula (16-22), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms (preferably an alkylene group having 2 to 9 carbon atoms); X ４ represents an oxygen atom or NH, and R ５３ ~R ５５ each independently represents an alkyl group having 1 to 10 carbon atoms, a monovalent group having 50 or less carbon atoms, represented by formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 6 carbon atoms; １ represents *-NH-CO-O-** (where * represents R in formula (2)) ４ and ** represents the bonding position of R ５ represents the bonding position with ５ represents an alkyl group having 1 to 25 carbon atoms, a monovalent group represented by formula (9):

[0260]

[0261] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５and R both represent a methyl group or a phenyl group. ) A monovalent group represented by the formula: a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond, or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group, and p represents 2 or 3; ionic lipid (1).

[0262] [Ionic lipid (1-3a)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ are each independently represented by formula (25):

[0263]

[0264] (In formula (25), * represents a bonding position, and R ６０ represents a methyl group, an ethyl group, a propyl group, or an isopropyl group.) or a divalent group represented by formula (26):

[0265]

[0266] (In formula (26), * represents R in formula (1) １ａ or R １ｂ ** represents the bonding position with R in formula (1). ２ａ or R ２ｂ and q represents 1 or 2; ２ａ and R ２ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently represented by formula (13):

[0267]

[0268] (In formula (13), * represents the bonding position with O in formula (1), ** represents the bonding position with Y in formula (1) ａ or Y ｂ represents a bonding position with s, s represents 0 or 1, t represents an integer of 0 to 2, u represents an integer of 0 to 2 (preferably 0), and u R ３２ each independently represents a substituent; na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently represented by the formula (15):

[0269]

[0270] (In formula (15), * represents a bonding position, and R ３５ represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a cyclohexyl group.), a monovalent group having 50 or less carbon atoms, represented by formula (16-20):

[0271]

[0272] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 or 3 carbon atoms or an alkenediyl group having 2 or 3 carbon atoms (preferably an alkylene group having 2 or 3 carbon atoms); X ４ represents an oxygen atom, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms, a monovalent group having 50 or less carbon atoms, represented by formula (16-22):

[0273]

[0274] (In formula (16-22), * represents a bonding position, R ３６ represents an alkylene group having 2 or 3 carbon atoms or an alkenediyl group having 2 or 3 carbon atoms (preferably an alkylene group having 2 or 3 carbon atoms); X４ represents an oxygen atom, and R ５３ ~R ５５ each independently represents an alkyl group having 1 to 10 carbon atoms, a monovalent group having 50 or less carbon atoms, represented by formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 4 carbon atoms; X １ represents *-NH-CO-O-** (where * represents R in formula (2)) ４ and ** represents the bonding position of R ５ represents the bonding position with ５ represents an alkyl group having 1 to 25 carbon atoms, a monovalent group represented by formula (9):

[0275]

[0276] (In formula (9), * represents a bonding position, and R ２３ is a hydrogen atom or *-Si(R １３ ) (R １４ ) (R １５ ) group (where * represents the bonding position, R １３ represents a tert-butyl group, and R １４ and R １５ and R both represent a methyl group. ) represents a monovalent group represented by the formula: ), an alkenyl group having 2 to 20 carbon atoms, or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a hydroxyl group, and p represents 2. ) ; Ionic lipid (1).

[0277] [Ionic lipid (1-1b)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂeach independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 8 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently expressed by the formula (16-20):

[0278]

[0279] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms, an alkenediyl group having 2 to 9 carbon atoms, or an alkynediyl group having 2 to 9 carbon atoms; X ４ represents an oxygen atom, NH, or sulfur atom, and R ４９ and R ５０each independently represent an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms represented by the following formula: a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond; or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8; ionic lipid (1).

[0280] [Ionic lipid (1-2b)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２-); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently expressed by the formula (16-20):

[0281]

[0282] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 to 9 carbon atoms or an alkenediyl group having 2 to 9 carbon atoms (preferably an alkylene group having 2 to 9 carbon atoms); X ４ represents an oxygen atom or NH, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms or an alkenyl group having 2 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms represented by the following formula: a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, in which one ethylene group in the alkyl group may be replaced by one ester bond; or R ３ｃ -CO-(CH ２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group, and p represents 2 or 3; ionic lipid (1).

[0283] [Ionic lipid (1-3b)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ are each independently represented by formula (25):

[0284]

[0285] (In formula (25), * represents a bonding position, and R ６０ represents a methyl group, an ethyl group, a propyl group, or an isopropyl group.) or a divalent group represented by formula (26):

[0286]

[0287] (In formula (26), * represents R in formula (1) １ａ or R １ｂ ** represents the bonding position with R in formula (1). ２ａ or R ２ｂ and q represents 1 or 2; ２ａ and R ２ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently represented by formula (13):

[0288]

[0289] (In formula (13), * represents the bonding position with O in formula (1), ** represents the bonding position with Y in formula (1) ａ or Y ｂ represents a bonding position with s, s represents 0 or 1, t represents an integer of 0 to 2, u represents an integer of 0 to 2 (preferably 0), and u R ３２ each independently represents a substituent; na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently expressed by the formula (16-20):

[0290]

[0291] (In formula (16-20), * represents a bonding position, R ３６ represents an alkylene group having 2 or 3 carbon atoms or an alkenediyl group having 2 or 3 carbon atoms (preferably an alkylene group having 2 or 3 carbon atoms); X ４ represents an oxygen atom, and R ４９ and R ５０ each independently represents an alkyl group having 1 to 17 carbon atoms; a monovalent group having 50 or less carbon atoms represented by the formula: ３ｃ -CO-(CH２ ) ｐ - group (wherein, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a hydroxyl group, and p represents 2. ) ; Ionic lipid (1).

[0292] [Ionic lipid (1-1c)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 8 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently represented by the formula (15):

[0293]

[0294] (In formula (15), * represents a bonding position, and R ３５represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and R ３５ may be substituted with a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a hydrocarbon ring group having 3 to 12 carbon atoms.), a monovalent group having 50 or less carbon atoms represented by the following formula: or a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, wherein one ethylene group in the alkyl group may be replaced by one ester bond; ionic lipid (1).

[0295] [Ionic lipid (1-2c)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂare each independently represented by the formula (15):

[0296]

[0297] (In formula (15), * represents a bonding position, and R ３５ is an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon ring group having 3 to 12 carbon atoms, and the alkyl group may be substituted with a hydrocarbon ring group having 3 to 12 carbon atoms.), a monovalent group having 50 or less carbon atoms represented by: or a monovalent group which is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and one ethylene group in the alkyl group may be replaced with one ester bond; an ionic lipid (1).

[0298] [Ionic lipid (1-3c)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ are each independently represented by formula (25):

[0299]

[0300] (In formula (25), * represents a bonding position, and R ６０ represents a methyl group, an ethyl group, a propyl group, or an isopropyl group.) or a divalent group represented by formula (26):

[0301]

[0302] (In formula (26), * represents R in formula (1) １ａ or R １ｂ ** represents the bonding position with R in formula (1). ２ａ or R ２ｂ and q represents 1 or 2; ２ａ and R ２ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Yｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently represented by formula (13):

[0303]

[0304] (In formula (13), * represents the bonding position with O in formula (1), ** represents the bonding position with Y in formula (1) ａ or Y ｂ represents a bonding position with s, s represents 0 or 1, t represents an integer of 0 to 2, u represents an integer of 0 to 2 (preferably 0), and u R ３２ each independently represents a substituent; na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently represented by the formula (15):

[0305]

[0306] (In formula (15), * represents a bonding position, and R ３５ is an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, and the alkyl group may be substituted with a cyclohexyl group.), a monovalent group having 50 or less carbon atoms represented by, or an alkenyl group having 2 to 20 carbon atoms; ionic lipid (1).

[0307] [Ionic lipid (1-1d)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., piperidinediyl group); R ２ａ and R ２ｂare each independently an alkylene group having 1 to 8 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently a monovalent group that is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, and one ethylene group in the alkyl group may be replaced by one ester bond; an ionic lipid (1).

[0308] [Ionic lipid (1-2d)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., piperidinediyl group); R ２ａ and R ２ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂare each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently a monovalent group that is an alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 20 carbon atoms, and one ethylene group in the alkyl group is optionally replaced by one ester bond; an ionic lipid (1).

[0309] [Ionic lipid (1-3d)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (i.e., a methylene group or an ethylene group); ａ and X ｂ are each independently represented by formula (25):

[0310]

[0311] (In formula (25), * represents a bonding position, and R ６０ represents a methyl group, an ethyl group, a propyl group, or an isopropyl group.) or a divalent group represented by formula (26):

[0312]

[0313] (In formula (26), * represents R in formula (1) １ａ or R １ｂ ** represents the bonding position with R in formula (1). ２ａ or R ２ｂ and q represents 1 or 2; ２ａ and R ２ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group, or a trimethylene group); ａand Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently represented by formula (13):

[0314]

[0315] (In formula (13), * represents the bonding position with O in formula (1), ** represents the bonding position with Y in formula (1) ａ or Y ｂ represents a bonding position with s, s represents 0 or 1, t represents an integer of 0 to 2, u represents an integer of 0 to 2 (preferably 0), and u R ３２ each independently represents a substituent; na and nb are each independently 0 or 1; and R ３ａ and R ３ｂ are each independently an alkenyl group having 2 to 20 carbon atoms; an ionic lipid (1).

[0316] Specific examples of the ionic lipid (1) include compounds 1 to 198 listed in Tables 1-1 to 1-38 below (hereinafter collectively referred to as "Table 1"). Note that "TBS" in Table 1 means tert-butyldimethylsilyl, and "Me" means methyl.

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355] Specific examples of the ionic lipid (1) include O-Ph-P3C1, O-Ph-P4C1, SS-OP, O-Bn-P4C2, SS-EP, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, B-2, B-2-5, SS-EC, L-P4C2, and SS-OC, which are listed in Tables 2-1 to 2-3 below (hereinafter collectively referred to as "Table 2").

[0356]

[0357]

[0358]

[0359] In one embodiment of the present invention, the ionic lipid (1) is preferably at least one selected from the group consisting of the compounds listed in Tables 1 and 2, more preferably at least one selected from the group consisting of SS-OP, SS-EC, and compound 13, even more preferably SS-OP; a combination of SS-OP and SS-EC; SS-EC; or compound 13, particularly preferably SS-OP; or a combination of SS-OP and SS-EC, and most preferably SS-OP.

[0360] In another embodiment of the present invention, the ionic lipid (1) is preferably at least one selected from the group consisting of SS-OP, SS-EC, Compound 7, Compounds 9 to 13, Compound 23, Compound 33, Compound 34, Compound 37, Compound 58, Compound 155, Compound 159, Compounds 161 to 169, and Compounds 196 to 198, and more preferably at least one selected from the group consisting of SS-OP, Compounds 9 to 12, Compound 33, Compound 34, Compound 37, Compound 159, Compounds 161 to 169, and Compound 197.

[0361] In another embodiment of the present invention, the ionic lipid (1) is preferably at least one selected from the group consisting of SS-OP, SS-EC, Compound 9, Compound 10, Compound 11, Compound 12, Compound 33, Compound 34, Compound 37, Compound 159, Compound 161, Compound 162, Compound 163, Compound 164, Compound 165, Compound 166, Compound 167, Compound 168, Compound 169, and Compound 197, more preferably SS-OP; a combination of SS-OP and SS-EC; SS-EC; Compound 11; Compound 33; Compound 37; Compound 163, or Compound 197, even more preferably SS-OP; a combination of SS-OP and SS-EC; or Compound 11, and particularly preferably SS-OP or Compound 11.

[0362] The ionic lipid (1) listed in Table 2 can be produced by known methods (for example, the methods described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, WO2021 / 195529A2, WO2024 / 203577A1). In addition, compound 13 used in the examples was produced by the following synthesis method. Ionic lipids (1) listed in Table 1 other than compound 13 can also be produced in the same manner as the synthesis method of compound 13.

[0363] The abbreviations used in the synthesis method of Compound 13 have the following meanings: DMAP: 4-dimethylaminopyridine EDC hydrochloride: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride TBAF: tetra-n-butylammonium fluoride TBDPS-Cl: tert-butyldiphenylsilyl chloride THF: tetrahydrofuran

[0364] <Method for Synthesizing Compound 13> <Synthesis of Intermediate 11-2> Intermediate 11-2 was synthesized according to the following synthetic route.

[0365]

[0366] <Synthesis of Intermediate 11-1> 25.0 g (189 mmol) of (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol and 19.3 g (284 mmol) of imidazole were dissolved in 251 g of dimethylformamide at room temperature. 57.3 g (208 mmol) of TBDPS-Cl was added to the resulting mixture and allowed to react at room temperature for 1 hour. Chloroform was then added to the reaction solution, which was then washed with a 5 wt% aqueous solution of sodium hydrogen phosphate and ion-exchanged water, followed by dehydration with sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 56.0 g of Intermediate 11-1.

[0367] <Synthesis of Intermediate 11-2> 55.9 g (151 mmol) of Intermediate 11-1 and 503 g of 0.5 M phosphate buffer (pH 1.0) were dissolved in 530 g of THF at room temperature and reacted at 50°C for 12 hours. 1.0 M aqueous NaOH solution was added to adjust the pH to 7.0. Chloroform was added to the resulting mixture, which was then washed with 20 wt % saline, and sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 44.9 g of Intermediate 11-2.

[0368] <Synthesis of Intermediate 13-1> 6.90 g (20.9 mmol) of Intermediate 11-2, 5.38 g (46.0 mmol) of hexanoic acid, and 0.511 g (4.18 mmol) of DMAP were dissolved in 69.5 g of chloroform at room temperature. 12.0 g (62.7 mmol) of EDC hydrochloride was added to the resulting mixture and allowed to react at room temperature for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt % sodium bicarbonate water, and 20 wt % saline, followed by dehydration with the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 9.405 g of Intermediate 13-1.

[0369]

[0370] <Synthesis of Intermediate 13-2> 9.41 g (17.9 mmol) of intermediate 13-1 and 4.29 g (71.6 mmol) of acetic acid were dissolved in 28.2 g of THF at room temperature. 65.9 g (71.6 mmol) of a 1 M TBAF THF solution was added to the resulting mixture and reacted at room temperature for 18 hours. 94.3 g of ethyl acetate was then added to the reaction solution. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0) and 20 wt % saline, and then dehydrated by adding sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 3.10 g of intermediate 13-2.

[0371]

[0372] <Synthesis of Intermediate 13> 3.10 g (10.7 mmol) of Intermediate 13-2, 2.45 g (21.5 mmol) of glutaric anhydride, 3.25 g (32.1 mmol) of triethylamine, and 0.261 g (2.14 mmol) of DMAP were dissolved in 51.6 g of chloroform at room temperature and allowed to react for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt % aqueous sodium bicarbonate, and 20 wt % saline, followed by dehydration with the addition of sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 3.21 g of Intermediate 13.

[0373]

[0374] <Synthesis of Intermediate 1> Intermediate 1 was synthesized according to the following synthetic route.

[0375]

[0376] <Synthesis of Intermediate 1-A> 30.0 g (197 mmol) of 4-hydroxyphenylacetic acid and 5.00 g (19.9 mmol) of pyridinium p-toluenesulfonate were dissolved in 120 mL of dichloromethane at room temperature. A mixed solution of 83.0 g (987 mmol) of 3,4-dihydro-2H-pyran and 31.1 mL of dichloromethane was added dropwise to the resulting mixture at 20°C or below, and the mixture was allowed to react at room temperature for 2 hours. Subsequently, 12.0 g (98.2 mmol) of DMAP was added to the reaction mixture for neutralization. 301 mL of 2-propanol and 160 g of a 100 g / L aqueous NaOH solution were added to the resulting mixture, and the mixture was allowed to react at room temperature for 1 hour. The mixed solution was concentrated using an evaporator, and the concentrate was washed with chloroform and neutralized by adding 6 M hydrochloric acid. The resulting mixture was extracted with chloroform, and the organic layer was dehydrated by adding sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 47.0 g of intermediate 1-A.

[0377] <Synthesis of Intermediate 1-B> 36.0 g (95.6 mmol) of intermediate 57 synthesized according to the method described in WO 2016 / 121942, 49.7 g (210 mmol) of intermediate 1-A, and 4.68 mg (38.3 mmol) of DMAP were dissolved in 240 mL of chloroform at room temperature. 55.1 g (287 mmol) of EDC hydrochloride was added to the resulting mixture and allowed to react at room temperature for 2 hours. The reaction solution was then washed with a 5 wt% aqueous solution of sodium dihydrogen phosphate, a 9 wt% aqueous solution of sodium bicarbonate, and 20 wt% saline, followed by dehydration by the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 77.9 g of intermediate 1-B.

[0378] <Synthesis of Intermediate 1> 77.7 g (95.6 mmol) of Intermediate 1-B was dissolved in 325 mL of THF at room temperature. 319 mL of 2-propanol and 38.2 g (201 mmol) of p-toluenesulfonic acid monohydrate were added to the resulting mixture and reacted at 25°C or below for 1 hour. 25.0 g (205 mmol) of DMAP was then added to the reaction mixture for neutralization. After removing the DMAP by filtration, the filtrate was concentrated using an evaporator. The resulting residue was dissolved in 627 mL of chloroform, washed with 0.5 M phosphate buffer (pH = 6.5) and 0.5 M glycine buffer (pH = 9.5), and then dehydrated by adding sodium sulfate. After removing the sodium sulfate by filtration, 1.56 L of toluene was added to allow crystallization. The resulting crude product was washed with hexane and then vacuum-dried to obtain 40.3 g of Intermediate 1.

[0379] <Intermediate 1 １ H-NMR (600MHz, DMSO-d ６ )> δ: 1.07-1.11 (m, 4H), 1.16-1.25 (m, 2H), 1.42-1.59 (m, 8H), 1.79-1.88 (m, 4H), 2.25-2.59 (m, 4 H), 2.76-2.89 (m, 8H), 3.49 (s, 4H), 4.02-4.09 (m, 4H), 6.67-6.72 (m, 4H), 7.02-7.07 (m, 4H)

[0380] <Synthesis of Intermediate 1-1> 5.11 g (18.1 mmol) of oleic acid, 16.6 g (25.8 mmol) of Intermediate 1, and 630 mg (5.16 mmol) of DMAP were dissolved in 166 mL of chloroform at room temperature. 5.94 g (31.0 mmol) of EDC hydrochloride was added to the resulting mixture and allowed to react at room temperature for 2 hours. The reaction solution was then washed with a 5 wt% aqueous solution of sodium hydrogen phosphate, 0.5 M phosphate buffer (pH = 2.0), and 20 wt% saline, followed by dehydration with the addition of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator. The resulting residue was purified by silica gel column chromatography (chloroform / ethanol = 88 / 12) to obtain 8.21 g of Intermediate 1-1.

[0381]

[0382] Synthesis of Compound 13 Using Intermediate 1-1: 2.23 g of chloroform was added to 214.1 mg (0.235 mmol) of Intermediate 1-1 to dissolve it, and then 88.4 mg (0.220 mmol) of Intermediate 13, 5.4 mg (0.044 mmol) of DMAP, and 65.8 mg (0.34 mmol) of EDC hydrochloride were added and reacted at room temperature for 5.5 hours. The reaction solution was then washed with a 5 wt% aqueous solution of sodium hydrogen phosphate and 20 wt% saline, and then dehydrated by adding sodium sulfate. Chloroform was then added, and the sodium sulfate was removed by filtration. The filtrate was then concentrated using an evaporator to obtain 279 mg of Compound 13.

[0383]

[0384] The content of ionic lipid (1) in the frozen composition is preferably 20 to 75 mol%, more preferably 30 to 70 mol%, and even more preferably 35 to 60 mol% of the total lipids, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability. In this specification, the "content (mol%) of B relative to A" means "100 × amount (mol) of B / amount (mol) of A."

[0385] The content of ionic lipid (1) in the frozen composition is preferably 0.012 to 8.1 wt %, more preferably 0.018 to 7.56 wt %, and even more preferably 0.021 to 7.02 wt %, based on the total weight of the frozen composition, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability. In this specification, the "content (wt %) of B relative to A" means "100 × amount of B (g) / amount of A (g)."

[0386] <Nucleic acid> The nucleic acid encapsulated in lipid nanoparticles can be either RNA or DNA, for example, as described below.In addition, both RNA and DNA can be antisense oligonucleotides.Nucleic acid is preferably mRNA.In addition, if the N / P ratio and the content of ionic lipid (1) are specified, the content of nucleic acid in the frozen composition can be specified by these.

[0387] <Phospholipid> Phospholipids can be used as a lipid membrane constituent component of lipid nanoparticles. One type of phospholipid may be used alone, or two or more types may be used in combination.

[0388] Examples of phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), and lyso forms thereof.

[0389] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) include 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), Examples of such phosphocholine include 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

[0390] In the above specific example of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), the "phosphocholine" portion is replaced with phosphoethanolamine, phosphoserine, phosphoglycerol, or phosphatidic acid, respectively, to give 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), and 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA).

[0391] The phospholipid is preferably at least one selected from the group consisting of PC, PE, PS, and PG, and more preferably at least one selected from the group consisting of DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DOPS (1,2-dioleoyl-sn-glycero-3-phosphoserine), DLoPS (1,2-dilinoleoyl-sn-glycero-phosphoserine), and DOPG (1,2-dioleoyl-sn-glycero-3-phosphoglycerol).

[0392] In one embodiment of the present invention, the phospholipid is more preferably at least one selected from the group consisting of DOPC, DSPC, DOPG, DEPC, POPE, and DOPE, particularly preferably at least one selected from the group consisting of DOPC, DOPG, DEPC, POPE, and DOPE, and most preferably DOPC, DOPE, or POPE.

[0393] In another embodiment of the present invention, the phospholipid is more preferably at least one selected from the group consisting of DOPC, DSPC, DEPC, and DOPE, particularly preferably at least one selected from the group consisting of DOPC and DOPE, and most preferably DOPC or DOPE.

[0394] The content of phospholipids in the frozen composition is preferably 0 to 35 mol%, more preferably 2.5 to 35 mol%, and even more preferably 5 to 32.5 mol%, based on the total lipids, from the viewpoints of nucleic acid encapsulation efficiency, nucleic acid release efficiency within cells, and stability of lipid nanoparticles.

[0395] <Sterol> Sterol can be used as a component that adjusts the fluidity of the lipid membrane of the lipid nanoparticle. Only one type of sterol may be used, or two or more types may be used in combination.

[0396] Examples of sterols include cholesterol, lanosterol, phytosterol, zymosterol, zymostenol, desmosterol, stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol. The sterol is preferably at least one selected from the group consisting of cholesterol, lanosterol, and phytosterol, and more preferably cholesterol.

[0397] When a sterol is used, its content in the frozen composition is preferably 5 to 60 mol%, more preferably 7 to 55 mol%, and even more preferably 10 to 50 mol%, based on the total lipids, from the viewpoints of nucleic acid encapsulation efficiency, nucleic acid release efficiency within cells, and stability of lipid nanoparticles.

[0398] <PEG lipid> PEG lipid can be used as a stabilizer by coating the surface of lipid nanoparticles with hydrophilic polyethylene glycol (PEG) to suppress aggregation of particles, or to suppress interaction between biological components and particles when administered to a living body. Only one type of PEG lipid may be used, or two or more types may be used in combination.

[0399] The PEG region can be of any molecular weight, hi some embodiments, the PEG region has a number average molecular weight of 200 to 10,000, and can be linear or branched.

[0400] Examples of PEG lipids include phospholipid-PEG, ceramide-PEG, diacylglycerol-PEG, cholesterol-PEG.

[0401] The PEG lipid is preferably at least one selected from the group consisting of diacylglycerol-PEG having a PEG number-average molecular weight of 1,000 to 10,000, more preferably dimyristoylglycerol-PEG having a PEG number-average molecular weight of 1,000 to 10,000, and distearoylglycerol-PEG having a PEG number-average molecular weight of 1,000 to 10,000, and even more preferably dimyristoylglycerol-PEG having a PEG number-average molecular weight of 1,000 to 10,000.

[0402] When PEG lipids are used, their content in the frozen composition is preferably 0.1 to 6.0 mol%, more preferably 0.5 to 5.0 mol%, and even more preferably 0.7 to 3.0 mol%, relative to the total of other lipids, from the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability. Here, "other lipids" refers to lipids other than PEG lipids. For example, when ionic lipid (1) and PEG lipids are used as lipids, the "total of other lipids" refers to ionic lipid (1). Furthermore, for example, when ionic lipid (1), phospholipids, sterols, and PEG lipids are used as lipids, the "total of other lipids" refers to the sum of ionic lipid (1), phospholipids, and sterols.

[0403] <Other Components> The frozen composition of the present invention may contain components other than nucleic acids and lipids, as long as the effects of the present invention are not impaired. The other components may be used alone or in combination of two or more.

[0404] <Water (Ice)> The frozen composition of the present invention preferably contains water (ice). When the frozen composition of the present invention contains water (ice), the content of water (ice) is preferably 75 to 99 wt %, more preferably 80 to 99 wt %, and even more preferably 85 to 99 wt %, based on the total weight of the frozen composition.

[0405] <Cryoprotectant> One or more cryoprotectants may be used as other components. Examples of cryoprotectants include saccharides such as monosaccharides, sugar alcohols, disaccharides, oligosaccharides, and polysaccharides, as well as PVP, PVA, Poloxamer, Brij, TERGITOL, Thesit, and PEG. The saccharide is preferably a disaccharide, and more preferably sucrose.

[0406] When a cryoprotectant is used, the content of the cryoprotectant in the frozen composition is preferably 40.0 to 99.9% by weight, more preferably 50.0 to 99.9% by weight, even more preferably 60.0 to 99.9% by weight, particularly preferably 70.0 to 99.9% by weight, and most preferably 90.0 to 99.9% by weight, based on the total frozen composition excluding water (ice), from the viewpoints of the formation and stability of nucleic acid-encapsulated lipid nanoparticles, and cryoprotective ability.

[0407] When a cryoprotectant is used, its content in the frozen composition is preferably 2 to 50% by weight, more preferably 4 to 32% by weight, and even more preferably 6 to 16% by weight, based on the total weight of the frozen composition, from the viewpoints of the formation and stability of nucleic acid-encapsulated lipid nanoparticles, and cryoprotective ability.

[0408] The particle size of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition of the present invention is not particularly limited, but is preferably 10 nm to 200 nm, more preferably 30 nm to 150 nm. Thawing of the frozen composition for measuring the particle size is performed by leaving 20 mL or less of the frozen composition at 4 to 25°C in an atmospheric pressure atmosphere for 30 minutes to 2 hours. Measurement and calculation of the particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). The particle size of the nucleic acid-encapsulated lipid nanoparticles can be appropriately adjusted depending on the manufacturing method. In the present invention, "particle size" refers to the Z-average size (mean particle size) measured and calculated by dynamic light scattering.

[0409] The surface potential (zeta potential) of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition of the present invention is not particularly limited, but is preferably −15 mV to +15 mV, more preferably −10 mV to +10 mV. Measurement of the surface potential can be performed using a zeta potential measuring device such as a Zetasizer Nano. The surface potential of the nucleic acid-encapsulated lipid nanoparticles can be adjusted by the composition of the constituent components of the lipid nanoparticles.

[0410] The PdI of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition of the present invention is not particularly limited, but is preferably 0.01 to 0.30, more preferably 0.01 to 0.25. Thawing of the frozen composition for measuring the PdI is performed by leaving 20 mL or less of the frozen composition in an atmospheric pressure atmosphere at 4 to 25°C for 30 minutes to 2 hours. Measurement and calculation of the PdI can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern).

[0411] The nucleic acid encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition of the present invention is not particularly limited, but is preferably 75% to 100%, more preferably 80% to 100%, and even more preferably 85% to 100%. Thawing of the frozen composition for measuring the encapsulation rate is carried out by allowing 20 mL or less of the frozen composition to stand in an atmospheric pressure atmosphere at 4 to 25°C for 30 minutes to 2 hours. The encapsulation rate can be measured and calculated using the Ribogreen® assay.

[0412] <Method for producing a frozen composition> The present invention also provides a method for producing a frozen composition of nucleic acid-encapsulated lipid nanoparticles. In the production method of the present invention, the nucleic acid-encapsulated lipid nanoparticles contain an ionic lipid (1), and the molar ratio of amino groups in the ionic lipid (1) to phosphate groups in the nucleic acid is 7 or more and 250 or less, and the production method of the present invention comprises the following steps a), b), c), and d), or the following steps a), b'), and d), or the following steps a), b'), c), and d): a) mixing an alcohol solution containing the ionic lipid (1), a sterol, and a PEG lipid with a nucleic acid solution containing an acidic buffer solution having a pH of 1.0 to 6.5 as a solvent to prepare a suspension containing nucleic acid-encapsulated lipid nanoparticles, b) exchanging the dispersion medium of the suspension obtained in step a) for a buffer solution having a pH of 4.5 to 8.0 other than the acidic buffer solution used in step a) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles, b') exchanging the dispersion medium of the suspension obtained in step a) for a buffer solution having a pH of 4.5 to 8.0 and containing a cryoprotectant, other than the acidic buffer solution used in step a), to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant; c) mixing the suspension obtained in step b) or step b') with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant; d) freezing the suspension obtained in step b') or step c) at -10°C or below.

[0413] The manufacturing method of the present invention preferably comprises steps a), b), c), and d), or comprises steps a), b'), and d), and in step c), the suspension obtained in step b) is mixed with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant.

[0414] The explanation of the ionic lipid (1) and the N / P ratio in the production method of the present invention is the same as that described above for the frozen composition of the present invention. Hereinafter, each step in the production method of the present invention will be described in order.

[0415] <Step a)> In step a), an alcohol solution containing ionic lipid (1), sterol, and PEG lipid (hereinafter sometimes abbreviated as "alcohol solution") is mixed with a nucleic acid solution (hereinafter sometimes abbreviated as "nucleic acid solution") containing an acidic buffer solution having a pH of 1.0 to 6.5 as a solvent (hereinafter sometimes abbreviated as "acidic buffer") to prepare a suspension containing nucleic acid-encapsulated lipid nanoparticles. Any procedure that induces assembly can be used to prepare nucleic acid-encapsulated lipid nanoparticles. The alcohol solution preferably contains phospholipids.

[0416] The phospholipids, sterols, PEG lipids, and nucleic acids used in step a) are the same as those described above for the frozen composition of the present invention. Examples of the alcohol used in step a) include ethanol and tert-butanol.

[0417] The concentration of total lipids (i.e., the total of ionic lipid (1), phospholipid, sterol and PEG lipid) in the alcohol solution used in step a) is preferably 0.1 to 30 mM, more preferably 0.5 to 25 mM, and even more preferably 1 to 20 mM, from the viewpoint of lipid solubility and nucleic acid encapsulation.

[0418] The concentration of the ionic lipid (1) in the alcohol solution used in step a) is preferably 0.02 to 22.5 mM, more preferably 0.15 to 17.5 mM, and even more preferably 0.35 to 15 mM, from the viewpoint of lipid solubility.

[0419] The concentration of the sterol in the alcohol solution used in step a) is preferably 0.005 to 18 mM, more preferably 0.035 to 13.75 mM, and even more preferably 0.1 to 12.5 mM, from the viewpoint of lipid solubility.

[0420] The concentration of the phospholipid in the alcohol solution used in step a) is preferably 0 to 12 mM, more preferably 0.0125 to 8.75 mM, and even more preferably 0.05 to 8.125 mM, from the viewpoint of lipid solubility.

[0421] The concentration of the PEG lipid in the alcohol solution used in step a) is preferably 0.0001 to 1.8 mM, more preferably 0.0025 to 1.25 mM, and even more preferably 0.007 to 0.75 mM, from the viewpoint of lipid solubility.

[0422] In step a), an acidic buffer solution having a pH of 1.0 to 6.5 is used, preferably 3.0 to 6.5, more preferably 3.0 to 5.0.

[0423] Examples of acidic buffer solutions include tartaric acid / NaOH buffer solution, phthalic acid HK / HCl buffer solution, glycine / HCl buffer solution, trans-aconitic acid / NaOH buffer solution, formic acid / Na formate buffer solution, 3,3-dimethylglutaric acid / NaOH buffer solution, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer solution, phenylacetic acid / Na phenylacetic acid buffer solution, acetic acid / Na acetate buffer solution, succinic acid / NaOH buffer solution, phthalic acid HK / NaOH buffer solution, cacodylic acid Na / HCl buffer solution, maleic acid HNa / NaOH buffer solution, maleic acid / Tris / NaOH buffer solution, phosphate buffer solution, KH ２ P.O. ４ / NaOH buffer, 2-morpholinoethanesulfonic acid (MES) buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, Tris / maleic acid buffer, succinic acid buffer, tartrate buffer, citrate buffer, bis-tris buffer, glycylglycine buffer, and the like.

[0424] The acidic buffer solution is preferably a malic acid buffer solution, a citrate buffer solution, an acetate buffer solution, or an MES buffer solution (more preferably a malic acid buffer solution or a citrate buffer solution) having a pH of 1.0 to 6.5 (more preferably 3.0 to 6.5, and even more preferably 3.0 to 5.0).

[0425] From the viewpoint of nucleic acid stability, the buffer component concentration in the acidic buffer used in step a) is preferably 10 to 100 mM, more preferably 10 to 80 mM. Here, for example, when the acidic buffer is a malic acid buffer, the "buffer component concentration" refers to the total concentration of malic acid and its salts. Furthermore, for example, when the acidic buffer is a maleic acid / Tris / NaOH buffer, the "buffer component concentration" refers to the total concentration of maleic acid and its salts, and Tris and its salts. The "buffer component concentration" of other acidic buffers has the same meaning as the "buffer component concentration" of the malic acid buffer and the "buffer component concentration" of the maleic acid / Tris / NaOH buffer.

[0426] The nucleic acid concentration in the nucleic acid solution used in step a) is preferably 0.1 to 720 μg / mL, more preferably 0.5 to 350 μg / mL, and even more preferably 1 to 250 μg / mL, from the viewpoint of nucleic acid solubility.

[0427] When mixing the nucleic acid solution and the lipid alcohol solution, the volume ratio (nucleic acid solution:lipid alcohol solution) is preferably 1:1 to 20:1, more preferably 1.5:1 to 15:1, and even more preferably 2:1 to 8:1, from the viewpoint of the particle size of the nucleic acid-encapsulated lipid nanoparticles and the nucleic acid encapsulation rate.

[0428] Examples of the "assembly-inducing procedure" for preparing nucleic acid-encapsulated lipid nanoparticles include alcohol dilution using a microchannel or vortex, simple hydration, ultrasonic treatment, heating, vortexing, ether injection, French press method, cholic acid method, Ca ２＋ Examples of methods include known methods such as fusion, freeze-thaw, and reverse phase evaporation, and are preferably alcohol dilution using a microchannel or vortex, and more preferably alcohol dilution using a microchannel. Particle preparation by the alcohol dilution method using a microchannel can be performed using, for example, NanoAssemblr (manufactured by Precision NanoSystems).

[0429] <Step b)> In step b), the dispersion medium of the suspension obtained in step a) is exchanged for a buffer solution having a pH of 4.5 to 8.0 (hereinafter sometimes abbreviated as "another buffer solution") other than the acidic buffer solution used in step a) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles. The pH of the other buffer solution is preferably 5.0 to 7.6, more preferably 5.5 to 6.9.

[0430] Specific examples of other buffer solutions include tartaric acid / NaOH buffer solution, phthalic acid HK / HCl buffer solution, glycine / HCl buffer solution, trans-aconitic acid / NaOH buffer solution, formic acid / Na formate buffer solution, 3,3-dimethylglutaric acid / NaOH buffer solution, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer solution, phenylacetic acid / Na phenylacetic acid buffer solution, acetic acid / Na acetate buffer solution, succinic acid / NaOH buffer solution, phthalic acid HK / NaOH buffer solution, cacodylic acid Na / HCl buffer solution, maleic acid HNa / NaOH buffer solution, maleic acid / Tris / NaOH buffer solution, phosphate buffer solution, KH ２ P.O. ４ Examples of buffer solutions include Tris / NaOH buffer, MES buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, Tris / maleic acid buffer, succinic acid buffer, tartrate buffer, citrate buffer, Bis-Tris buffer, glycylglycine buffer, Tris / NaCl buffer, Tris / HCl buffer, and Tris-buffered saline.

[0431] Another buffer solution is malate buffer, citrate buffer, MES buffer, maleic acid / Tris / NaOH buffer, Tris / maleic acid buffer, or Tris-buffered saline (more preferably Tris / maleic acid buffer or Tris-buffered saline), preferably having a pH of 4.5 to 8.0 (more preferably 5.0 to 7.6, even more preferably 5.5 to 6.9).

[0432] The buffer component concentration in the other buffer solution is preferably 10 to 100 mM, more preferably 20 to 50 mM, from the viewpoint of nucleic acid encapsulation rate and nucleic acid-encapsulated lipid nanoparticle formation. Here, for example, when the other buffer solution is a citrate buffer solution, the "buffer component concentration" refers to the total concentration of citric acid and its salts. Furthermore, for example, when the other buffer solution is a maleic acid / Tris / NaOH buffer solution, the "buffer component concentration" refers to the total concentration of maleic acid and its salts, and Tris and its salts. The "buffer component concentration" of another buffer solution other than the citrate buffer solution and the maleic acid / Tris / NaOH buffer solution has the same meaning as the "buffer component concentration" of the citrate buffer solution and the "buffer component concentration" of the maleic acid / Tris / NaOH buffer solution.

[0433] The nucleic acid concentration in the suspension obtained in step b) is not particularly limited as long as it is a concentration at which the nucleic acid-encapsulated lipid nanoparticles can be maintained after the exchange of another buffer solution, but from the viewpoint of the particle size of the nucleic acid-encapsulated lipid nanoparticles, it is preferably 0.25 to 3300 μg / mL, more preferably 2.5 to 1500 μg / mL, and even more preferably 3 to 800 μg / mL. In step b), another buffer solution may be used in an amount that will result in the above-mentioned nucleic acid concentration.

[0434] The exchange of the dispersion medium of the suspension obtained in step a) with another buffer solution can be carried out, for example, by concentrating the suspension obtained in step a) and diluting it with another buffer solution. The concentration can be carried out, for example, by ultrafiltration. Ultrafiltration can be carried out, for example, by applying centrifugal force using an Amicon Ultra 15 (a centrifugal filter unit manufactured by Merck) or by tangential flow filtration. Here, "tangential flow filtration" refers to a method of filtration in which suspended matter in the membrane feed water is filtered while suppressing the deposition of the suspended matter on the membrane surface by creating a flow parallel to the membrane surface. This can be carried out, for example, using a "KrosFlo (registered trademark) KR2i TFF System" manufactured by REPLIGEN. The dilution can be carried out by adding a buffer solution or by dialysis. The order of the concentration and dilution is not particularly limited; concentration may be carried out first, followed by dilution, or dilution may be carried out first, followed by concentration, and then dilution. Multiple concentrations and multiple dilutions may also be carried out. Furthermore, examples of methods for exchanging a dispersion medium without concentration or dilution include diafiltration. Here, "diafiltration" refers to a method in which another buffer solution is transferred to the suspension at the same pace as filtration, thereby constantly exchanging the buffer solution from the suspension. This can be performed using, for example, the "KrosFlo (registered trademark) KR2i TFF System" manufactured by REPLIGEN.

[0435] <Step b')> In step b'), the dispersion medium of the suspension obtained in step a) is exchanged for a buffer solution having a pH of 4.5 to 8.0 and containing a cryoprotectant (hereinafter sometimes abbreviated as "another buffer solution containing a cryoprotectant") other than the acidic buffer solution used in step a), to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant. The cryoprotectant used in step b') is the same as that described above for the frozen composition of the present invention.

[0436] Examples of the other buffer solution containing a cryoprotectant in step b') include the other buffer solution in step b) to which sucrose has been added. Specific examples thereof include tartaric acid / NaOH / sucrose buffer, HK phthalate / HCl / sucrose buffer, glycine / HCl / sucrose buffer, trans-aconitic acid / NaOH / sucrose buffer, formic acid / Na formate / sucrose buffer, 3,3-dimethylglutaric acid / NaOH / sucrose buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl / sucrose buffer, phenylacetic acid / Na phenylacetate / sucrose buffer, acetic acid / Na acetate / sucrose buffer, succinic acid / NaOH / sucrose buffer, HK phthalate / NaOH / sucrose buffer, Na cacodylate / HCl / sucrose buffer, HNa maleate / NaOH / sucrose buffer, maleic acid / Tris / NaOH / sucrose buffer, phosphate / sucrose buffer, KH ２ P.O. ４ Examples of such buffers include tartaric acid / NaOH / sucrose buffer, MES / sucrose buffer, malic acid / sucrose buffer, phthalic acid / sucrose buffer, maleic acid / sucrose buffer, Tris / maleic acid / sucrose buffer, succinic acid / sucrose buffer, tartaric acid / sucrose buffer, citric acid / sucrose buffer, BisTris / sucrose buffer, glycylglycine / sucrose buffer, Tris / NaCl / sucrose buffer, Tris / HCl / sucrose buffer, and Tris-buffered saline / sucrose buffer. Here, "tartaric acid / NaOH / sucrose buffer" refers to a buffer obtained by adding sucrose to a tartaric acid / NaOH buffer. The other specific examples mentioned above also have the same meaning as "tartaric acid / NaOH / sucrose buffer."

[0437] Another buffer solution containing a cryoprotectant is a malic acid / sucrose buffer, a citrate / sucrose buffer, an MES / sucrose buffer, a maleic acid / Tris / NaOH / sucrose buffer, a Tris / maleic acid / sucrose buffer, or a Tris / NaCl / sucrose buffer (more preferably an MES / sucrose buffer, a Tris / NaCl / sucrose buffer, or a Tris / maleic acid / sucrose buffer), each preferably having a pH of 4.5 to 8.0 (more preferably 5.0 to 7.6, even more preferably 5.5 to 6.9).

[0438] The concentration of the buffer component in the separate buffer solution containing a cryoprotectant is preferably 10 to 100 mM, more preferably 20 to 50 mM, from the viewpoint of the nucleic acid encapsulation rate and the formation of nucleic acid-encapsulated lipid nanoparticles. The explanation for the buffer component concentration in the separate buffer solution containing a cryoprotectant in step b') is the same as the explanation for the buffer component concentration in the separate buffer solution in step b).

[0439] The concentration of the cryoprotectant in the separate buffer solution containing the cryoprotectant is preferably 20 to 320 mg / mL, more preferably 80 to 160 mg / mL, from the viewpoint of protecting the nucleic acid-encapsulated lipid nanoparticles from the stress of freezing and thawing.

[0440] The concentration of the cryoprotectant in the suspension obtained in step b') is preferably 20 to 320 mg / mL, more preferably 40 to 240 mg / mL, and even more preferably 80 to 160 mg / mL, from the viewpoint of protecting the nucleic acid-encapsulated lipid nanoparticles from the stress of freezing and thawing.

[0441] The nucleic acid concentration in the suspension obtained in step b') is not particularly limited as long as it is a concentration at which the nucleic acid-encapsulated lipid nanoparticles can be maintained after exchange with another buffer solution containing a cryoprotectant, but from the viewpoint of the particle size of the nucleic acid-encapsulated lipid nanoparticles, it is preferably 0.25 to 3300 μg / mL, more preferably 2.5 to 1500 μg / mL, and even more preferably 3 to 800 μg / mL. In step b'), another buffer solution containing a cryoprotectant can be used in an amount that will result in the above-mentioned nucleic acid concentration.

[0442] The exchange of the dispersion medium of the suspension obtained in step a) with another buffer solution containing a cryoprotectant can be carried out, for example, by concentrating the suspension obtained in step a) and diluting it with another buffer solution containing a cryoprotectant. The concentration and dilution can be carried out, for example, by ultrafiltration, dialysis, tangential flow filtration, etc. The order of concentration and dilution is not particularly limited, and concentration may be carried out first and then dilution may be carried out, or dilution may be carried out first, then concentration, and then dilution may be carried out. Furthermore, multiple concentrations and multiple dilutions may be carried out.

[0443] <Step c)> In step c), the suspension obtained in step b) or step b') is mixed with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant. The cryoprotectant used in step c) is the same as that described above for the frozen composition of the present invention.

[0444] In step c), a solution of the cryoprotectant may be used. Examples of solvents for this solution include water, ethanol, and dimethyl sulfoxide. This solvent is preferably water, and the cryoprotectant solution is preferably an aqueous solution of the cryoprotectant. In one embodiment of the present invention, from the viewpoint of protecting the nucleic acid-encapsulating lipid nanoparticles from the stress of freezing and thawing, the concentration of the cryoprotectant in the solution is preferably 40 to 1040 mg / mL, more preferably 40 to 640 mg / mL, even more preferably 60 to 640 mg / mL, particularly preferably 60 to 480 mg / mL, and most preferably 80 to 480 mg / mL.

[0445] The concentration of the cryoprotectant in the suspension obtained in step c) is preferably 40 to 320 mg / mL, more preferably 60 to 240 mg / mL, and even more preferably 80 to 480 mg / mL, from the viewpoint of protecting the nucleic acid-encapsulated lipid nanoparticles from the stress of freezing and thawing.

[0446] <Step d)> In step d), the suspension obtained in step b') or step c) is frozen at or below -10°C. The freezing temperature is preferably -80°C to -10°C, more preferably -25°C to -10°C.

[0447] The freezing in step d) can be carried out in any suitable container known in the pharmaceutical art, such as a polypropylene (PP) tube, a glass container (e.g., a glass vial), a two-chamber container, etc. Preferably, the container is a glass container.

[0448] The suspension obtained in step b') or step c) is added to a container (e.g., a PP tube or a glass container). The volume of the suspension added to the container is preferably 0.01 to 20 mL, more preferably 0.1 to 15 mL, and even more preferably 1 to 10 mL.

[0449] In the production method of the present invention, the ratio of (particle size of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) to (particle size of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained in step b') or step c) is preferably 0.8 to 1.5, more preferably 0.8 to 1.2. Thawing of the frozen composition for measuring the particle size is performed by allowing 20 mL or less of the frozen composition to stand in an air atmosphere at 4 to 25°C for 30 minutes to 2 hours. The measurement and calculation of the particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). In the present invention, "particle size" refers to the Z-average size (mean particle size) measured and calculated by dynamic light scattering.

[0450] In the manufacturing method of the present invention, the particle size of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) is not particularly limited, but is preferably 10 nm or more and 200 nm or less, more preferably 30 nm or more and 150 nm or less.

[0451] In the production method of the present invention, the PdI of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) is preferably 0.01 or more and 0.30 or less, more preferably 0.01 or more and 0.25 or less. Thawing of the frozen composition for measuring the PdI is performed by allowing 20 mL or less of the frozen composition to stand in an atmospheric pressure atmosphere at 4 to 25°C for 30 minutes to 2 hours. Measurement and calculation of the PdI can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern).

[0452] In the production method of the present invention, the ratio of (nucleic acid encapsulation rate of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) to (nucleic acid encapsulation rate of nucleic acid-encapsulated lipid nanoparticles in the suspension obtained in step b') or step c) is preferably 0.7 or more and 1.0 or less, more preferably 0.85 or more and 1.0 or less. Thawing of the frozen composition for measuring the encapsulation rate is carried out by allowing 20 mL or less of the frozen composition to stand in an atmospheric pressure atmosphere at 4 to 25°C for 30 minutes to 2 hours. The encapsulation rate can be measured and calculated using the Ribogreen® assay.

[0453] In the manufacturing method of the present invention, the nucleic acid encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) is not particularly limited, but is preferably 75% or more and 100% or less, more preferably 80% or more and 100% or less, and even more preferably 85% or more and 100% or less.

[0454] <Storage of frozen materials containing nucleic acid-encapsulated lipid nanoparticles> The frozen composition of the present invention and the frozen composition obtained by the production method of the present invention are preferably stored at a temperature of −10°C or below, more preferably −80°C to −10°C, and even more preferably −25°C to −15°C.

[0455] <Method for producing pharmaceutical composition> The present invention also provides a method for producing a pharmaceutical composition, which comprises the method for producing the frozen composition of the present invention.

[0456] <Method for introducing nucleic acid into cells> The present invention also provides: (I) a method for introducing nucleic acid contained in a suspension into cells, comprising the step of contacting cells ex vivo with a suspension obtained by thawing the frozen composition of the present invention or a frozen composition produced by the production method of the present invention, or a suspension obtained by exchanging the dispersal medium of the suspension with another dispersal medium; and (II) a method for introducing nucleic acid contained in a suspension into target cells in a living organism, comprising the step of administering to the living organism a suspension obtained by thawing the frozen composition of the present invention or a frozen composition produced by the production method of the present invention, or a suspension obtained by exchanging the dispersal medium of the suspension with another dispersal medium.

[0457] Hereinafter, the above-mentioned method (I) and the above-mentioned method (II) may be collectively referred to as the "introduction method of the present invention." Furthermore, "a suspension obtained by thawing the frozen composition of the present invention or the frozen composition produced by the production method of the present invention, or a suspension obtained by exchanging the dispersion medium of the suspension for another dispersion medium" used in the introduction method of the present invention may be hereinafter abbreviated as the "suspension of the present invention."

[0458] The suspension obtained by thawing the frozen composition of the present invention or the frozen composition produced by the production method of the present invention can have its dispersion medium exchanged for another dispersion medium by concentrating it and diluting it with another dispersion medium, for example. The concentration and dilution can be performed, for example, by ultrafiltration, dialysis, tangential flow filtration, etc. The order of concentration and dilution is not particularly limited, and concentration may be performed first and then dilution, or dilution may be performed first, then concentration, and then dilution. Multiple concentrations and multiple dilutions may also be performed. Examples of other dispersion media include the buffer solutions described in steps a) and b).

[0459] The suspension of the present invention (i.e., a suspension obtained by thawing the frozen composition of the present invention or the frozen composition produced by the production method of the present invention, or a suspension obtained by exchanging the dispersant of the suspension with another dispersant) may be concentrated or diluted before use in the introduction method of the present invention.

[0460] The method of introduction of the present invention allows any nucleic acid to be introduced into cells. Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, while any one of mono-, tri-, or tri-stranded nucleic acids can be used, single- or double-stranded nucleic acids are preferred. The nucleic acid may also be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, other oligomers with non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs)), or other oligomers with special linkages (provided that the oligomer contains nucleotides with configurations that allow base pairing or base attachment, such as those found in DNA or RNA). Furthermore, the nucleic acid may be, for example, a nucleic acid having a known modification added thereto, a nucleic acid labeled as known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides have been replaced with an analogue, a nucleic acid having an intramolecular nucleotide modification, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid having an intercurrent compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-anomeric nucleic acid, etc.), etc.

[0461] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc., preferably plasmid DNA, cDNA, and antisense DNA, more preferably plasmid DNA. Circular DNA such as plasmid DNA can also be digested with appropriate restriction enzymes and used as linear DNA.

[0462] The type of RNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., and preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.

[0463] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0464] The suspension of the present invention used in the introduction method of the present invention can be administered in vivo, for example, for the purpose of preventing and / or treating a disease. Therefore, the nucleic acid used in the present invention is preferably one that has preventive and / or therapeutic activity against a specific disease (prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0465] The suspension of the present invention used in the introduction method of the present invention can be used as a drug delivery system for selectively delivering nucleic acids and the like into specific cells, and is useful, for example, for DNA vaccines and gene therapy drugs for tumors that involve the introduction of antigen genes into dendritic cells, and nucleic acid pharmaceuticals that suppress the expression of target genes using RNA interference.

[0466] The step of contacting the suspension of the present invention with cells in vitro will be specifically described below.

[0467] The cells are suspended in an appropriate medium and cultured under appropriate conditions several days before contact with the suspension of the present invention. The cells may or may not be in a growth phase at the time of contact with the suspension of the present invention.

[0468] The culture medium at the time of contact may be a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 30% by weight or less, more preferably 20% by weight or less. If the medium contains excessive proteins such as serum, there is a possibility that contact between the suspension of the present invention and the cells is inhibited.

[0469] The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the type of cell, etc., but is usually 1×10 ４ ~1 x 10 ７ cells / mL range.

[0470] The suspension of the present invention is added to cells. The amount of the suspension of the present invention to be added is not particularly limited and can be appropriately set taking into account the number of cells, etc. The total lipid concentration when contacting the nucleic acid-encapsulated lipid nanoparticles or the like with cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved, but is typically 1 to 300 nmol / mL, preferably 10 to 200 nmol / mL, and the nucleic acid concentration is typically 0.01 to 100 μg / mL, preferably 0.05 to 10 μg / mL.

[0471] After adding the suspension of the present invention to the cells, the cells are cultured. ２ The concentration and other factors are appropriately set taking into consideration the type of cells. When the cells are mammalian cells, the temperature is usually about 37°C, the humidity is about 95%, and CO ２ The concentration is about 5%. The incubation time can also be set appropriately taking into account conditions such as the type of cells used, but is usually in the range of 0.1 to 96 hours, preferably 0.2 to 72 hours, and more preferably 0.5 to 48 hours. If the incubation time is too short, the nucleic acid may not be sufficiently introduced into the cells, and if the incubation time is too long, the cells may become weak.

[0472] The nucleic acid is introduced into the cells by the above-mentioned culture, and preferably the culture medium is replaced with a fresh medium or fresh medium is added to the medium and the culture is continued. When the cells are mammalian cells, the fresh medium preferably contains serum or nutrient factors.

[0473] Furthermore, as described above, the suspension of the present invention can be used to introduce nucleic acids into cells not only in vitro but also in vivo. That is, by administering the suspension of the present invention to a subject, the nucleic acid-encapsulated lipid nanoparticles contained therein reach and contact target cells, and the nucleic acid in the nucleic acid-encapsulated lipid nanoparticles is introduced into cells in vivo. The subjects to which the suspension of the present invention can be administered are not particularly limited, and examples include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), and fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), and plants. The subjects to which the suspension of the present invention is administered are preferably humans or other mammals.

[0474] The type of target cell is not particularly limited, and by using the suspension of the present invention, it is possible to introduce nucleic acids into cells in various tissues (e.g., liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph node, tumor, etc.).

[0475] The method of administering the suspension of the present invention to a subject (e.g., vertebrates, invertebrates, etc.) is not particularly limited as long as it allows the nucleic acid-encapsulated lipid nanoparticles contained in the suspension of the present invention to reach and contact the target cells, and can be appropriately selected from known administration methods (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) taking into account the type and site of the target cells, etc. The dosage of the suspension of the present invention is not particularly limited as long as it is within a range that allows for the introduction of the compound into cells, and can be appropriately selected taking into account the type of subject, administration method, type and site of the target cells, etc.

[0476] Examples of the present invention will be described in more detail below, but the present invention is not limited to these examples.

[0477] In the following examples, ionic lipid (1) is shown by the name listed in Tables 1 and 2. The ionic lipid "D-Lin-MC3-DMA" (CAS number: 1224606-06-7) used in the comparative examples is hereinafter abbreviated as "MC3." MC3 is an ionic lipid that does not have an S—S bond and is different from ionic lipid (1).

[0478] The meanings of the abbreviations used in the following examples are as follows: Chol: cholesterol DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (number average molecular weight (Mn) of PEG: 2000) DEPC: 1,2-dierucoyl-sn-glycero-3-phosphocholine DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPG: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine MES: 2-morpholinoethanesulfonic acid PBS: phosphate buffered saline DDW: deionized distilled water tBuOH: tert-butanol EtOH: ethanol

[0479] Example 1 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0480] 2075 μL of a nucleic acid solution (nucleic acid concentration: 3.3 μg / mL) obtained by diluting mRNA (CleanCap® FLuc mRNA (L-7602), hereinafter referred to as "FLuc mRNA") with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 725 μL of a lipid ethanol solution were each weighed into a syringe. Using a NanoAssemblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0481] After adding 14,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) to the resulting suspension, the resulting suspension was transferred to an Amicon Ultra 15 (Merck centrifugal filter unit) and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4 (a centrifugal filter unit manufactured by Merck), diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 50 μg / mL) (step b)).

[0482] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at -80°C to obtain a frozen composition (ionic lipid (1) content: 0.9 wt%, nucleic acid content: 5.0 μg, N / P ratio: 241, cryoprotectant (sucrose) content: 14 wt%, water (ice) content: 85 wt%) (step d).

[0483] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0484] Example 2 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0485] 2075 μL of nucleic acid solution (nucleic acid concentration: 6.7 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 725 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0486] After adding 14,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) to the resulting suspension, the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 100 μg / mL) (step b)).

[0487] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.9 wt %, nucleic acid content: 10 μg, N / P ratio: 120, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0488] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0489] Example 3 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0490] 2075 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 725 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0491] After adding 14,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) to the resulting suspension, the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0492] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.9 wt %, nucleic acid content: 20 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0493] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0494] Example 4 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 4.0 mM.

[0495] 1325 μL of nucleic acid solution (nucleic acid concentration: 26.7 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 475 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0496] To the resulting suspension, 7000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 1000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0497] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.2 wt %, nucleic acid content: 20 μg, N / P ratio: 15, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 86 wt %) (step d).

[0498] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0499] Example 5 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 4.0 mM.

[0500] 1325 μL of nucleic acid solution (nucleic acid concentration: 53.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 475 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0501] To the resulting suspension, 7000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 1000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 400 μg / mL) (step b)).

[0502] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.2 wt %, nucleic acid content: 40 μg, N / P ratio: 8, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0503] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0504] Comparative Example 1 (Preparation of a frozen composition containing nucleic acid-encapsulated lipid nanoparticles) MC3 was used as the ionic lipid, and DSPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of MC3:DSPC:chol:DMG-PEG2000 used was 50:10:38.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0505] 2075 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 725 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0506] After adding 14,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) to the resulting suspension, the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0507] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at -80°C to obtain a frozen composition (ionic lipid content: 1.0 wt%, nucleic acid content: 20 μg, N / P ratio: 62, cryoprotectant (sucrose) content: 14 wt%, water (ice) content: 84 wt%) (step d).

[0508] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0509] Comparative Example 2 (Preparation of a frozen composition containing nucleic acid-encapsulated lipid nanoparticles) MC3 was used as the ionic lipid, and DSPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of MC3:DSPC:chol:DMG-PEG2000 used was 50:10:38.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 4.0 mM.

[0510] 2075 μL of nucleic acid solution (nucleic acid concentration: 6.7 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 725 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0511] After adding 14,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) to the resulting suspension, the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 100 μg / mL) (step b)).

[0512] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at -80°C to obtain a frozen composition (ionic lipid content: 0.3 wt%, nucleic acid content: 10 μg, N / P ratio: 31, cryoprotectant (sucrose) content: 14 wt%, water (ice) content: 85 wt%) (step d).

[0513] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0514] Comparative Example 3 (Preparation of a frozen composition containing nucleic acid-encapsulated lipid nanoparticles) MC3 was used as the ionic lipid, and DSPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of MC3:DSPC:chol:DMG-PEG2000 used was 50:10:38.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 4.0 mM.

[0515] 1775 μL of nucleic acid solution (nucleic acid concentration: 33.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 625 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0516] To the resulting suspension, 11,200 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 400 μg / mL) (step b)).

[0517] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). The resulting suspension was placed in a screw tube and frozen at -80°C to obtain a frozen composition (ionic lipid content: 0.2 wt%, nucleic acid content: 40 μg, N / P ratio: 6, cryoprotectant (sucrose) content: 14 wt%, water (ice) content: 86 wt%) (step d).

[0518] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0519] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained by thawing the frozen compositions of Examples 1 to 5 and Comparative Examples 1 to 3 were analyzed. The mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained in step c) of Examples 1 to 5 and Comparative Examples 1 to 3 was also analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate (referred to as "encapsulation rate" in the tables below) was measured by the Ribogreen (registered trademark) assay. The results are shown in Table 3. In the tables below, the mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained by thawing the frozen compositions is listed in the "After freezing and thawing" column, and the mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained in step c) is listed in the "Step c)" column. The particle size is shown as the Z-average size (referred to as "Z-ave" in the following tables).

[0520]

[0521] The frozen compositions of Example 3, Example 5, Comparative Example 1, or Comparative Example 3 were thawed and further diluted with Tris-buffered saline (pH: 7.6) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 2 μg / mL). The nucleic acid-encapsulated lipid nanoparticles in the obtained suspension were allowed to act on HeLa cells, and their cytotoxicity was evaluated. Specifically, HeLa cells were cultured at a concentration of 5 × 10 in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% by volume FBS and 1% by volume penicillin-streptomycin solution). ３ The cells were seeded into the wells of a 96-well flat-bottom transparent black plate at 100 μL per well. A suspension containing nucleic acid-encapsulated lipid nanoparticles obtained by thawing the frozen composition was added to the wells so that the amount of nucleic acid in the medium was 7.8 to 125 ng. The 50% survival dose after 24 hours of incubation was evaluated using Cell Counting Kit-8 (Dojindo Laboratories). The results are shown in Tables 4-1 and 4-2.

[0522]

[0523]

[0524] As shown in Table 4-1, the N / P ratios of the nucleic acid-encapsulated lipid nanoparticles of Example 3 and Comparative Example 1, which have high N / P ratios, are similar (approximately 60). However, the nucleic acid-encapsulated lipid nanoparticles of Example 1 exhibited a higher 50% viable dose than those of Comparative Example 1. Furthermore, as shown in Table 4-2, the N / P ratios of the nucleic acid-encapsulated lipid nanoparticles of Example 5 and Comparative Example 3, which have low N / P ratios, are similar (6 to 8). The nucleic acid-encapsulated lipid nanoparticles of Example 5 and Comparative Example 3 both exhibited a 50% viable dose of 125 ng or more.

[0525] The frozen compositions of Examples 3 to 5 or Comparative Examples 1 to 3 were thawed and further diluted with Tris-buffered saline (pH: 7.6) to obtain suspensions containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 2 μg / mL). The nucleic acid-encapsulated lipid nanoparticles in the obtained suspensions were allowed to act on HeLa cells, and their gene expression activity was evaluated. Specifically, HeLa cells were cultured at a concentration of 5 × 10 in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (containing 10% by volume FBS and 1% by volume penicillin-streptomycin solution). ３ The resulting suspension containing nucleic acid-encapsulated lipid nanoparticles was seeded into the wells of a 96-well, flat-bottom, transparent black plate at 100 μL / cell. The resulting suspension containing nucleic acid-encapsulated lipid nanoparticles was added to the wells so that the nucleic acid concentration in the medium was 0.031 μg / 10 μL. Luciferin was added to the wells so that the concentration in the medium was 0.1 mM, and the amount of luciferase protein introduced after 24 hours was evaluated using the luminescence intensity. HeLa cells cultured in a medium containing nucleic acid-encapsulated lipid nanoparticles and luciferin were measured for luminescence at 560 nm using a plate reader. The results are shown in Figures 1 and 2.

[0526] As shown in Figure 1, the gene expression activity of Example 3, in which the N / P ratio was 60, was 1.9 times that of Example 5, in which the N / P ratio was 8. On the other hand, as shown in Figure 2, the gene expression activity of Comparative Example 1, in which the N / P ratio was 62, was 0.2 times that of Comparative Example 3, in which the N / P ratio was 6. Furthermore, the gene expression activity of the nucleic acid-encapsulated lipid nanoparticles of Example 3 was 38.8 times that of the nucleic acid-encapsulated lipid nanoparticles of Comparative Example 1.

[0527] Table 4 and Figures 1 and 2 demonstrate that the nucleic acid-encapsulated lipid nanoparticles in the frozen composition of the present invention have both low toxicity and high gene expression activity.

[0528] Comparative Example 4 (Preparation of a suspension containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and chol. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 3.0 mM.

[0529] 975 μL of nucleic acid solution (nucleic acid concentration: 5.0 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 433 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0530] To the resulting suspension, 1590 μL of Tris-buffered saline (buffer component concentration: 50 mM, pH: 7.6) was added, and the resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes) to concentrate to approximately 250 μL. The above procedure was repeated twice to concentrate to 200 μL, yielding a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 10 μg / mL, N / P ratio: 60) (step b)).

[0531] Example 6 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and chol. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0532] 2225 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with acidic malate buffer (buffer component concentration: 20 mM, pH: 3.0) and 775 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, nucleic acid-encapsulated lipid nanoparticles were prepared under the conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 4 mL / min (step a)).

[0533] To the resulting suspension, 12,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0534] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). 25 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.9 wt %, nucleic acid content: 2.5 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0535] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0536] Example 7 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and chol. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0537] 2225 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 775 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0538] To the resulting suspension, 12,000 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0539] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). 25 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.9 wt %, nucleic acid content: 2.5 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0540] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0541] Example 8 (Method for preparing a frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and chol. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0542] 2225 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 775 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0543] To the resulting suspension, 12,000 μL of Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugal conditions (25 ° C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with Tris / maleic acid buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugal conditions (25 ° C, 1,000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0), and again concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0544] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). 25 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 0.9 wt %, nucleic acid content: 2.5 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0545] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0546] Example 9 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0547] 1613 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 638 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0548] To the resulting suspension, 7000 μL of Tris / NaCl / sucrose buffer (buffer component concentration: 25 mM, sucrose concentration: 400 mM, pH: 7.4) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2000 μL by ultrafiltration under centrifugal conditions (25 ° C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with Tris / NaCl / sucrose buffer (buffer component concentration: 20 mM, pH: 7.4) and again concentrated to approximately 500 μL by ultrafiltration under centrifugal conditions (25 ° C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4 and diluted to 4 mL with Tris / NaCl / sucrose buffer (buffer component concentration: 25 mM, sucrose concentration: 137 mg / mL, pH: 7.4). This was again concentrated to approximately 240 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). This was then diluted with Tris / NaCl / sucrose buffer (buffer component concentration: 25 mM, sucrose concentration: 137 mg / mL, pH: 7.4) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (880 μL, nucleic acid concentration: 200 μg / mL) (step b')).

[0549] The resulting suspension was placed in 35 μL aliquots in Eppendorf tubes and frozen at −80° C. to obtain a frozen composition (ionic lipid (1) content: 1.9 wt %, nucleic acid content: 7 g, N / P ratio: 60, cryoprotectant (sucrose) content: 12 wt %, water (ice) content: 84 wt %) (step d)).

[0550] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0551] Example 10 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 6.0 mM.

[0552] 3148 μL of nucleic acid solution (nucleic acid concentration: 56 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 1270 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, nucleic acid-encapsulated lipid nanoparticles were prepared at a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0553] To the resulting suspension, 4000 μL of Tris / NaCl buffer (buffer component concentration: 50 mM, pH: 7.4) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 1500 μL by ultrafiltration under centrifugal conditions (25° C., 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with Tris / NaCl buffer (buffer component concentration: 50 mM, pH: 7.4) and again concentrated to approximately 500 μL by ultrafiltration under centrifugal conditions (25° C., 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 4, diluted to 4 mL with Tris / NaCl buffer (buffer component concentration: 50 mM, pH: 7.4), and again concentrated to approximately 250 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with Tris / NaCl buffer (buffer component concentration: 50 mM, pH: 7.4) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 200 μg / mL) (step b)).

[0554] To the resulting suspension, an aqueous sucrose solution was added so that the final sucrose concentration was 160 mg / mL (step c). 60 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −20° C. to obtain a frozen composition (ionic lipid (1) content: 0.5 wt %, nucleic acid content: 88 μg, N / P ratio: 33, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 84 wt %) (step d).

[0555] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand at 25° C. for 30 minutes in an atmospheric pressure atmosphere to obtain a suspension.

[0556] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained by thawing the frozen compositions of Examples 6 to 10 were analyzed. The results are shown in the "After freezing and thawing" row of Table 5. Similarly, the particle size, PdI, and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained in step b') or step c) of Examples 6 to 10 and the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained in Comparative Example 4 were analyzed. The results are shown in the "Step b')" or "Step c)" row of Table 5. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate (referred to as "encapsulation rate" in the tables below) was measured by Ribogreen (registered trademark) assay. Note that particle size is shown as Z-average size (referred to as "Z-ave" in the tables below).

[0557]

[0558] Each of the frozen compositions of Examples 6 to 9 was thawed and further diluted with Tris-buffered saline (pH: 7.6) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 2 μg / mL). The suspension of Comparative Example 4 was diluted with Tris-buffered saline (pH: 7.6) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 2 μg / mL). The nucleic acid-encapsulated lipid nanoparticles in the obtained suspension were allowed to act on HeLa cells, and their gene expression activity was evaluated. Specifically, HeLa cells were cultured at a concentration of 3 × 10 in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (containing 10% by volume FBS and 1% by volume penicillin-streptomycin solution). ４ The cells were seeded into wells of a 24-well flat-bottom transparent black plate at a concentration of 0.12 μg / 600 μL. The resulting suspension containing nucleic acid-encapsulated lipid nanoparticles was added to the wells so that the nucleic acid concentration in the medium was 0.12 μg / 60 μL. Luciferin was added to the wells so that the concentration in the medium was 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using the amount of luminescence. HeLa cells cultured in a medium containing nucleic acid-encapsulated lipid nanoparticles and luciferin were placed in an incubator-type luminometer (ATTO Corporation, "WSL-1565 Kronos HT"), and the cumulative amount of luminescence was measured every hour. The results are shown in Figure 3. Note that "1.2.E+08" and the like in Figure 3 represent "1.2 x 10 ８ " etc.

[0559] As shown in Figure 3, the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained by thawing the frozen compositions of Examples 6 to 9 exhibited gene expression activity equal to or greater than that of the nucleic acid-encapsulated lipid nanoparticles in the unfrozen suspension of Comparative Example 4.

[0560] Example 11 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0561] 2400 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 900 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0562] To the resulting suspension, 8300 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 15 and diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0). This was again concentrated to approximately 230 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (230 μL, nucleic acid concentration: 100 μg / mL) (step b)).

[0563] To the resulting suspension, an aqueous sucrose solution was added so that the final concentration of sucrose was 160 mg / mL (step c). 35 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.5 wt %, nucleic acid content: 1.75 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0564] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0565] Example 12 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-EC was used as the ionic lipid (1), and DOPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-EC:DOPE:chol:DMG-PEG2000 used was 59:29.6:9.9:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0566] 2400 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 900 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0567] To the resulting suspension, 8300 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 15 and diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0). This was again concentrated to approximately 230 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (230 μL, nucleic acid concentration: 100 μg / mL) (step b)).

[0568] To the resulting suspension, an aqueous sucrose solution was added so that the final sucrose concentration was 160 mg / mL (step c). 35 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.7 wt %, nucleic acid content: 1.75 μg, N / P ratio: 73, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0569] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0570] Example 13 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP and SS-EC were used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:SS-EC:DOPC:chol:DMG-PEG2000 used was 30:18.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0571] 2400 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 900 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0572] To the resulting suspension, 8300 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 15 and diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0). This was again concentrated to approximately 230 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (230 μL, nucleic acid concentration: 100 μg / mL) (step b)).

[0573] To the resulting suspension, an aqueous sucrose solution was added so that the final sucrose concentration was 160 mg / mL (step c). 35 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.5 wt %, nucleic acid content: 1.75 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0574] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0575] Example 14 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 13 was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 13:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0576] 2400 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 900 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0577] To the resulting suspension, 8300 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). The obtained concentrate was transferred to an Amicon Ultra 15 and diluted to 4 mL with MES buffer (buffer component concentration: 20 mM, pH: 6.0). This was again concentrated to approximately 230 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with MES buffer (buffer component concentration: 20 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (230 μL, nucleic acid concentration: 100 μg / mL) (step b)).

[0578] To the resulting suspension, an aqueous sucrose solution was added so that the final sucrose concentration was 160 mg / mL (step c). 35 μL of the resulting suspension was placed in an Eppendorf tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.5 wt %, nucleic acid content: 1.75 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0579] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0580] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained by thawing the frozen compositions of Examples 11 to 14 were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate (referred to as "encapsulation rate" in the tables below) was measured by the Ribogreen (registered trademark) assay. The results are shown in Table 6. Particle size is shown as Z-average size (referred to as "Z-ave" in the tables below).

[0581]

[0582] Each of the frozen compositions of Examples 11 to 14 was thawed and further diluted with Tris-buffered saline (pH: 7.6) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 2 μg / mL). The obtained nucleic acid-encapsulated lipid nanoparticles were allowed to act on HeLa cells, and their gene expression activity was evaluated. Specifically, HeLa cells were cultured at a concentration of 3 × 10 in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (containing 10% by volume FBS and 1% by volume penicillin-streptomycin solution). ４ The cells were seeded into wells of a 24-well flat-bottom transparent black plate at a concentration of 0.12 μg / 600 μL. The resulting suspension containing nucleic acid-encapsulated lipid nanoparticles was added to the wells so that the nucleic acid concentration in the medium was 0.12 μg / 60 μL. Luciferin was added to the wells so that the concentration in the medium was 0.1 mM, and the amount of luciferase protein introduced was evaluated over time using the amount of luminescence. HeLa cells cultured in a medium containing nucleic acid-encapsulated lipid nanoparticles and luciferin were placed in an incubator-type luminometer (Kronos), and the cumulative amount of luminescence was measured every hour. The results are shown in Figure 4. Note that "6.00.E+07" and the like in Figure 4 represent "6.00 x 10 ７ " etc.

[0583] 4, the nucleic acid-encapsulated lipid nanoparticles in the suspensions obtained by thawing the frozen compositions of Examples 11 to 14 all exhibited high gene expression activity. The nucleic acid-encapsulated lipid nanoparticles of Examples 11 and 14 had particularly high gene expression activity.

[0584] Example 15 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0585] 11,700 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 4,100 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0586] To the resulting suspension, 11,400 μL of Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugal conditions (25 ° C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0) and again concentrated to approximately 2,000 μL by ultrafiltration under centrifugal conditions (25 ° C, 1,000 g, 3 minutes). The obtained concentrate was diluted to 15 mL with Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0), and again concentrated to approximately 1000 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 3 minutes). This was then diluted with Tris / maleic acid buffer (buffer component concentration: 25 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (1315 μL, nucleic acid concentration: 8.8 μg / mL) (step b)).

[0587] To the resulting suspension, an aqueous sucrose solution was added so that the final sucrose concentration was 160 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. or −20° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.04 wt %, nucleic acid content: 0.88 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 86 wt %) (step d).

[0588] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0589] Example 16 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol:DMG-PEG2000 used was 48.5:7.5:42.5:1.5. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0590] 11,700 μL of nucleic acid solution (nucleic acid concentration: 13.3 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 4,100 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0591] To the resulting suspension, 11,400 μL of Tris / maleic acid / NaCl buffer (buffer component concentration: 25 mM, pH: 6.0) was added, and the resulting suspension was transferred to an Amicon Ultra 15 and concentrated to approximately 2,000 μL by ultrafiltration under centrifugal conditions (25 ° C, 1,000 g, 3 minutes). The resulting concentrate was diluted to 15 mL with Tris / maleic acid / NaCl buffer (buffer component concentration: 50 mM, pH: 6.0) and again concentrated to approximately 2,000 μL by ultrafiltration under centrifugal conditions (25 ° C, 1,000 g, 3 minutes). The obtained concentrate was diluted to 15 mL with Tris / maleic acid / NaCl buffer (buffer component concentration: 50 mM, pH: 6.0), and again concentrated to approximately 1000 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 3 minutes). This was then diluted with Tris / maleic acid / NaCl buffer (buffer component concentration: 50 mM, pH: 6.0) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (1315 μL, nucleic acid concentration: 8.8 μg / mL) (step b)).

[0592] To the resulting suspension, an aqueous sucrose solution was added so that the final sucrose concentration was 160 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. or −20° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.04 wt %, nucleic acid content: 0.88 μg, N / P ratio: 60, cryoprotectant (sucrose) content: 14 wt %, water (ice) content: 85 wt %) (step d).

[0593] After freezing, the frozen composition was stored at −20° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0594] Comparative Example 5 (Preparation of a suspension containing nucleic acid-encapsulating lipid nanoparticles) SS-OP was used as the ionic lipid (1), and DOPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP:DOPC:chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP, DOPC, and chol. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 4.0 mM.

[0595] 1986 μL of nucleic acid solution (nucleic acid concentration: 6.7 μg / mL) obtained by diluting FLuc mRNA with acidic malate buffer (buffer component concentration: 20 mM, pH: 3.0) and 663 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under the following conditions: a nucleic acid solution:lipid ethanol solution flow ratio of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C (step a)).

[0596] After adding 10,600 μL of MES buffer (buffer component concentration: 20 mM, pH: 6.5) to the resulting suspension, the resulting suspension was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugal conditions (25 ° C, 1000 g, 5 minutes) to concentrate to approximately 2000 μL. The resulting concentrate was diluted to 15 mL with PBS (pH: 7.4) and again subjected to ultrafiltration under centrifugal conditions (25 ° C, 1000 g, 5 minutes) to concentrate to approximately 2000 μL. The above operation was repeated twice, concentrating to 600 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 10 μg / mL, N / P ratio: 60) (step b)). After preparation, the suspension was stored at 4 ° C.

[0597] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained in step c) of Examples 15 and 16 were analyzed. Similarly, the particle size, PdI, and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) of Examples 15 and 16 were analyzed 1 day after freezing (hereinafter referred to as "Day 1"), 7 days after freezing (hereinafter referred to as "Day 7"), and 30 days after freezing (hereinafter referred to as "Day 30"), respectively. Similarly, for Comparative Example 4, the particle size, PdI, and mRNA encapsulation rate of the nucleic acid-encapsulated lipid nanoparticles in the suspension were analyzed 1 day after the suspension was prepared (hereinafter referred to as "Day 1"), 7 days after the suspension was prepared (hereinafter referred to as "Day 7"), and 14 days after the suspension was prepared (hereinafter referred to as "Day 14"). Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate (referred to as "encapsulation rate" in the tables below) was measured by the Ribogreen (registered trademark) assay. The results are shown in Table 7. Particle size is shown as Z-average size (referred to as "Z-ave" in the tables below).

[0598]

[0599] As shown in Table 7, the frozen compositions of Examples 15 and 16 showed no significant changes in the physical properties of the nucleic acid-encapsulated lipid nanoparticles even after thawing after storage for 30 days, demonstrating high storage stability.

[0600] The frozen compositions of Example 9 (freezing temperature: -80°C), Example 15 (freezing temperature: -20°C or -80°C), and Example 16 (freezing temperature: -20°C or -80°C) were stored at -20°C after freezing as described above. The frozen compositions were thawed one day after freezing (hereinafter referred to as "1 day"), one week after freezing (hereinafter referred to as "1 week"), two weeks after freezing (hereinafter referred to as "2 weeks"), and one month after freezing (hereinafter referred to as "1 month") and further diluted with Tris-buffered saline (pH: 7.6) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 0.12 μg / 60 μL). The resulting nucleic acid-encapsulated nanoparticles were applied to HeLa cells, and their gene expression activity was evaluated.

[0601] Similarly, the suspension containing nucleic acid-encapsulated lipid nanoparticles obtained in Comparative Example 5 was prepared as described above and stored at 4 ° C. One day after the preparation date (hereinafter referred to as "1 day"), one week after the preparation date (hereinafter referred to as "1 week"), and two weeks after the preparation date (hereinafter referred to as "2 weeks"), the frozen composition was diluted with PBS (pH: 7.4) to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (nucleic acid concentration: 0.12 μg / 60 μL). The obtained nucleic acid-encapsulated nanoparticles were applied to HeLa cells, and their gene expression activity was evaluated.

[0602] Specifically, HeLa cells were cultured at a density of 3 × 10 in a D-MEM medium (high glucose) (containing L-glutamine and phenol red) (containing 10% by volume FBS and 1% by volume penicillin-streptomycin solution). ４The cells were seeded at 600 μL / cell into a 24-well flat-bottom transparent black plate. The suspension of nucleic acid-encapsulated nanoparticles obtained as described above was added to the culture medium. 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 the amount of luminescence. HeLa cells cultured in a culture medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubator-type luminometer (Kronos), and the cumulative amount of luminescence was measured every hour. The results are shown in Figure 5. Note that the bar graphs in Figure 5 for Examples 9, 15, and 16 and Comparative Example 5 show the results for 1 day, 1 week, 2 weeks, and 1 month, from left to right. Furthermore, "1.6.E+08" and the like in Figure 5 represent "1.6 x 10 ８ ", etc. In addition, the freezing temperatures of Examples 9, 15 and 16 are also shown in FIG.

[0603] As shown in Figure 5, the nucleic acid-encapsulated lipid nanoparticles in the frozen compositions of Examples 9, 15, and 16 maintained high gene expression activity even after one month of storage, demonstrating high storage stability. On the other hand, in the comparative example without frozen storage, a decrease in the gene expression activity of the nucleic acid-encapsulated lipid nanoparticles was observed one week after preparation, and this activity further decreased two weeks after preparation, indicating low storage stability.

[0604] Example 17 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 7 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 7:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0605] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0606] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 (Repligen, MWCO: 8-10 kD) and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4, and ultrafiltered under centrifugation conditions (25 ° C., 1000 g, 3 minutes), and concentrated to approximately 100 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) (step b)).

[0607] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.7 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0608] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0609] Example 18 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 9 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 9:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0610] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0611] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 200 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (200 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0612] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.3 wt %, nucleic acid content: 20 μg, N / P ratio: 16, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 92 wt %) (step d).

[0613] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0614] Example 19 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 10 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 10:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0615] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0616] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 200 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (200 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0617] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.3 wt %, nucleic acid content: 20 μg, N / P ratio: 16, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 92 wt %) (step d).

[0618] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0619] Example 20 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 11 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 11:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0620] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0621] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 500 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 100 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) (step b)).

[0622] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.6 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0623] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0624] Example 21 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 12 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 12:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0625] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0626] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 200 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (200 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0627] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.3 wt %, nucleic acid content: 20 μg, N / P ratio: 16, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 92 wt %) (step d).

[0628] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0629] Example 22 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 23 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 23:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0630] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0631] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes) to concentrate to approximately 100 μL, and a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) was obtained (step b)).

[0632] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.6 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0633] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0634] Example 23 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 33 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 33:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0635] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0636] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 200 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (200 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0637] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.3 wt %, nucleic acid content: 20 μg, N / P ratio: 16, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 92 wt %) (step d).

[0638] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0639] Example 24 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 34 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of Compound 34:POPE:Chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0640] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0641] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes) to concentrate to approximately 100 μL, and a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) was obtained (step b)).

[0642] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.6 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0643] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0644] Example 25 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 37 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 37:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0645] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0646] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes) to concentrate to approximately 100 μL, and a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) was obtained (step b)).

[0647] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.6 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0648] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0649] Example 26 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 58 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 58:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0650] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0651] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes) to concentrate to approximately 100 μL, and a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) was obtained (step b)).

[0652] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.6 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0653] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0654] Example 27 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 155 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 155:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0655] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0656] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes) to concentrate to approximately 100 μL, and a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) was obtained (step b)).

[0657] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.7 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0658] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0659] Example 28 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 159 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 159:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0660] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0661] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 200 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (200 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0662] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.3 wt %, nucleic acid content: 20 μg, N / P ratio: 16, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 92 wt %) (step d).

[0663] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0664] Example 29 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 161 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 161:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0665] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0666] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes), and concentrated to approximately 200 μL to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles (200 μL, nucleic acid concentration: 200 μg / mL) (step b)).

[0667] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.3 wt %, nucleic acid content: 20 μg, N / P ratio: 16, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 92 wt %) (step d).

[0668] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0669] Example 30 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 162 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 162:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0670] 975 μL of nucleic acid solution (nucleic acid concentration: 13.47 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0671] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C, 1000 g, 3 minutes) to concentrate to approximately 100 μL, and a suspension containing nucleic acid-encapsulated lipid nanoparticles (100 μL, nucleic acid concentration: 101 μg / mL) was obtained (step b)).

[0672] To the resulting suspension, Tris / maleic acid / sucrose buffer (buffer component concentration: 50 mM, pH: 6.0, sucrose concentration: 160 mg / mL) was added so that the final sucrose concentration was 80 mg / mL (step c). 200 μL of the resulting suspension was placed in a screw tube and frozen at −80° C. for 24 hours to obtain a frozen composition (ionic lipid (1) content: 0.6 wt %, nucleic acid content: 10 μg, N / P ratio: 64, cryoprotectant (sucrose) content: 7 wt %, water (ice) content: 91 wt %) (step d).

[0673] After freezing, the frozen composition was stored at −80° C., and before use, the frozen composition was thawed by leaving it to stand in an atmospheric pressure atmosphere in a refrigerator at 4° C. for 30 minutes to obtain a suspension.

[0674] Example 31 (Preparation of frozen composition containing nucleic acid-encapsulated lipid nanoparticles) Compound 163 was used as the ionic lipid (1), and POPE, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of compound 163:POPE:chol:DMG-PEG2000 used was 49:15:34:2. The lipids were mixed so that the total lipid concentration in the lipid ethanol solution was 8.0 mM.

[0675] 975 μL of nucleic acid solution (nucleic acid concentration: 53.33 μg / mL) obtained by diluting FLuc mRNA with an acidic citrate buffer (buffer component concentration: 20 mM, pH: 5.0) and 325 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssemblr® ultrafast nanomedicine production device, a suspension containing nucleic acid-encapsulated lipid nanoparticles was prepared under conditions of a nucleic acid solution:lipid ethanol solution flow ratio of 3:1 and a total flow rate of 12 mL / min (step a)).

[0676] The resulting suspension was transferred to a Spectra / Por Float-A-Lyzer G2 and dialyzed for 3 hours in a beaker containing 400 mL of Tris / maleic acid buffer (buffer component concentration: 50 mM, pH: 6.0) in a chamber at 4 ° C. After 3 hours, the buffer was replaced with a new one, and the mixture was dialyzed again for 3 hours in a chamber at 4 ° C. After that, the buffer was replaced with a new one again, and the mixture was dialyzed for 16 hours in a chamber at 4 ° C. The resulting suspension was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation c...

Claims

1. A frozen composition comprising nucleic acid-encapsulating lipid nanoparticles, wherein the nucleic acid-encapsulating lipid nanoparticles have the formula (1): (In formula (1), R １ａ and R １ｂ each independently represent an alkylene group having 1 to 6 carbon atoms, X ａ and X ｂ each independently represent an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R ２ａ and R ２ｂ each independently represent an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms, Y ａ and Y ｂ each independently represent an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z ａ and Z ｂ each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom, na and nb are each independently 0 or 1, R ３ａ and R ３ｂ each independently represent (i) a monovalent group having 10 to 50 carbon atoms having at least one unsaturated bond selected from the group consisting of one carbonyl group and at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond (provided that it excludes monovalent groups including residues of fat-soluble vitamins having a hydroxyl group and residues of sterol derivatives having a hydroxyl group), (ii) a monovalent group having 10 to 50 carbon atoms having at least two carbonyl groups (provided that it excludes monovalent groups including residues of fat-soluble vitamins having a hydroxyl group and residues of sterol derivatives having a hydroxyl group), (iii) formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents the bonding position, R ４ represents an alkylene group having 1 to 10 carbon atoms, X １ represents a carbamate bond, a carbonate bond, or an amide bond, and R ５ represents an alkyl group having 1 to 25 carbon atoms, and R ５ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group. ) A monovalent group represented by, (iv) Formula (3): *-R ６ -CO-O-R ７ (In Formula (3), * represents a bonding position, and R ６ represents an alkylene group having 1 to 10 carbon atoms, and R ７ represents an alkyl group having 1 to 25 carbon atoms substituted with at least one halogen atom. ) A monovalent group represented by, (v) Formula (4): (In Formula (4), * represents a bonding position, and R ８ and R ９ each independently represent an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R １０ to R １２ each independently represent a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ ) group (in the above formula, * represents a bonding position, and R １３ to R １５ each independently represent an alkyl group having 1 to 4 carbon atoms or a phenyl group. ) A monovalent group represented by, (vi) Formula (5): (In Formula (5), * represents a bonding position, and X ２ represents a nitrogen atom or Formula (6): (In Formula (6), * represents the bonding position with R １６ , and ** represents the bonding position with R １７ or R １８ . ) Represents a trivalent group represented by, when X ２ is a nitrogen atom, R １６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and X ２ When is a trivalent group represented by the formula (6), R １６ represents an alkylene group having 1 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and X ２ When is a nitrogen atom, R １７ and R １８ each independently represent an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and R １７ and R １８ may each independently be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and, X ２ When is a trivalent group represented by the formula (6), R １７ and R １８ each independently represent an alkyl group having 1 to 10 carbon atoms, and R １７ and R １８ may each independently be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group. ), a monovalent group represented by (vii) formula (7): (In formula (7), * represents the bonding position, and R １９ is a hydrogen atom, a benzyl group, *-Si(R １３ )(R １４ )(R １５ ) group (in the above formula, * represents the bonding position, and R １３ to R １５ each independently represent an alkyl group having 1 to 4 carbon atoms or a phenyl group. ), or *-CO-R ２０ group (in the above formula, * represents the bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms. ). ), a monovalent group represented by (viii) formula (8): (In formula (8), * represents the bonding position, and R ２１ and R ２２ each independently represent a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ )(wherein, * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group.) represents.) A monovalent group represented by, (ix) Formula (9): (In Formula (9), * represents a bonding position, and R ２３ is a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ )(wherein, * represents a bonding position, and R １３ ~R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group.) represents.) A monovalent group represented by, (x) Formula (10): (In Formula (10), * represents a bonding position. R ２４ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２５ represents an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, or an alkynyl group having 2 to 30 carbon atoms.) A monovalent group represented by, (xi) Formula (11): (In Formula (11), * represents a bonding position, and R ２６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２７ and R ２８ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms.) A monovalent group represented by, or (xii) Formula (12): (In Formula (12), * represents a bonding position, R ２９ represents an alkylene group having 1 to 10 carbon atoms, and R ３０ and R ３１ Each independently represents an alkyl group having 1 to 10 carbon atoms.) A monovalent group represented by, (xiii) A monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, wherein one ethylene group in the alkyl group may be replaced by one ester bond, or, (xiv) R ３ｃ -CO-(CH ２ )) ｐ - group (in the above formula, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8.) represents, and R ３ａ and R ３ｂ may be the same as or different from each other.) A frozen composition containing an ionic lipid represented by, and the molar ratio of the amino group of the ionic lipid represented by the formula (1) to the phosphate group of the nucleic acid is 7 or more and 250 or less.

2. R ３ａ and R ３ｂ are each independently (i) a monovalent group having 10 to 50 carbon atoms having one carbonyl group and at least one unsaturated bond selected from the group consisting of an olefinic carbon-carbon double bond and a carbon-carbon triple bond (however, excluding monovalent groups including residues of fat-soluble vitamins having a hydroxyl group and residues of sterol derivatives having a hydroxyl group), (ii) a monovalent group having 10 to 50 carbon atoms having at least two carbonyl groups (however, excluding monovalent groups including residues of fat-soluble vitamins having a hydroxyl group and residues of sterol derivatives having a hydroxyl group), (iii) the monovalent group represented by the formula (2), (vii) the monovalent group represented by the formula (7), (ix) the monovalent group represented by the formula (9), (xiii) a monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, wherein one ethylene group in the alkyl group may be replaced by one ester bond, or (xiv) R ３ｃ -CO-(CH ２ ) ｐ - group (in the above formula, R ３ｃ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8). The frozen composition according to claim 1.

3. The frozen composition according to claim 1, wherein the nucleic acid is RNA or DNA.

4. The frozen composition according to claim 1, wherein the nucleic acid is mRNA.

5. The frozen composition according to claim 1, wherein the particle diameter of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition is 10 nm or more and 200 nm or less.

6. The frozen composition according to claim 1, wherein the PdI of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition is 0.01 or more and 0.30 or less.

7. The frozen composition according to claim 1, wherein the nucleic acid encapsulation rate of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition is 75% or more and 100% or less.

8. A method for producing a frozen composition containing nucleic acid-encapsulating lipid nanoparticles, wherein the nucleic acid-encapsulating lipid nanoparticles have the formula (1): (In formula (1), R １ａ and R １ｂ each independently represent an alkylene group having 1 to 6 carbon atoms, X ａ and X ｂ each independently represent an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R ２ａ and R ２ｂ each independently represent an alkylene group having 1 to 8 carbon atoms or an oxydialkylene group having 2 to 8 carbon atoms, Y ａ and Y ｂ each independently represent an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z ａ and Z ｂ each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a hetero atom, na and nb are each independently 0 or 1, R ３ａ and R ３ｂ each independently represent (i) a monovalent group having 10 to 50 carbon atoms having at least one unsaturated bond selected from the group consisting of one carbonyl group and an olefinic carbon-carbon double bond and a carbon-carbon triple bond (provided that a monovalent group including a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group is excluded), (ii) a monovalent group having 10 to 50 carbon atoms having at least two carbonyl groups (provided that a monovalent group including a residue of a fat-soluble vitamin having a hydroxyl group and a residue of a sterol derivative having a hydroxyl group is excluded), (iii) formula (2): *-R ４ -X １ -R ５ (2) (In formula (2), * represents a bonding position, R ４ represents an alkylene group having 1 to 10 carbon atoms, X １ represents a carbamate bond, a carbonate bond, or an amide bond, and R ５ represents an alkyl group having 1 to 25 carbon atoms, and R ５ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group. A monovalent group represented by (iv) Formula (3): *-R ６ -CO-O-R ７ (In Formula (3), * represents a bonding position, and R ６ represents an alkylene group having 1 to 10 carbon atoms, and R ７ represents an alkyl group having 1 to 25 carbon atoms substituted with at least one halogen atom.) A monovalent group represented by (v) Formula (4): (In Formula (4), * represents a bonding position, and R ８ and R ９ each independently represent an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R １０ to R １２ each independently represent a hydrogen atom, a benzyl group, or a *-Si(R １３ )(R １４ )(R １５ ) group (in the above formula, * represents a bonding position, and R １３ to R １５ each independently represent an alkyl group having 1 to 4 carbon atoms or a phenyl group.) A monovalent group represented by (vi) Formula (5): (In Formula (5), * represents a bonding position, and X ２ represents a nitrogen atom or Formula (6): (In Formula (6), * represents a bonding position with R １６ , and ** represents a bonding position with R １７ or R １８ .) represents a trivalent group represented by, and when X ２ is a nitrogen atom, R １６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and X ２ When is a trivalent group represented by the formula (6), R １６ represents an alkylene group having 1 to 10 carbon atoms, and R １６ may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and X ２ When is a nitrogen atom, R １７ and R １８ each independently represent an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and R １７ and R １８ each independently may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group, and, X ２ When is a trivalent group represented by the formula (6), R １７ and R １８ each independently represent an alkyl group having 1 to 10 carbon atoms, and R １７ and R １８ each independently may be substituted with a substituent selected from the group consisting of a halogen atom and a hydroxyl group. ), a monovalent group represented by (vii) formula (7): (In formula (7), * represents a bonding position, and R １９ is a hydrogen atom, a benzyl group, *-Si(R １３ )(R １４ )(R １５ ) group (in the above formula, * represents a bonding position, and R １３ to R １５ each independently represent an alkyl group having 1 to 4 carbon atoms or a phenyl group. ), or *-CO-R ２０ group (in the above formula, * represents a bonding position, R ２０ represents an alkyl group having 1 to 9 carbon atoms. ). ), a monovalent group represented by (viii) formula (8): (In formula (8), * represents a bonding position, and R ２１ and R ２２ each independently are a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ )(wherein * represents the bonding position, and R １３ to R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group.).), a monovalent group represented by (ix) formula (9): (In formula (9), * represents the bonding position, and R ２３ is a hydrogen atom, a benzyl group, or *-Si(R １３ )(R １４ )(R １５ )(wherein * represents the bonding position, and R １３ to R １５ each independently represents an alkyl group having 1 to 4 carbon atoms or a phenyl group.).), a monovalent group represented by (x) formula (10): (In formula (10), * represents the bonding position. R ２４ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２５ represents an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, or an alkynyl group having 2 to 30 carbon atoms.).), a monovalent group represented by (xi) formula (11): (In formula (11), * represents the bonding position, and R ２６ represents an alkylene group having 1 to 10 carbon atoms, an alkenediyl group having 2 to 10 carbon atoms, or an alkynediyl group having 2 to 10 carbon atoms, and R ２７ and R ２８ each independently represents an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms.).), or (xii) formula (12): (In formula (12), * represents the bonding position, R ２９ represents an alkylene group having 1 to 10 carbon atoms, and R ３０ and R ３１ Each independently represents an alkyl group having 1 to 10 carbon atoms.) A monovalent group represented by, (xiii) A monovalent group which is an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms, wherein one ethylene group in the alkyl group may be replaced by one ester bond, or, (xiv) R ３ｃ -CO-(CH ２ ) ｐ - group (in the above formula, R ３ｃ represents the residue of a fat-soluble vitamin having a hydroxyl group or the residue of a sterol derivative having a hydroxyl group, and p represents an integer from 1 to 8.) represents, and R ３ａ and R ３ｂ may be the same as or different from each other.) contains an ionic lipid represented by, The molar ratio of the amino group of the ionic lipid represented by formula (1) to the phosphate group of the nucleic acid is 7 or more and 250 or less, and The method includes The following steps a), b), c), and d), The following steps a), b'), and d), or The following steps a), b'), c), and d): a) Mixing an alcohol solution containing an ionic lipid, a sterol, and a PEG lipid represented by formula (1) with a nucleic acid solution containing an acidic buffer having a pH of 1.0 to 6.5 as a solvent to prepare a suspension containing nucleic acid-encapsulated lipid nanoparticles; b) Exchanging the dispersion medium of the suspension obtained in step a) with a buffer having a pH of 4.5 to 8.0, which is different from the acidic buffer used in step a), to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles; b') Exchanging the dispersion medium of the suspension obtained in step a) with a buffer having a pH of 4.5 to 8.0, which is different from the acidic buffer used in step a), and containing a cryoprotectant, to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant; c) Mixing the suspension obtained in step b) or step b') with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulated lipid nanoparticles and a cryoprotectant; d) Freezing the suspension obtained in step b') or step c) at -10°C or lower.

9. The method according to claim 8, comprising step a), step b), step c), and step d), or comprising step a), step b'), and step d), and in step c), mixing the suspension obtained in step b) with a cryoprotectant to obtain a suspension containing nucleic acid-encapsulating lipid nanoparticles and a cryoprotectant.

10. The method according to claim 8, wherein the ratio of (the particle diameter of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d)) / (the particle diameter of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained in step b' or step c)) is 0.8 or more and 1.5 or less.

11. The method according to claim 8, wherein the PdI of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d) is 0.01 or more and 0.25 or less.

12. The method according to claim 8, wherein the ratio of (the nucleic acid encapsulation rate of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained by thawing the frozen composition obtained in step d)) / (the nucleic acid encapsulation rate of the nucleic acid-encapsulating lipid nanoparticles in the suspension obtained in step b' or step c)) is 0.8 or more and 1.0 or less.

13. A method for manufacturing a pharmaceutical composition, comprising the method according to any one of claims 8 to 12.

14. A method for introducing the nucleic acid contained in the suspension into cells in vitro, comprising a step of contacting a suspension obtained by thawing the frozen composition according to any one of claims 1 to 7 or the frozen composition manufactured by the method according to any one of claims 8 to 12, or a suspension obtained by exchanging the dispersion medium of the suspension with another dispersion medium, with the cells.

15. A method for introducing the nucleic acid contained in the suspension into target cells of a living body, the method comprising a step of administering to the living body a suspension obtained by thawing the freezing composition according to any one of claims 1 to 7 or the freezing composition produced by the method according to any one of claims 8 to 12, or a suspension obtained by exchanging the dispersion medium of the suspension with another dispersion medium.

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