Method for producing nucleic acid-encapsulated lipid nanoparticles, method for producing pharmaceutical composition containing said lipid nanoparticles, and method for introducing nucleic acid into cell or target cell

Abstract

The purpose of the present invention is to provide a method for producing nucleic acid-encapsulated lipid nanoparticles, whereby it becomes possible to encapsulate an arbitrary nucleic acid with high efficiency and in a simple manner. Provided is a method for producing nucleic acid-encapsulated lipid nanoparticles, the method comprising the following steps: (a) a step for mixing an alcohol solution containing an ionic lipid, a sterol and a PEG lipid with an acidic buffer solution exhibiting the buffering effect thereof at pH 1 to 6.5, to prepare a suspension of lipid nanoparticles each having no nucleic acid contained therein; and (b) a step for mixing the suspension of the lipid nanoparticles which has been obtained at step a with an aqueous solution containing a nucleic acid and optionally containing 0 to 25 v / v% of an alcohol without freeze-drying the suspension, and then optionally incubating the resultant mixture at 0 to 95°C for 0 to 60 minutes, to produce nucleic acid-encapsulated lipid nanoparticles.

Description

Method for producing nucleic acid-encapsulated lipid nanoparticles, method for producing pharmaceutical compositions containing the same, and method for introducing nucleic acid into cells or target cells

[0001] The present invention relates to a method for producing nucleic acid-encapsulated lipid nanoparticles, which includes a step of preparing lipid nanoparticles that do not contain nucleic acid and then adding nucleic acid, a method for producing a pharmaceutical composition containing the same, and a method for introducing nucleic acid into cells or target cells.

[0002] Effective and safe nucleic acid delivery carriers are required for the practical application of nucleic acid therapy using oligonucleic acids such as siRNA and gene therapy using mRNA, pDNA, etc. 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] Cationic liposomes using cationic lipids containing quaternary amines are positively charged and can form complexes (lipoplexes) with negatively charged nucleic acids through electrostatic interactions, enabling the delivery of nucleic acids into cells. Furthermore, by utilizing the electrostatic interaction between quaternary amines and nucleic acids, a lyophilized composition of nucleic acid-free cationic liposomes can be prepared and rehydrated with an aqueous solution of nucleic acid to form lipoplexes, which has been shown to be useful as a gene transfer reagent (see, for example, Patent Documents 1 and 2).

[0004] However, it is difficult to control the particle size of lipoplexes prepared by such methods, and they have problems such as cytotoxicity due to the positively charged cationic lipids.

[0005] For this reason, lipid nanoparticles (also called lipid nanoparticles, or LNPs) have been developed using ionic lipids that have tertiary amines in their molecules, which are positively charged under acidic conditions but uncharged near neutral conditions. These LNPs are currently the most commonly used non-viral nucleic acid delivery carriers (see, for example, Non-Patent Document 1).

[0006] As for lipid nanoparticles using an ionic lipid having a tertiary amine in the molecule, there is an example in which a degradable group is attached to the ionic lipid (see, for example, Patent Document 3).

[0007] Although various non-viral carriers have been developed, nucleic acids are generally unstable compounds, and therefore stability as formulations remains an issue.

[0008] Japanese Patent No. 4919397 Japanese Patent No. 4598908 Japanese Patent No. 6093710 International Publication No. 2017 / 218704 International Publication No. 2021 / 060440

[0009] Gene Therapy 6:271-281, 1999Biol. Pharm. Bull. 41, 1291-1294 (2018)

[0010] Incidentally, one method for improving the stability of a formulation has been to freeze-dry lipid nanoparticles encapsulating nucleic acids and then rehydrate them at the time of use to reconstitute the lipid nanoparticles (Patent Document 4 and Non-Patent Document 2).

[0011] These methods are useful for increasing the storage stability of lipid nanoparticles encapsulating specific nucleic acids, but they pose problems when used to more simply encapsulate any nucleic acid into lipid nanoparticles.

[0012] A simple method for encapsulating any nucleic acid in lipid nanoparticles includes the method described in Patent Documents 1 and 2, in which a freeze-dried composition not containing nucleic acid is prepared and then rehydrated with an aqueous solution of nucleic acid.

[0013] However, lipid nanoparticles obtained using ionic lipids having tertiary amines in the molecule have a surface charge ranging from weakly negative to neutral after preparation, and therefore do not interact electrostatically with nucleic acids. Therefore, nucleic acid-encapsulated lipid nanoparticles cannot be prepared by the method disclosed in Patent Documents 1 and 2, in which a freeze-dried composition not containing nucleic acids is prepared and then rehydrated with an aqueous solution of nucleic acid.

[0014] In this regard, Patent Document 5 shows that any nucleic acid can be encapsulated in lipid nanoparticles with high efficiency and ease by preparing lipid nanoparticles that do not contain nucleic acids in an acidic buffer, adding a cryoprotectant, lyophilizing the nanoparticles, and then rehydrating the nanoparticles with an aqueous solution containing nucleic acids. However, since the above technique requires a lyophilization step, there is room for improvement in terms of simplicity.

[0015] In view of the above problems, the present invention aims to provide a method for producing nucleic acid-encapsulated lipid nanoparticles that can encapsulate any nucleic acid with high efficiency and ease, which was not possible with conventional technology, a method for producing a pharmaceutical composition containing the same, and a method for introducing nucleic acid into cells or target cells.

[0016] In view of the above problems, the present inventors have made extensive efforts and found that any nucleic acid can be encapsulated in lipid nanoparticles with high efficiency and ease by preparing lipid nanoparticles that do not contain nucleic acids in an acidic buffer and adding an aqueous solution containing nucleic acids. Furthermore, when a gene transfer experiment into cells was carried out using lipid nanoparticles prepared by this method, it was unexpectedly found that the gene transfer efficiency was improved compared to conventional techniques, and thus the present invention was completed.

[0017] That is, the present invention encompasses the following: [1] A method for producing nucleic acid-encapsulated lipid nanoparticles, comprising the following steps: a) mixing an alcohol solution containing an ionic lipid, a sterol, and a PEG lipid with an acidic buffer solution having a buffering effect at pH 1 to 6.5 to prepare a suspension of lipid nanoparticles that does not contain nucleic acid, and b) mixing the lipid nanoparticle suspension obtained in step a, without lyophilization, with an aqueous solution containing nucleic acid and optionally containing 0 to 25 v / v % alcohol, and optionally incubating the mixture at 0 to 95°C for 0 to 60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles.

[0018] [2] A method for producing nucleic acid-encapsulated lipid nanoparticles according to [1], comprising the following step c after step b: c) a step of exchanging the external aqueous phase of the obtained nucleic acid-encapsulated lipid nanoparticles with a neutral buffer solution by dialysis, ultrafiltration or dilution.

[0019] [3] The method for producing nucleic acid-encapsulated lipid nanoparticles according to [1] or [2], further comprising the step of freezing the nucleic acid-free lipid nanoparticles at -80 to 0°C and then thawing them at 0 to 95°C in step a.

[0020] [4] The method for producing nucleic acid-encapsulated lipid nanoparticles according to any one of [1] to [3], further comprising a step of exchanging the external aqueous phase with another acidic buffer solution having a buffering effect at pH 1 to 6.5 by dialysis, ultrafiltration, or dilution after preparing the lipid nanoparticle suspension in step a.

[0021] [5] The method for producing nucleic acid-encapsulated lipid nanoparticles according to any one of [1] to [4], wherein in step a, the alcohol solution further contains a phospholipid.

[0022] [6] A method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of [1] to [5], wherein the ionic lipid is a compound represented by formula (1):

[0023]

[0024] (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 or an oxydialkylene group having 8 or less 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; ａ and n ｂ are each independently 0 or 1; ３ａ and R ３ｂare each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (3): R ９ —O—CO—(CH ２ )a- (3) (In formula (3), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.

[0025] [7] The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of [1] to [5], wherein the ionic lipid is a compound represented by formula (2):

[0026]

[0027] (wherein, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen atoms; R １ represents an aliphatic hydrocarbon group having 8 or less carbon atoms; １ represents an ester bond, an amide bond, a carbamate bond, an N-alkylcarbamate bond, a carbonate bond or a urea bond; k represents 0 or 1; R ｘ and R ｙ each independently represents an alkylene group having 2 to 5 carbon atoms; ２ represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond; R ２ represents an alkylene group having 8 or less carbon atoms or is absent, and Y (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) has a group containing at least one bond selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) represents a group containing at least one bond selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue, and a residue of a sterol derivative.

[0028] [8] A method for introducing a nucleic acid into a cell, the method comprising the step of contacting the nucleic acid-encapsulated lipid nanoparticles produced by the method according to any one of [1] to [7] with the cell in vitro.

[0029] [9] A method for introducing nucleic acid into target cells, comprising the step of administering to a living body the nucleic acid-encapsulated lipid nanoparticles produced by the method according to any one of [1] to [7].

[0030]

[10] A method for producing a pharmaceutical composition, comprising the method according to any one of [1] to [7].

[0031] In the method for producing nucleic acid-encapsulated lipid nanoparticles of the present invention, lipid nanoparticles not containing nucleic acid are prepared, and then an aqueous nucleic acid solution is added without lyophilization, so lipid nanoparticles encapsulating any nucleic acid can be produced efficiently and simply. Furthermore, the nucleic acid-encapsulated lipid nanoparticles prepared using the production method of the present invention have a higher nucleic acid transfer efficiency than conventional techniques, are advantageous for gene transfer in cells and living organisms, and are particularly useful as pharmaceutical compositions.

[0032] 8 is a diagram evaluating the effect of a buffer solution on the encapsulation rate. 9 is a diagram evaluating the effect of a buffer solution on particle diameter. 10 is a diagram evaluating the effect of sucrose concentration and incubation temperature on the encapsulation rate. Incubation temperatures are shown in ° C. 11 is a diagram evaluating the effect of sucrose concentration and incubation temperature on particle diameter. Incubation temperatures are shown in ° C. 12 is a diagram evaluating the effect of sucrose concentration and incubation temperature on in vitro activity. Incubation temperatures are shown in ° C. 13 is a diagram evaluating the effect of incubation time on the encapsulation rate. 14 is a diagram evaluating the effect of incubation time on particle diameter. 15 is a diagram evaluating the effect of incubation time on in vitro activity. 16 is a diagram showing the area under the curve (AUC) of FIG. 10. 17 is a diagram evaluating the effect of buffer solution pH on the encapsulation rate. 18 is a diagram evaluating the effect of buffer solution pH on particle diameter. 19 is a diagram evaluating the effect of buffer solution salt concentration on the encapsulation rate. 20 is a diagram evaluating the effect of buffer solution salt concentration on particle diameter. 16 is a diagram comparing the encapsulation rate of nucleic acid-encapsulated nanoparticles of the present invention with that of a freeze-dried product. 17 is a diagram comparing the particle diameters of nucleic acid-encapsulated nanoparticles of the present invention with that of a freeze-dried product. 18 is a diagram comparing the in vitro activity of nucleic acid-encapsulated nanoparticles of the present invention with that of a freeze-dried product. 19 is a diagram showing the area under the curve (AUC) of FIG. 16. 20 is a diagram comparing the in vivo activity of nucleic acid-encapsulated nanoparticles of the present invention with that of a freeze-dried product. 21 is a diagram evaluating the effect of mixing ratio and mixing mode on the encapsulation rate. 22 is a diagram evaluating the effect of mixing ratio and mixing mode on particle size. 23 is a diagram evaluating the effect of mixing ratio and mixing mode on in vitro activity. 24 is a diagram evaluating the effect of buffer solution on the encapsulation rate. 25 is a diagram evaluating the effect of buffer solution on particle size. 26 is a diagram evaluating the effect of incubation temperature on the encapsulation rate. 27 is a diagram evaluating the effect of incubation temperature on particle size. 28 is a diagram evaluating the effect of incubation temperature on in vitro activity. 29 is a diagram evaluating the effect of incubation time on the encapsulation rate. 29 is a diagram evaluating the effect of incubation time on particle size. 1 is a graph evaluating the effect of incubation time on in vitro activity, FIG. 2 is a graph evaluating the effect of buffer pH on encapsulation rate, and FIG. 3 is a graph evaluating the effect of buffer pH on particle diameter.1 is a diagram evaluating the effect of buffer pH on in vitro activity. FIG. 2 is a diagram showing the relationship between the pH at the time of mixing with mRNA and the Zeta potential at each pH. FIG. 3 is a diagram evaluating the effect of buffer salt concentration on encapsulation rate. FIG. 4 is a diagram evaluating the effect of buffer salt concentration on particle size. FIG. 5 is a diagram comparing the encapsulation rate of nucleic acid-encapsulated nanoparticles of the present invention and a freeze-dried product. FIG. 6 is a diagram comparing the particle size of nucleic acid-encapsulated nanoparticles of the present invention and a freeze-dried product. FIG. 7 is a diagram comparing the in vivo activity of nucleic acid-encapsulated nanoparticles of the present invention and a freeze-dried product. FIG. 8 is a diagram comparing the encapsulation rate of nucleic acid-encapsulated nanoparticles of the present invention and nucleic acid-encapsulated nanoparticles (MF) prepared using a microchannel. FIG. 9 is a diagram showing the expression distribution in nucleic acid-encapsulated nanoparticles (MF) prepared using a microchannel. FIG. 10 is a diagram showing the expression distribution in nucleic acid-encapsulated nanoparticles of the present invention. FIG. 11 is a diagram comparing the in vitro activity of nucleic acid-encapsulated nanoparticles of the present invention and a freeze-dried product.

[0033] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these.

[0034] The present invention relates to a method for producing nucleic acid-encapsulated lipid nanoparticles by mixing a lipid solution containing no nucleic acid but containing an ionic lipid, a sterol, and a PEG lipid with an acidic buffer solution having a buffering effect at pH 1 to 6.5 to prepare lipid nanoparticles that do not contain nucleic acid, and then adding an aqueous solution of nucleic acid.

[0035] Lipid nanoparticles refer to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are aligned toward the aqueous phase at the interface. "Amphipathic lipid" refers to a lipid having both hydrophilic and hydrophobic groups. Examples of amphipathic lipids include ionic lipids, phospholipids, and PEG lipids. The lipid nanoparticles used in the present invention contain ionic lipids, sterols, and PEG lipids as membrane constituents, and may further contain phospholipids. The particle size of the lipid nanoparticles is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Measurement of particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be adjusted appropriately depending on the manufacturing method. In the present invention, particle size refers to the average particle size (number average) measured by dynamic light scattering. In the present invention, "total lipid" refers to the total amount of lipids. Lipids include ionic lipids, sterols, PEG lipids, and phospholipids.In the present invention, "does not contain nucleic acid" or "does not contain nucleic acid" means that it does not substantially contain nucleic acid, and the content of nucleic acid is below the detection limit.In the present invention, "nucleic acid-encapsulated lipid nanoparticles" refers to lipid nanoparticles in which nucleic acid is encapsulated inside the lipid nanoparticles.

[0036] Ionic Lipids Ionic lipids that can be used in the present invention are those that are composed of a tertiary amino group and a hydrophobic group and can form lipid nanoparticles. Specific examples of ionic lipids include 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), and 2,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA). oleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), heptadecan-9-yl Examples of the compound include 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (Lipid 8), and a compound of the following formula (1) or formula (2). Preferred are the compound of the following formula (1) or formula (2), DODMA, MC3, Lipid 5, and Lipid 8, and most preferred is the compound of the following formula (1) or formula (2). Formula (1)

[0037]

[0038] (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 ２ｂeach independently represents an alkylene group or an oxydialkylene group having 8 or less 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; ａ and n ｂ are each independently 0 or 1; ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (3): R ９ —O—CO—(CH ２ )a- (3) (In formula (3), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10. A compound represented by the formula:

[0039] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 4, and more preferably 1 to 2. Specific examples of alkylene groups having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. R １ａ and R １ｂ are preferably each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each are an ethylene group.

[0040] R１ａ is R １ｂ may be the same as or different from, but preferably, R １ａ is R １ｂ is the same group as

[0041] 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, and preferably each independently represents a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups.

[0042] The alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0043] A preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is: X １ It is shown as follows.

[0044]

[0045] X １ R ５represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0046] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups is preferably 4 or 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups include an aziridylene group, an azetidylene group, a pyrrolidine group, a piperidylene group, an imidazolidylene group, and a piperaziylene group, preferably a pyrrolidine group, a piperidylene group, and a piperaziylene group, and most preferably a piperidylene group.

[0047] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is X ２ It is shown as follows.

[0048]

[0049] X ２ In the formula, p is 1 or 2. When p is 1, X ２ is a pyrrolidine group, and when p is 2, X ２ is a piperidylene group. Preferably, p is 2.

[0050] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X ３ It is shown as follows.

[0051]

[0052] X３ The w in is 1 or 2. When w is 1, X ３ is an imidazolidylene group, and when w is 2, X ３ is a piperazylene group.

[0053] X ａ is X ｂ may be the same as or different from, but preferably, X ａ is X ｂ is the same group as

[0054] R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, and preferably each independently represents an alkylene group having 8 or less carbon atoms.

[0055] The alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear. The number of carbon atoms contained in the alkylene group is preferably 6 or less, and most preferably 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group. Preferred are a methylene group, an ethylene group, a trimethylene group, and a tetramethylene group, and most preferably an ethylene group.

[0056] In this specification, an "oxydialkylene group having 8 or less carbon atoms" refers to an alkylene group (alkylene-O-alkylene, in other words, an "alkyleneoxyalkylene group") bonded via an ether bond, in which the total number of carbon atoms in the two alkylene groups is 8 or less. Here, the two alkylene groups may be the same or different, but are preferably the same. Specific examples of oxydialkylene groups having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydi(trimethylene) group (i.e., a trimethyleneoxytrimethylene group), and an oxydi(tetramethylene) group (i.e., a tetramethyleneoxytetramethylene group). Preferred are an oxydimethylene group, an oxydiethylene group, and an oxydi(trimethylene) group, and most preferred is an oxydiethylene group.

[0057] R ２ａ is R ２ｂ may be the same as or different from, but preferably, R ２ａ is R ２ｂ is the same group as

[0058] Y ａ and Y ｂ are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or a carbamate bond, more preferably each independently an ester bond or an amide bond, and most preferably each an ester bond. ａ and Y ｂ The bond direction of Y is not restricted. ａ and Y ｂ is an ester bond, preferably -Z ａ -CO-O-R ２ａ - and -Z ｂ -CO-O-R ２ｂ - structure.

[0059] Y ａ Is Y ｂ may be the same as or different from, but preferably, Y ａ Is Y ｂ is the same group as

[0060] 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. The aromatic compound preferably contains 6 to 12 carbon atoms, and most preferably 6 to 7 carbon atoms. The aromatic compound preferably contains one aromatic ring.

[0061] Examples of the aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms include, for example, a benzene ring, a naphthalene ring, and an anthracene ring as aromatic hydrocarbon rings, and 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 as aromatic hetero rings. Of these, a benzene ring, a naphthalene ring, and an anthracene ring are preferred, and a benzene ring is most preferred.

[0062] The aromatic ring may have 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, 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 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 having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl 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 having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, a methyl group having 1 to 4 ... Examples of the alkyl group include an aryl group having 0 carbon atoms and an aryloxy group having 6 to 10 carbon atoms, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, 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.

[0063] Z ａ and Z ｂ Preferred specific structures of Z １ Examples include:

[0064]

[0065] In the formula, s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R ４ each independently represents a substituent.

[0066] Z １ In the formula, s is preferably an integer of 0 to 1, and more preferably 0. １ t is preferably an integer of 0 to 2, more preferably 1. １ The u in the formula (1) is preferably an integer of 0 to 2, and more preferably an integer of 0 to 1.

[0067] Z １ R ４is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the synthesis process of the ionic lipid. Examples of the substituent include 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 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 having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, Examples of such an aryloxy group include those having 6 to 10 carbon atoms, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, 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. ４ When there are multiple R ４ may be the same or different.

[0068] Z ａ is Z ｂ may be the same as or different from, but preferably, Z ａ is Z ｂ is the same group as

[0069] n ａ and n ｂ are each independently 0 or 1.

[0070] n ａ is n ｂ may be the same as or different from, but preferably, n ａ is n ｂ is the same as

[0071] R ３ａand R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having a cyclopropane ring and having 3 to 40 carbon atoms, or a group represented by the formula (3): R ９ —O—CO—(CH ２ )a- (3) (In formula (3), R ９ represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, and a represents an integer of 2 to 10.) Preferably, each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and most preferably, each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0072] The residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride is a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group with a hydroxyl group of *-O-CO-CH ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure in which the hydroxyl group of a sterol derivative is replaced by *-O-CO-CH. * represents the bonding position with a fat-soluble vitamin. The residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride is a residue derived from the reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride. ２ -CH ２ - or *-O-CO-CH ２ -CH ２ -CH ２ represents a group having a structure where a - is replaced with *. The symbol * represents the bonding position with the sterol derivative.

[0073] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0074] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and preferably cholesterol or cholestanol.

[0075] The aliphatic hydrocarbon group having 1 to 40 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, with carbon-carbon double bonds being preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., with alkyl or alkenyl groups being preferred. Specific examples of the aliphatic hydrocarbon group having 1 to 40 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl 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, 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 tetracontyl group, a dodecenyl group, a tridecenyl group, a tetracont ... Adecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosenyl group, docosenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosenyl group Examples of such groups include a 1-hexyl group, a 1-hexylnonyl group, a 1-octylundecyl group, a 1-decylundecyl group, and a 1-decylundecyl group.The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0076] In one embodiment of the present invention, R ３ａ and R ３ｂ The aliphatic hydrocarbon group having 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) represented by the formula (1) is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). Specific examples of the aliphatic hydrocarbon group include a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.

[0077] R ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the formula (I) means an alkyl group having 3 to 40 carbon atoms and having at least one cyclopropane ring in the alkyl chain. The number of carbon atoms in the alkyl group, 3 to 40, does not include the number of carbon atoms in the cyclopropane ring. The number of cyclopropane rings in the alkyl group is preferably one. ３ａ and R ３ｂ The alkyl group having 3 to 40 carbon atoms and a cyclopropane ring in the formula (4):

[0078]

[0079] (In formula (4), b and c are each independently an integer, and the sum of b and c is 2 to 39.) Preferably, b is an integer of 1 to 20, and c is an integer of 1 to 19. More preferably, b is an integer of 2 to 18, even more preferably an integer of 3 to 17, and even more preferably an integer of 4 to 12. More preferably, c is an integer of 3 to 15, even more preferably an integer of 3 to 11, and even more preferably an integer of 3 to 9. Examples of the group represented by formula (4) include 7-(2-octylcyclopropyl)heptyl.

[0080] In formula (3), R９The aliphatic hydrocarbon group having 2 to 20 carbon atoms represented by the formula (I) may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, with carbon-carbon double bonds being preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, even more preferably 13 to 19, and most preferably 13 to 17. Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, etc., with alkyl groups or alkenyl groups being preferred, and alkyl groups being more preferred. Specific examples of the aliphatic hydrocarbon group having 2 to 20 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl 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, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, and a heptadecenyl group. Examples of the alkyl group include an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosenyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group, a tetradecadienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-ethylnonyl group, a 1-butylnonyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, and a 3-octylundecyl group.The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0081] In formula (3), a is preferably an integer of 3 to 9, more preferably an integer of 3 to 7, even more preferably an integer of 5 to 7, and most preferably 5 or 7.

[0082] 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

[0083] 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 R ２ａ 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

[0084] Suitable examples of the ionic lipid represented by formula (1) (sometimes abbreviated as "ionic lipid (1)" in this specification) include the following ionic lipids: [Ionic lipid (1-1)] 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 an acyclic 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., a piperidylene group); R ２ａ and R ２ｂare each independently an alkylene group having 8 or less 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; 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 hetero atom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); n ａ and n ｂ are each independently 0 or 1; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0085] [Ionic lipid (1-2)] 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 an acyclic 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., a piperidylene group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less 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; 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 -); n ａ and n ｂ are each independently 0 or 1; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0086] [Ionic lipid (1-3)] 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, X １ :

[0087]

[0088] (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ :

[0089]

[0090] (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less 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; ａ and Z ｂ are each independently, Z １ :

[0091]

[0092] (wherein s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R ４ each independently represents a substituent; ａ and n ｂ are each independently 0 or 1; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group); an ionic lipid (1).

[0093] Specific examples of ionic lipid (1) include the following: O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, B-2, B-2-5, TS-P4C2, L-P4C2, and O-P4C2.

[0094] Specific examples of the ionic lipid (1) include Lipid 1 to Lipid 20 described in WO2021 / 195529A2.

[0095]

[0096]

[0097]

[0098] The ionic lipid (1) can be produced by known methods (for example, the methods described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, and WO2021 / 195529A2).

[0099] Formula (2)

[0100]

[0101] (wherein, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen atoms; R １ represents an aliphatic hydrocarbon group having 8 or less carbon atoms; １ represents an ester bond, an amide bond, a carbamate bond, an N-alkylcarbamate bond, a carbonate bond or a urea bond; k represents 0 or 1; R ｘ and R ｙ each independently represents an alkylene group having 2 to 5 carbon atoms; ２ represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond; R ２ represents an alkylene group having 8 or less carbon atoms or is absent, and Y (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) has a group containing at least one bond selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) represents a group containing at least one bond selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue, and a residue of a sterol derivative.

[0102] A preferred example of X can be represented by the following general formula (5): α -N(R β )-(5) (wherein, R α is an aliphatic hydrocarbon group, R β is an aliphatic hydrocarbon group, an aliphatic group containing one or more heteroatoms other than nitrogen, or an aliphatic group containing one or more tertiary amines, or R α and R β may be bonded to form a 3- to 8-membered nitrogen-containing alicyclic ring)

[0103] More preferred examples of X include a dialkylamino group (the two alkyl groups of the dialkylamino group each independently have 1 to 8 carbon atoms), a 3- to 6-membered cyclic amino group which may have a heteroatom, or -N(R a )-R ｂ and R ａ is an alkyl group having 1 to 8 carbon atoms, and R ｂis -(CH ２ ) q-O-R ｃ and R ｃ is hydrogen or an alkyl group having 1 to 8 carbon atoms; and q is an integer of 2 to 4.

[0104] The number of carbon atoms in the two alkyl groups in the dialkylamino group is preferably each independently 1 to 5, more preferably each independently 1 to 4. The alkyl group may be linear, branched, or cyclic. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, and a cyclopentyl group. Preferably, they are each independently a methyl group, an ethyl group, a propyl group, or an isopropyl group, and more preferably they are each independently a methyl group or an ethyl group.

[0105] The 3- to 6-membered cyclic amino group which may have a heteroatom means a group in which substituents of an amino group are bonded to form a ring, the number of atoms forming the ring is 3 to 6, and the group may contain a heteroatom such as oxygen. The 3- to 6-membered cyclic amino group which may have a heteroatom is preferably a 5- or 6-membered cyclic amino group which may have a heteroatom, more preferably a 6-membered cyclic amino group which may have a heteroatom. The cyclic amino group is one in which the ring contains a nitrogen atom and a methylene group (-CH ２ An amino group formed only from 1-(-) is preferred, and may contain an oxygen atom. Specifically, it is a 1-pyrrolidinyl group, a 1-piperidyl group, or a morpholino group (4-morpholinyl group), and preferably a 1-piperidyl group or a morpholino group.

[0106] R １ The aliphatic hydrocarbon group having 8 or less carbon atoms in the formula (I) is preferably an alkylene group, an alkenylene group, or an alkynylene group, and more preferably an alkylene group or an alkenylene group.

[0107] The alkylene group having 8 or less carbon atoms may be either linear or branched. The number of carbon atoms in the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), isobutylene group (-C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -), pentamethylene group, or hexamethylene group, and more preferably methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), a tetramethylene group, or an isobutylene group (—C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -).

[0108] The alkenylene group having 8 or less carbon atoms may be either linear or branched. The number of carbon atoms in the alkenylene group is preferably 6 or less, more preferably 4 or less. The alkenylene group having 8 or less carbon atoms is preferably a propenylene group (-CH ２ CH=CH-, -CH=CHCH ２ -), butenylene group, isopropenylene group, isobutenylene group, pentenylene group, or hexenylene group, and more preferably a propenylene group (-CH ２ CH=CH-, -CH=CHCH ２ -), butenylene group, isopropenylene group, or isobutenylene group.

[0109] L １is an ester bond, an amide bond, a carbamate bond, an N-alkylcarbamate bond, a carbonate bond or a urea bond, and is preferably an ester bond, an amide bond, a carbamate bond, an N-methylcarbamate bond or a carbonate bond.

[0110] As used herein, an N-alkylcarbamate bond is defined as —NR １０ —CO—O— or —O—CO—NR １０ represents -, and R １０ represents an alkyl group having 1 to 8 carbon atoms. The alkyl group having 1 to 8 carbon atoms may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, and a cyclopentyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0111] k represents 0 or 1. Here, when k is 0, R １ -L １ does not exist, that is, X and R ｘ The same applies when l, m, n, etc. are 0.

[0112] R ｘ and R ｙ The alkylene group having 2 to 5 carbon atoms may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 2 to 4, more preferably 2. Specifically, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), or an isobutylene group (—C(CH ３ ) ２ CH２ -, -CH ２ C(CH ３ ) ２ -), preferably an ethylene group or a trimethylene group, more preferably an ethylene group.

[0113] L ２ is an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond, preferably an ester bond or an amide bond, more preferably an ester bond.

[0114] R ２ represents an alkylene group having 8 or less carbon atoms, or is absent. ２ does not exist, L ２ and Y are directly bonded. ２ The alkylene group having 8 or less carbon atoms in the above formula may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), isobutylene group (-C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -), pentamethylene group, or hexamethylene group, and more preferably methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), a tetramethylene group, or an isobutylene group (—C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -).

[0115] R ａ and R ｃ The alkyl group having 1 to 8 carbon atoms in the formula (I) may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specific examples of the alkyl group having 1 to 8 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, and a 2-methylbutyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0116] A preferred example of Y can be represented by the following general formula (6).

[0117]

[0118] (In the formula, R ３ represents an alkylene group having 8 or less carbon atoms; ３ represents an ester bond or a carbonate bond; １ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R ｅ -L ａ -R ７ ', or -R ｅ -L ａ -Z'-L ｂ -R ７ ', S ２ is -R ｅ '-L ａ -R ７ '' or -R ｅ '-L ａ -Z''-L ｂ -R ７ '', and R ｅ and R ｅ L ′ each independently represents an alkylene group having 8 or less carbon atoms; ａ represents an ester bond or a carbonate bond; ｂrepresents an ester bond or a carbonate bond; l represents 0 or 1; Z, Z', and Z'' each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; L ｘ represents an ester bond or a carbonate bond; R ４ represents an alkylene group having 8 or less carbon atoms or is absent, R ５ represents hydrogen or an alkyl group having 1 to 8 carbon atoms; ３ is -R ６ '-L ４ '-R ７ "'" represents, m represents 0 or 1, R ６ and R ６ L ′ each independently represents an alkylene group having 8 or less carbon atoms or is absent; ４ and L ４ R ′ each independently represents an ester bond or a carbonate bond; ７ , R ７ ', R ７ '' and R ７ "'" each independently represent an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ )p-C(=O)-R ｆ represents R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group; p represents 2 or 3; and n represents 0 or 1.

[0119] R ４ represents an alkylene group having 8 or less carbon atoms, or is absent. ４ does not exist, R ５ and S ３ and the carbon atom to which L is bonded. ｘ means that the bond is direct.

[0120] R ６ and R ６ R' each independently represents an alkylene group having 8 or less carbon atoms, or is absent. ６ does not exist when m = 0, Lｘ and L ４ means that m is directly bonded, and when m=1, R ５ and S ３ and the carbon atom to which L is bonded. ４ means that the bond is directly bonded. ６ ' does not exist, R ４ and R ５ and the carbon atom to which L is bonded. ４ ' means direct binding.

[0121] R ３ , R ４ , R ６ , R ６ ', R ｅ and R ｅ The alkylene group having 8 or less carbon atoms in ' may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), isobutylene group (-C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -), pentamethylene group, or hexamethylene group, and more preferably methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), a tetramethylene group, or an isobutylene group (—C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -).

[0122] R ５ and S １The alkyl group having 1 to 8 carbon atoms in the formula (I) may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specific examples of the alkyl group having 1 to 8 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, and a 2-methylbutyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0123] L ｘ , L ３ , L ４ , L ４ ', L ａ and L ｂ is an ester bond or a carbonate bond, preferably an ester bond.

[0124] Z, Z', and Z'' each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. Here, the divalent group refers to a divalent group having a structure obtained by removing two hydrogen atoms from the above aromatic compound. The above aromatic compound preferably has 6 to 12 carbon atoms, more preferably 6 to 7. The above aromatic compound preferably has 1 aromatic ring. Z, Z', and Z'' may be the same or different, but preferably Z, Z', and Z'' are the same.

[0125] The aromatic ring of the aromatic compound 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. The aromatic ring of the aromatic compound is preferably a benzene ring, a naphthalene ring, or an anthracene ring, and more preferably a benzene ring.

[0126] The aromatic ring of the aromatic compound may have a substituent. Examples of the substituent include 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 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, 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 having 1 to 4 carbon atoms, 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. Preferred examples of the substituent include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, 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.

[0127] Z, Z′ and Z″ are preferably each independently a group represented by formula (7):

[0128]

[0129] (Wherein, t represents an integer of 0 to 3, u represents an integer of 0 to 3, v represents an integer of 0 to 4, and v R ８ each independently represents a substituent, * represents L ３ or L ａ represents the bonding position with. t is preferably 0 or 1, more preferably 1. u is preferably an integer of 0 to 2, more preferably 0. v is preferably an integer of 0 to 2, more preferably 0 or 1, and even more preferably 0.

[0130] R ８ Examples of R include 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 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, 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 having 1 to 4 carbon atoms, 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. ８Preferred examples of the aryloxy group include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, a propanoyloxy group, a butanoyloxy group, a pentanoyloxy group, a hexanoyloxy group, a heptanoyloxy group, an octanoyloxy group, a nonanoyloxy group, a decanoyloxy group, an undecanoyloxy group, a dodecanoyloxy group, a tridecanoyloxy group, a tetradecanoyloxy group, a pentadecanoyloxy group, a hexadecanoyloxy group, a heptadecanoyloxy group, and an octadecanoyloxy group. Examples of R include an aryloxy group, an octadecenoyloxy group, an octadecadienoyloxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, 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. ８ If there are multiple R ８ may be the same or different from each other.

[0131] R ７ , R ７ ', R ７ '' and R ７ "'" each independently represents an aliphatic hydrocarbon group having 10 to 37 carbon atoms or -(CH ２ )p-C(=O)-R ｆ represents R ｆ 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 2 or 3. ７ , R ７ ', R ７ '' and R ７ The "''' may be the same or different.

[0132] In this specification, 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 a fat-soluble vitamin, and 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 a sterol derivative.

[0133] The aliphatic hydrocarbon group having 10 to 37 carbon atoms may be either linear or branched. The aliphatic hydrocarbon group preferably has 12 to 37 carbon atoms, more preferably 13 to 37 carbon atoms, and even more preferably 15 to 37 carbon atoms.

[0134] The aliphatic hydrocarbon group may be saturated or unsaturated. When the aliphatic hydrocarbon group is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds is preferably 1 to 6, more preferably 1 to 3, and even more preferably 1 to 2. The unsaturated bond may be either a carbon-carbon double bond or a carbon-carbon triple bond, but is preferably a carbon-carbon double bond. The aliphatic hydrocarbon group is preferably an alkyl group or an alkenyl group.

[0135] Examples of the aliphatic hydrocarbon group having 10 to 37 carbon atoms include 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, a henicosyl group, a docosyl group, a decenyl group, an undecenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosyl group, a heneicosenyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group, a tetradecadienyl group, a pentadecadien ... Examples of such alkyl groups include a xadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, a henicosadienyl group, a heneicosadienyl group, a docosadienyl group, an octadecatrienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, a docosahexaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, a 1-decylundecyl group, a 1-dodecyltridecyl group, a 1-tetradecylpentadecyl group, a 1-hexadecylheptadecyl group, and a 1-octadecylnonadecyl group.

[0136] The aliphatic hydrocarbon group having 10 to 37 carbon atoms is preferably 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, a heneicosenyl group, a docosyl group, a decenyl group, an undecenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an icosenyl group, a heneicosenyl group, a heneicosenyl group, a docosenyl group, a dodecadecyl group, or a dodecadecyl group. enyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, heneicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group , 1-decylundecyl group, 1-dodecyltridecyl group, 1-tetradecylpentadecyl group, 1-hexadecylheptadecyl group, and 1-octadecylnonadecyl group, and particularly preferred are pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group, docosyl group, heptadecenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, and nonadecadienyl group. nyl group, icosadienyl group, henicosadienyl group, heneicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group, 1-decylundecyl group, 1-dodecyltridecyl group, 1-tetradecylpentadecyl group, 1-hexadecylheptadecyl group, and 1-octadecylnonadecyl group.

[0137] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0138] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, etc. The sterol derivative having a hydroxyl group is preferably cholesterol or cholestanol. ｆ is preferably a residue of a fat-soluble vitamin having a hydroxyl group. p is preferably 2.

[0139] l, m, and n represent 0 or 1. Preferred combinations of l, m, and n are l=0, m=0, n=0, l=0, m=0, n=1, l=0, m=1, n=1, and l=1, m=0, n=0, and particularly preferred are l=0, m=0, n=0, l=0, m=1, n=1, and l=1, m=0, n=0.

[0140] Suitable examples of the ionic lipid represented by formula (2) (sometimes abbreviated as "ionic lipid (2)" in this specification) include the following ionic lipids: X is a dialkylamino group (the number of carbon atoms in the two alkyl groups of the dialkylamino group is each independently 1 to 8), a 3- to 6-membered cyclic amino group which may have a heteroatom, or -N(R ａ )-R ｂ represents R ａ represents an alkyl group having 1 to 8 carbon atoms, and R ｂ is -(CH ２ ) q-O-R ｃ represents R ｃ represents hydrogen or an alkyl group having 1 to 8 carbon atoms, q is an integer of 2 to 4; R １ is an alkylene group having 6 or less carbon atoms or an alkenylene group having 6 or less carbon atoms; L １is an ester bond, an amide bond, a carbamate bond or a carbonate bond, k is 0 or 1; R ｘ and R ｙ are each independently an alkylene group having 2 to 4 carbon atoms; ２ is an ester bond or an amide bond; R ２ is an alkylene group having 6 or less carbon atoms or is absent; Y is a group represented by the formula (Y):

[0141]

[0142] (In the formula, R ３ represents an alkylene group having 6 or less carbon atoms; ３ represents an ester bond, S １ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R ｅ -L ａ -R ７ ', or -R ｅ -L ａ -Z'-L ｂ -R ７ ', S ２ is -R ｅ '-L ａ -R ７ '' or -R ｅ '-L ａ -Z''-L ｂ -R ７ '', and R ｅ and R ｅ L ′ each independently represents an alkylene group having 6 or less carbon atoms; ａ represents an ester bond; ｂ represents an ester bond; l represents 0 or 1; L ｘ represents an ester bond or a carbonate bond; R ４ represents an alkylene group having 6 or less carbon atoms or is absent, R ５ represents hydrogen or an alkyl group having 1 to 8 carbon atoms; ３ is -R ６ '-L ４ '-R ７ "'" represents, m represents 0 or 1, R ６ and R６ L ′ each independently represents an alkylene group having 6 or less carbon atoms, or is absent; ４ and L ４ Both R and R′ represent an ester bond. ７ , R ７ ', R ７ '' and R ７ "'" each independently represent an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ )p-C(=O)-R ｆ represents R ｆ 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 2 or 3, and n represents 0 or 1; Z, Z', and Z'' are each independently a group represented by the formula (Z):

[0143]

[0144] (Wherein, t represents an integer of 0 or 1, u represents an integer of 0 to 2, v represents an integer of 0 to 2, and v R ８ each independently represent 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 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, 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 having 1 to 4 carbon atoms, 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.) is a group represented by the formula (I); an ionic lipid (2).

[0145] Preferred specific examples of the ionic lipid (2) include the compounds described in Production Examples 1 to 46 below, but the present invention is not intended to be limited thereto. The compounds described in Production Examples 1 to 46 are referred to as Compounds 1 to 46, respectively.

[0146] The ionic lipid (2) is preferably at least one selected from the group consisting of these, more preferably at least one selected from the group consisting of compounds 5, 7, 8, 9, 10, 11, 15, 19, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 34, 35, 36, and 42, even more preferably at least one selected from the group consisting of compounds 5, 7, 8, 9, 10, 11, 15, 21, 22, 24, 31, 32, 34, 35, and 36, and most preferably compound 31 or compound 34.

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] Next, a method for producing ionic lipid (2) will be described. Ionic lipid (2) has an -S-S- (disulfide) bond. Therefore, the method for producing ionic lipid (2) is as follows: (A) X-(R １ -L １ ) ｋ -R Ｘ Thiol represented by —SH and HS—R ｙ -L ２ -R ２ (B) a method in which necessary portions are sequentially synthesized from a starting compound containing an -S-S- bond to finally obtain ionic lipid (2), etc. The method for producing ionic lipid (2) is preferably method (B).

[0154] Method (B) will be explained below, but the method for producing ionic lipid (2) is not limited to this.

[0155] The raw materials, reagents used in each step of the following production methods, and the resulting compounds may each form a salt. Examples of such salts include the same salts as those of the compounds of the present invention described above.

[0156] When the compound obtained in each step is a free compound, it can be converted into the desired salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted into the free compound or another desired type of salt by a known method.

[0157] The compound obtained in each step can be used in the next reaction either as a reaction solution or as a crude product, or can be appropriately purified from the reaction mixture by a common purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0158] When the raw materials and reagent compounds for each step are commercially available, the commercially available products can be used as they are.

[0159] In the reaction of each step, the reaction time is usually 1 minute to 48 hours, preferably 10 minutes to 22 hours, unless otherwise specified.

[0160] In the reactions of each step, the reaction temperature is usually −78° C. to 300° C., preferably −78° C. to 150° C., unless otherwise specified.

[0161] In the reactions of each step, unless otherwise specified, the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 8 equivalents, relative to the substrate. When a reagent is used as a catalyst, the reagent is used in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.4 equivalents, relative to the substrate. When a reagent also serves as a reaction solvent, the reagent is used in the amount of the solvent.

[0162] Unless otherwise specified, the reactions in each step are carried out without solvent or by dissolving or suspending in an appropriate solvent. Specific examples of the solvent include those described in the examples, as well as methanol, ethanol, isopropanol, tert-butyl alcohol, tetrahydrofuran, toluene, cyclohexane, hexane, heptane, N,N-dimethylformamide, N-methylpyrrolidone, chloroform, dichloromethane, acetonitrile, dimethyl sulfoxide, ethyl acetate, acetone, and water. Two or more of the above solvents may be mixed in an appropriate ratio.

[0163] When a base is used in the reaction of each step, examples of the base include the bases described in the examples, as well as sodium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, sodium hydrogencarbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), imidazole, piperidine, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium hydride.

[0164] When an acid or an acidic catalyst is used in the reaction of each step, examples of the acid or acidic catalyst include those described in the Examples, as well as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, and boron trifluoride diethyl ether complex.

[0165] In each step, protection or deprotection of a functional group is carried out using known reagents and methods (for example, the reagents and methods described in GREENE'S PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th ed., WILEY-INTERSCIENCEM) or in accordance with the methods described in the Examples.

[0166] Examples of the protecting group for a hydroxyl group of an alcohol or the like or a phenolic hydroxyl group include ether-type protecting groups such as tetrahydropyranyl ether, methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, and t-butyldiphenylsilyl ether; carboxylic acid ester-type protecting groups such as acetate ester; sulfonic acid ester-type protecting groups such as methanesulfonate ester; and carbonate-type protecting groups such as t-butyl carbonate.

[0167] The protecting group can be removed by a known method, such as a method using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, or a trialkylsilyl halide, or a reduction method.

[0168] In each step, when a carbonation reaction or a carbamate reaction is carried out, examples of the reagents used include N,N'-disuccinimidyl carbonate (DSC), 4-nitrophenyl chloroformate (pNPCl), and a base (basic salt, organic base, etc.). When carbonation is carried out, an additive such as 4-dimethylaminopyridine (DMAP) may be further added.

[0169] In each step, when a mesylation reaction is carried out, methanesulfonyl chloride and a base (basic salts, organic bases, etc.) are used as reagents.

[0170] When an esterification reaction, amidation reaction, or urea reaction is carried out in each step, examples of the reagent used include activated carboxylic acids such as acyl halides such as acid chlorides, acid anhydrides, and activated esters. Examples of carboxylic acid activators include carbodiimide condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), triazine condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and combinations thereof. When a carbodiimide condensing agent is used, an additive such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), or 4-dimethylaminopyridine (DMAP) may be further added to the reaction.

[0171] In each step, when a nucleophilic substitution reaction such as amination is carried out, a nucleophile (e.g., an amine) and a base (e.g., a basic salt, an organic base) are used as reagents. An additive such as potassium iodide (KI) or tetrabutylammonium iodide (TBAI) may also be added to the reaction.

[0172] Ionic lipid (2) can be produced, for example, by the following method: Furthermore, a salt of ionic lipid (2) can be obtained by appropriately mixing with an inorganic acid or an organic acid.

[0173] Manufacturing method A: k = 1, l = 0, m = 0, n = 0, R ｘ and R ｙ When both are ethylene groups

[0174]

[0175] Manufacturing method B: k = 1, l = 0, m = 1, n = 1, R ｘ and R ｙ When both are ethylene groups

[0176]

[0177] Manufacturing method C: k = 1, l = 1, m = 0, n = 0, R ｘ and R ｙ are both ethylene groups, and S １ When is hydrogen or an alkyl group

[0178]

[0179] Manufacturing method D: k = 0, l = 0, m = 0, n = 0, R ｘ and R ｙ When both are ethylene groups

[0180]

[0181] In the above formula, F １ ~F １０ each independently represents a reactive functional group; P １ ~P ４ each independently represents a protecting group.

[0182] Specific production methods are described below in Production Examples 1 to 46. Those skilled in the art can produce the desired ionic lipid (2) by appropriately selecting raw materials and carrying out reactions in accordance with the methods described in Production Examples 1 to 46.

[0183] Phospholipids Phospholipids can be used as lipid membrane components of lipid nanoparticles. Examples of phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphatidylethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphatidylserine (PS), 1,2-diacyl-sn-glycero-3-phosphatidylglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), or lyso forms thereof, specifically, 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), 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), Examples include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and these PCs can be appropriately converted into PE, PS, PG, or PA and used.

[0184] The phospholipid used in the present invention is preferably PC or PE, more preferably DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), or POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), and particularly preferably DOPC, DSPC, or DEPC.

[0185] Sterols can be used as a component that adjusts the fluidity of the lipid membrane of lipid nanoparticles. Examples of sterols include cholesterol, lanosterol, phytosterol, zymosterol, zymostenol, desmosterol, stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol, preferably cholesterol, lanosterol, or phytosterol, and more preferably cholesterol.

[0186] PEG lipids are used to coat the surface of lipid nanoparticles with hydrophilic polyethylene glycol (PEG) to stabilize the particles by preventing aggregation and to prevent interaction between the particles and biological components when administered to a living body. The PEG region may have any molecular weight. In some embodiments, the PEG region has a molecular weight of 200 to 10,000 Da and may be linear or branched.

[0187] Examples of PEG lipids include PEG-phospholipids, PEG-ceramides, PEG-diacylglycerols, and PEG-cholesterols. Diacylglycerol PEGs having a molecular weight of 1,000 to 10,000 are preferred, and dimyristoylglycerol PEGs or distearoylglycerol PEGs having a molecular weight of 1,000 to 10,000 are more preferred.

[0188] Acidic buffers: Buffers with buffering properties in the acidic range can be used. Specifically, HCl / KCl buffer, p-toluenesulfonic acid / p-toluenesulfonic acid Na salt buffer, tartaric acid / NaOH buffer, citric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / formic acid Na buffer, citric acid / citric acid Na buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer, phenylacetic acid / phenylacetic acid Na salt buffer, acetic acid / acetate Na buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, cacodylate Na / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH2PO4 / NaOH buffer, imidazole / HCl buffer, and s-collidine. Examples include (2,4,6-trimethylpyridine) / HCl buffer, triethanolamine HCl / NaOH buffer, 5,5-diethylbarbituric acid Na / HCl buffer, N-methylmorpholine / HCl buffer, pyrophosphate Na / HCl buffer, MES buffer, malic acid buffer, ADA buffer, PIPES buffer, ACES buffer, HEPES, BES, Bis-Tris buffer, Bis-Tris propane buffer, anhydrous sodium carbonate buffer, glycylglycine buffer, MOPS, MOPSO, and TES.

[0189] Preferably, it is an acidic buffer solution having a buffering effect at pH 1 to 6.5, more preferably pH 3 to 6.5, and examples thereof include tartaric acid / NaOH buffer solution, citric 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, citric acid / Na citrate buffer solution, 3,3-dimethylglutaric acid / NaOH buffer solution, and 3,3-dimethylglutaric acid / NaOH / 0.1M Examples of the buffer include NaCl buffer, phenylacetic acid / phenylacetic acid Na salt buffer, acetic acid / Na acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, cacodylic acid Na / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH2PO4 / NaOH buffer, MES buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartrate buffer, citrate buffer, bis-tris buffer, and glycylglycine buffer, with malic acid buffer or MES buffer being more preferred.

[0190] In the method of the present invention, a suspension of nucleic acid-free lipid nanoparticles is prepared by mixing an alcohol solution containing ionic lipids, sterols, and PEG-lipids with an acidic buffer solution having a pH of 1 to 6.5. Any assembly-inducing procedure can be used to prepare lipid nanoparticles. Examples of alcohols include ethanol and tert-butanol.

[0191] Examples of the "assembly-inducing operation" for preparing 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 known methods include fusion, freeze-thaw, and reverse-phase evaporation. Preferred is the alcohol dilution method using a microchannel or vortex, and more preferred is the alcohol dilution method using a microchannel. Particle preparation using the alcohol dilution method using a microchannel can be performed, for example, using NanoAssemblr (Precision NanoSystems). The buffer solution in the external aqueous phase of the prepared lipid nanoparticles can be exchanged by ultrafiltration, dialysis, dilution, or other procedures. In a preferred embodiment, after preparing a lipid nanoparticle suspension, the external aqueous phase can be exchanged for another acidic buffer solution with a buffering action at pH 1 to 6.5 (preferably pH 3 to 6.5) by ultrafiltration, dialysis, dilution, or other procedures. Furthermore, sugars such as monosaccharides, sugar alcohols, disaccharides, oligosaccharides, and polysaccharides can be added to the lipid nanoparticle suspension. Disaccharides are preferred as sugars, and sucrose is more preferred. The final sugar concentration is preferably 0 to 320 mg / mL, more preferably 0 to 160 mg / mL.

[0192] Storage Conditions for Lipid Nanoparticles Lipid nanoparticles not containing nucleic acids may be stored as a liquid at a temperature range of 0°C to 50°C, or may be stored frozen at a temperature range of -80°C to 0°C. When stored as a liquid, the storage temperature is preferably 0°C to 30°C, more preferably 0°C to 10°C, and most preferably 0°C to 5°C, from the viewpoint of the stability of the components. When stored frozen, the storage temperature is preferably -80°C to -10°C, and more preferably -80°C to -20°C, from the viewpoint of the stability of the components. When stored frozen, the nanoparticles are thawed at 0°C to 100°C before use. From the viewpoint of the stability of the components, the melting temperature is preferably 0°C to 95°C, more preferably 0°C to 50°C, even more preferably 0°C to 30°C, and most preferably 0°C to 10°C. Freezing and thawing are preferably performed under atmospheric pressure.

[0193] Nucleic Acid Addition Step The nucleic acid addition step is a step in which nucleic acid-encapsulated lipid nanoparticles are prepared by mixing the nucleic acid-free lipid nanoparticles prepared in the previous step with an aqueous solution containing nucleic acid without lyophilization. Note that "without lyophilization" means that the nucleic acid addition step is performed immediately after the nucleic acid-free lipid nanoparticle preparation step. The nucleic acid-free lipid nanoparticle preparation step involves storing the nucleic acid-free lipid nanoparticles as a liquid, or freezing and storing the nucleic acid-free lipid nanoparticles, and then thawing them. Nucleic acid-encapsulated lipid nanoparticles can be prepared by adding an aqueous solution containing nucleic acid such that the ratio of total lipid to nucleic acid contained in the lipid nanoparticles is, for example, 1 to 1,000 nmol / μg (total lipid / nucleic acid), preferably 50 to 500 nmol / μg, and mixing by pipetting or vortexing. Furthermore, alcohol can be added to increase the nucleic acid encapsulation rate. Examples of alcohol that can be used include methanol, ethanol, n-butanol, and t-butanol, with ethanol being preferred. The concentration of alcohol in the nucleic acid-containing aqueous solution is preferably 0-50 v / v%, more preferably 0-30 v / v%, and even more preferably 0-25 v / v%. Furthermore, in the nucleic acid addition step, to further enhance the nucleic acid encapsulation efficiency, the nucleic acid-containing aqueous solution and / or alcohol can be added to the liquid or frozen lipid nanoparticles that do not contain nucleic acids, followed by incubation. Incubation conditions are, for example, 0-100°C for 0-120 minutes, preferably 0-95°C for 0-60 minutes. An acidic buffer solution containing nucleic acids is preferably used as the nucleic acid-containing aqueous solution. The acidic buffer solution can be a buffer with buffering activity in the acidic range, preferably an acidic buffer with buffering activity at pH 1-6.5, more preferably pH 3-6.5. Specifically, the acidic buffer solution used in the preparation of lipid nanoparticles includes the same acidic buffer solutions as those exemplified above, such as MES buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartaric acid buffer, and citrate buffer.

[0194] Step of exchanging the external aqueous phase with a neutral buffer solution: By exchanging the external aqueous phase of the nucleic acid-encapsulated lipid nanoparticles obtained in the nucleic acid addition step with a neutral buffer solution, nucleic acid-encapsulated lipid nanoparticles that can be used as nucleic acid transfer agents can be prepared. Methods for exchanging the external aqueous phase with a neutral buffer solution include dialysis, ultrafiltration, and dilution. Examples of neutral buffer solutions include phosphate-buffered saline (PBS), Tris-HCl buffer, ADA, PIPES, PIPES sesquisodium, ACES, MOPS, BES, MOPSO, TES, HEPES, TAPSO, POPSO, and HEPSO. The pH of the neutral buffer solution is 6 to 8.

[0195] By contacting cells with the nucleic acid-encapsulated lipid nanoparticles prepared by the method of the present invention, the nucleic acid can be introduced into the cells in vivo and / or ex vivo. Thus, the present invention provides a method for introducing the nucleic acid into the cells in vivo and / or ex vivo.

[0196] Any nucleic acid can be introduced into cells using the methods of the present invention. Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. While any single-, double-, or triple-stranded nucleic acid 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.

[0197] 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.

[0198] 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.

[0199] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0200] The nucleic acid-encapsulated lipid nanoparticles produced by the 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.

[0201] The nucleic acid-encapsulated lipid nanoparticles produced by the method of the present invention can be used as a drug delivery system for selectively delivering nucleic acids, etc. into specific cells, and are useful, for example, in DNA vaccines and gene therapy drugs for tumors that involve antigen gene transfer into dendritic cells, and nucleic acid pharmaceuticals that suppress the expression of target genes using RNA interference. Thus, the present invention provides a method for producing a pharmaceutical composition.

[0202] The particle size of the lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Measurement of the particle size can be performed using a particle size distribution analyzer such as Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be adjusted appropriately depending on the method for producing the lipid nanoparticles.

[0203] The surface potential (zeta potential) of lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably −15 to +15 mV, more preferably −10 to +10 mV. In previous gene transfer methods, particles with a positively charged surface potential have primarily been used. This is useful as a method for promoting electrostatic interactions with heparin sulfate on the negatively charged cell surface and facilitating cellular uptake. However, a positive surface charge may inhibit the release of nucleic acids from the carrier due to intracellular interactions with the delivered nucleic acid, or inhibit protein synthesis due to interactions between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface charge within the above range. Surface charge can be measured using a zeta potential measuring device such as a Zetasizer Nano. The surface charge of lipid nanoparticles can be adjusted by the composition of the lipid nanoparticle's components.

[0204] The step of contacting cells with lipid nanoparticles encapsulating nucleic acids in vitro will be specifically described below.

[0205] The cells are suspended in an appropriate medium and cultured under appropriate conditions several days before contact with the lipid nanoparticles. The cells may or may not be in a proliferative phase at the time of contact with the lipid nanoparticles.

[0206] 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 lipid nanoparticles and the cells may be inhibited.

[0207] 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.

[0208] To the cells prepared in this manner, for example, a suspension of lipid nanoparticles encapsulating the nucleic acid described above is added. The amount of the suspension added is not particularly limited and can be set appropriately taking into account the number of cells, etc. The concentration of the lipid nanoparticles when contacted with the cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved, but the lipid concentration is usually 1 to 300 nmol / mL, preferably 10 to 200 nmol / mL, and the nucleic acid concentration is usually 0.01 to 100 μg / mL, preferably 0.05 to 10 μg / mL.

[0209] After adding the above-mentioned suspension 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.

[0210] The nucleic acid is introduced into the cells by the above-mentioned culture, and preferably the medium is replaced with a fresh medium or fresh medium is added to the medium and the culture is continued. When the cells are derived from a mammal, the fresh medium preferably contains serum or nutrient factors.

[0211] Furthermore, as described above, by using lipid nanoparticles encapsulating nucleic acids, it is possible to introduce nucleic acids into cells not only in vitro but also in vivo. That is, by administering lipid nanoparticles encapsulating nucleic acids to a subject, the lipid nanoparticles reach and contact target cells, and the nucleic acids encapsulated in the lipid nanoparticles are introduced into cells in vivo. The subjects to which the lipid nanoparticles 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 lipid nanoparticles encapsulating nucleic acids are administered are preferably humans or other mammals.

[0212] The type of target cell is not particularly limited, and by using lipid nanoparticles encapsulating nucleic acids, 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 nodes, tumors, etc.).

[0213] The method of administering lipid nanoparticles into which nucleic acids and / or compounds other than nucleic acids have been introduced to a subject (e.g., vertebrates, invertebrates, etc.) is not particularly limited as long as the lipid nanoparticles reach and contact target cells and the compound introduced into the lipid nanoparticles can be introduced into the cells. Taking into consideration the type of compound to be introduced, the type and site of the target cells, etc., a known administration method (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected. The dosage of the lipid nanoparticles is not particularly limited as long as it is within a range that allows for the introduction of the compound into the cells, and can be appropriately selected taking into consideration the type of subject, administration method, type of compound to be introduced, type and site of the target cells, etc.

[0214] When lipid nanoparticles encapsulating nucleic acids are used as nucleic acid transfer agents, they can be formulated according to conventional methods.

[0215] When the nucleic acid transfer agent of the present invention is provided as a research reagent, the nucleic acid transfer agent may be provided as a lipid nanoparticle encapsulating a nucleic acid as is, or as a sterile solution or suspension in, for example, water or other physiologically acceptable liquid (e.g., a water-soluble solvent (e.g., malic acid buffer, etc.), an organic solvent (e.g., ethanol, methanol, DMSO, tert-butanol, etc.), or a mixture of a water-soluble solvent and an organic solvent, etc.). The nucleic acid transfer agent of the present invention may optionally contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0216] Furthermore, when the nucleic acid transfer agent is provided as a pharmaceutical, the nucleic acid transfer agent of the present invention can be manufactured as an oral agent (e.g., tablet, capsule, etc.) or a parenteral agent (e.g., injection, spray, etc.), preferably a parenteral agent (more preferably, an injection), by using the lipid nanoparticles encapsulating the nucleic acid as they are or together with known pharmaceutically acceptable additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.) and mixing them in a unit dosage form required for generally accepted formulation practice.

[0217] The nucleic acid transfer agent of the present invention can be formulated for use in children as well as for adults.

[0218] Examples of the present invention will be described in more detail below, but the present invention is not limited to these examples.

[0219] In the following examples, the ionic lipids represented by formula (1) or formula (2) are shown by the names listed in the above table. Nucleic acid-encapsulated lipid nanoparticles are sometimes referred to as "nucleic acid-encapsulated nanoparticles." The abbreviations used in the following examples have the following meanings: Chol: cholesterol; DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (PEG number average molecular weight (Mn): 2000); DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; DEPC: 1,2-dierucoyl-sn-glycero-3-phosphocholine; MES: 2-morpholinoethanesulfonic acid; PBS: phosphate buffered saline; DDW: deionized distilled water; DCM: dichloromethane; THF: tetrahydrofuran; IPA: 2-propanol; DHP: 3,4-dihydro-2H-pyran; PPTS: pyridinium p-toluenesulfonate; THP: 2-tetrahydropyranyl; DMAP: 4-dimethylaminopyridine. DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate DSC: di(N-succinimidyl) carbonate EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride TEA: triethylamine TBAI: tetrabutylammonium iodide DIPEA: N,N-diisopropylethylamine NPM: 4-phenylmorpholine MsCl: methanesulfonyl chloride Ms: methanesulfonyl pNPCl: 4-nitrophenyl chloroformate

[0220] Example 1 Effect of buffer solution SS-OP and MC3 were used as ionic lipids. When SS-OP was used as the ionic lipid, the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio, and when MC3 was used as the ionic lipid, the lipid composition was MC3:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5 in molar ratio.

[0221] 2100 μL of acidic malate buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 5) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 5) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 5) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0222] The particle solution (total lipid 20 mM, 15 μL) was diluted with MES (pH 5), phthalic acid (pH 5), maleic acid (pH 5), succinic acid (pH 5), DL-malic acid (pH 5), DL-tartaric acid (pH 5), or citric acid (pH 5) buffer (nucleic acid concentration 1.5 μg / 135 μL, 135 μL) containing luciferase-encoded mRNA (CleanCap (registered trademark) FLuc mRNA - (L-7602)) and a vortex mixer under stirring conditions, and then incubated at 37 ° C. for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0223] The resulting nucleic acid-encapsulated nanoparticles were analyzed for particle size, polydispersity index (PdI), and mRNA encapsulation rate. Particle size and PdI were measured by dynamic light scattering using a Zetasizer®. The mRNA encapsulation rate was measured by the Ribogreen® assay. As a result, mRNA was encapsulated in all buffers: MES, phthalic acid, maleic acid, succinic acid, DL-malic acid, DL-tartaric acid, and citric acid, and no difference in encapsulation rate was observed (see Table 3, Figures 1 and 2). Particle size is shown as Z-Ave (Z-average) and Number Mean (same below).

[0224]

[0225] Example 2 Effect of Sucrose Concentration and Incubation Temperature SS-OP was used as the ionic lipid. The lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio.

[0226] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution or DNase / RNase-free distilled water was added to the concentrate to a final sucrose concentration of 0 mg / mL, 160 mg / mL, or 320 mg / mL to prepare a particle solution.

[0227] Particle solution (total lipid 20 mM, 15 μL) stored at 4 ° C (hereinafter also referred to as liquid) or particle solution frozen at -80 ° C and then thawed at 4 ° C (hereinafter also referred to as frozen-thawed particles) was mixed with a solution containing luciferase-encoding mRNA (CleanCap (registered trademark) FLuc mRNA - (L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 5), 135 μL) under stirring conditions using a vortex mixer while diluting, and for the mixture of each sucrose concentration, using an aluminum block-type incubator, the liquid sample was incubated at 95 ° C, 75 ° C, 55 ° C, 35 ° C, and room temperature for 5 minutes. Of these, the one with a sucrose concentration of 160 mg / mL was added, and the sample was incubated on ice for 5 minutes. The frozen-thawed particles were incubated at 95 ° C, 35 ° C, and room temperature for 5 minutes. After incubation, 150 μL of PBS was added to the solution to obtain nucleic acid-encapsulated nanoparticles.

[0228] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer®. The mRNA encapsulation rate was measured by the Ribogreen® assay. As a result, mRNA was encapsulated at all incubation temperatures, and no difference in encapsulation rate was observed. Furthermore, mRNA was encapsulated at all sucrose concentrations, and no difference in encapsulation rate was observed. The particle size of the resulting nucleic acid-encapsulated nanoparticles incubated at 95°C was large, and no difference in particle size was observed at other temperatures (75°C, 55°C, 35°C, or room temperature) (see Table 4, Figures 3 and 4).

[0229] The nucleic acid-encapsulated nanoparticles thus obtained were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. 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% FBS and 1% penicillin-streptomycin solution). ３The cells were seeded into a 96-well flat-bottom transparent plate at 100 μL of cells / 100 μL. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration was 0.02 μg / 4 μL. After 16 hours, the amount of luciferase protein introduced was evaluated based on the amount of luminescence. 100 μL of Steady-Glo (registered trademark) was added, and the cells were lysed by shaking for 5 minutes. The amount of luminescence was quantified using a GloMax 20 / 20 luminometer. As a result, introduction of luciferase protein was confirmed at both incubation temperatures, and no difference in in vitro activity was observed. Furthermore, higher activity was observed with the frozen-thawed particles compared to the liquid formulation (see Figure 5).

[0230]

[0231] Example 3 Effect of Incubation Time SS-OP was used as the ionic lipid. The lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio.

[0232] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0233] The particle solution (total lipid 20 mM, 15 μL) was diluted and mixed with a solution containing luciferase-encoded mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 6), 135 μL) under stirring conditions using a vortex mixer, and incubated at room temperature for 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles. Alternatively, for comparison, 150 μL of PBS was first added to the particle solution (total lipid 20 mM, 15 μL), and then luciferase-encoded mRNA (same as above) was added to a final concentration of 1.5 μg / 300 μL.

[0234] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated at all incubation times, and no difference in encapsulation rate was observed. Furthermore, a low encapsulation rate was obtained when PBS was first added to the particle solution (PBS → mRNA) (see Figures 6 and 7).

[0235] The nucleic acid-encapsulated nanoparticles thus obtained were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. 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% FBS and 1% penicillin-streptomycin solution). ３The cells were seeded into a 96-well flat-bottom transparent plate at a concentration of 100 μL per 100 μL. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration in the medium was 0.02 μg / 4 μL. Luciferin was added to the 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 medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubator-type luminometer (Kronos), and the cumulative luminescence was measured every hour for 2 minutes. As a result, the nucleic acid introduction efficiency did not change with incubation time. Furthermore, when PBS was first added to the particle solution (PBS → mRNA), the nucleic acid introduction efficiency was low (see Figures 8 and 9).

[0236] Example 4: Effect of pH SS-OP, SS-OC, MC3, or DODAP was used as the ionic lipid. When SS-OP or SS-OC was used, DOPC, chol, and DMG-PEG2000 were used as other lipids. When MC3 or DODAP was used, DSPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP or SS-OC: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 or SS-OC, DOPC, and chol. The molar ratio of MC3 or DODAP:DSPC:chol:DMG-PEG2000 used was 50:10:38.5:1.5.

[0237] 2100 μL of acidic malate buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production system (manufactured by Precision NanoSystems), LNPs were prepared at a flow rate of 7:1 (acidic buffer solution:lipid ethanol solution), a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. After adding 9000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) and concentrated again by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to approximately 500 μL. This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0238] The particle solution (total lipid 20 mM, 15 μL) was mixed with a solution containing luciferase-encoding mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 6), 135 μL) while diluting under stirring conditions using a vortex mixer, and the mixtures of each pH and ionic lipid combination were incubated at 37 ° C. for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0239] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by Ribogreen (registered trademark) assay. As a result, SS-OP and MC3 were encapsulated up to pH 6, and the encapsulation rate decreased at pH 6.5 and pH 7. On the other hand, SS-OC and DODAP were encapsulated up to pH 5.5, and the encapsulation rate decreased at pH 6, pH 6.5, and pH 7 (see Figures 10 and 11).

[0240] Example 5 Effect of Salt Concentration SS-OP and MC3 were used as ionic lipids. When SS-OP was used as the ionic lipid, the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio, and when MC3 was used as the ionic lipid, the lipid composition was MC3:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5 in molar ratio.

[0241] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0242] The particle solution (total lipid 20 mM, 15 μL) was mixed with a solution containing luciferase-encoded mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer conditions NaCl 0 mM, 1 mM, 10 mM, 30 mM, 50 mM, 100 mM, 150 mM or 300 mM MES (pH 6), 135 μL) and diluted under stirring conditions with a vortex mixer, and the mixtures of each salt concentration and ionic lipid combination were incubated at 37 ° C. for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0243] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, the mRNA encapsulation rate decreased with increasing salt concentration of the buffer solution (see Figures 12 and 13).

[0244] Example 6: Effect of mixing ratio and mixing mode SS-OP was used as the ionic lipid. The lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio.

[0245] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0246] The particle solution or a solution obtained by diluting the particle solution with MES (pH 6) buffer (lipid concentration: 300 nmol / 15 μL, 300 nmol / 75 μL, or 300 nmol / 135 μL) was mixed with MES (pH 6) buffer containing luciferase-encoding mRNA (CleanCap® FLuc mRNA - (L-7602)) (nucleic acid concentration: 3 μg / 135 μL, 3 μg / 75 μL, or 3 μg / 15 μL) under vortex mixing or by pipetting followed by tapping, and the mixture was incubated at 37°C for 5 minutes in an aluminum block incubator. 150 μL of PBS was added to the incubated solution to obtain nucleic acid-encapsulated nanoparticles.

[0247] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer®. The mRNA encapsulation rate was measured by the Ribogreen® assay. As a result, a high encapsulation rate was obtained under stirring conditions using a vortex mixer at all mixing ratios. However, when mixing was performed by tapping after pipetting, the encapsulation rate decreased as the volume of the nucleic acid aqueous solution decreased (see Figures 19, 20, and 21).

[0248] The nucleic acid-encapsulated nanoparticles thus obtained were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. 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% FBS and 1% penicillin-streptomycin solution). ３The cells were seeded into a 96-well flat-bottom transparent plate at 100 μL per 100 μL of nucleic acid-encapsulated particles. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated based on the amount of luminescence. 100 μL of Steady-Glo® was added, and the cells were lysed by shaking for 5 minutes. The amount of luminescence was quantified using a GloMax 20 / 20 luminometer. As a result, no difference in in vitro activity was observed under vortex mixer agitation conditions, but in vitro activity decreased when the mixture was mixed by tapping after pipetting and the volume of the aqueous nucleic acid solution was small (see Figure 21).

[0249] Example 7 Effect of buffer solution SS-OP and MC3 were used as ionic lipids. When SS-OP was used as the ionic lipid, the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio, and when MC3 was used as the ionic lipid, the lipid composition was MC3:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5 in molar ratio.

[0250] 2100 μL of acidic malate buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 5) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 5) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 5) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0251] The particle solution (total lipid 20 mM, 15 μL) was diluted with MES (pH 5), phthalic acid (pH 5), maleic acid (pH 5), succinic acid (pH 5), DL-malic acid (pH 5), DL-tartaric acid (pH 5), or citric acid (pH 5) buffer (nucleic acid concentration 3 μg / 135 μL, 135 μL) containing luciferase-encoded mRNA (CleanCap (registered trademark) FLuc mRNA - (L-7602)) and a vortex mixer under stirring conditions, and then incubated at 37 ° C. for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0252] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated in all buffer solutions: MES, phthalic acid, maleic acid, succinic acid, DL-malic acid, DL-tartaric acid, and citric acid, and no difference in the encapsulation rate was observed (see Figures 22 and 23).

[0253] Example 8 Effect of Incubation Temperature SS-OP was used as the ionic lipid. The lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio.

[0254] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution or DNase / RNase-free distilled water was added to the concentrate to a final sucrose concentration of 0 mg / mL, 160 mg / mL, or 320 mg / mL to prepare a particle solution.

[0255] The particle solution (total lipid 20 mM, 15 μL) was mixed with a solution containing luciferase-encoding mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 3 μg / 135 μL, buffer condition MES (pH 5), 135 μL) while diluting under stirring conditions using a vortex mixer, and then incubated for 5 minutes at 95°C, 75°C, 55°C, or 37°C using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0256] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated at all incubation temperatures, and no difference in encapsulation rate was observed. The particle size of the resulting nucleic acid-encapsulated nanoparticles tended to increase with increasing incubation temperature (see Figures 24 and 25).

[0257] The nucleic acid-encapsulated nanoparticles thus obtained were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. 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% FBS and 1% penicillin-streptomycin solution). ３ The cells were seeded into a 96-well flat-bottom transparent plate at 100 μL of cells / 100 μL. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration in the medium was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated based on the amount of luminescence. 100 μL of Steady-Glo (registered trademark) was added, and the cells were lysed by shaking for 5 minutes, and the amount of luminescence was quantified using a GloMax 20 / 20 luminometer. As a result, introduction of luciferase protein was confirmed at both incubation temperatures, and no difference in in vitro activity was observed (see Figure 26).

[0258] Example 9 Effect of Incubation Time SS-OP was used as the ionic lipid. The lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio.

[0259] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0260] The particle solution (total lipid 20 mM, 15 μL) was diluted and mixed with a solution containing luciferase-encoded mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 3 μg / 135 μL, buffer condition MES (pH 6), 135 μL) under stirring conditions using a vortex mixer, and incubated at room temperature for 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles. Alternatively, for comparison, 150 μL of PBS was first added to the particle solution (total lipid 20 mM, 15 μL), and then luciferase-encoded mRNA (same as above) was added to a final concentration of 1.5 μg / 300 μL.

[0261] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated at all incubation times, and no difference in encapsulation rate was observed. Furthermore, a low encapsulation rate was obtained when PBS was first added to the particle solution (PBS → mRNA) (see Figures 27 and 28).

[0262] The nucleic acid-encapsulated nanoparticles thus obtained were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. 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% FBS and 1% penicillin-streptomycin solution). ３ The cells were seeded into a 96-well flat-bottom transparent plate at 100 μL per cell. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated using the luminescence intensity. 100 μL of Steady-Glo® was added, and the cells were lysed by shaking for 5 minutes. The luminescence intensity was quantified using a GloMax 20 / 20 luminometer. As a result, introduction of luciferase protein was confirmed at all incubation times, and no difference in in vitro activity was observed. Furthermore, a decrease in in vitro activity was observed when PBS was first added to the particle solution (PBS → mRNA) (see Figure 29).

[0263] Example 10 Effect of pH SS-OP, SS-OC, MC3, or DODAP was used as the ionic lipid. When SS-OP or SS-OC was used, DOPC, chol, and DMG-PEG2000 were used as other lipids. When MC3 or DODAP was used, DSPC, chol, and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP or SS-OC: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 or SS-OC, DOPC, and chol. The molar ratio of MC3 or DODAP:DSPC:chol:DMG-PEG2000 used was 50:10:38.5:1.5.

[0264] 2100 μL of acidic malate buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production system (manufactured by Precision NanoSystems), LNPs were prepared at a flow rate of 7:1 (acidic buffer solution:lipid ethanol solution), a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. After adding 9000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) and concentrated again by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to approximately 500 μL. This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0265] The particle solution (total lipid 20 mM, 15 μL) was mixed with a solution containing luciferase-encoding mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 3 μg / 135 μL, buffer condition MES (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7), 135 μL) while diluting under stirring conditions using a vortex mixer, and the mixtures of each pH and ionic lipid combination were incubated at 37°C for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0266] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, SS-OP, SS-OC, MC3, and DODAP were all encapsulated up to pH 6, and the encapsulation rate decreased at pH 6.5 and pH 7 (see Figures 30 and 31).

[0267] Of the obtained nucleic acid-encapsulated nanoparticles, particles prepared using SS-OP were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. 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% FBS and 1% penicillin-streptomycin solution). ３ The cells were seeded into a 96-well flat-bottom transparent plate at 100 μL per cell. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated based on the amount of luminescence. 100 μL of Steady-Glo® was added, and the cells were lysed by shaking for 5 minutes. The amount of luminescence was quantified using a GloMax 20 / 20 luminometer. The results showed that the highest in vitro activity was observed at pH 6, with a slight decrease in activity at pH 6.5, and low activity at pH 5, pH 5.5, and pH 7 (see Figure 32).

[0268] Of the resulting nucleic acid-encapsulated nanoparticles, the zeta potential of particles prepared using SS-OP was analyzed. The zeta potential was measured by electrophoretic light scattering using a Zetasizer (registered trademark) using MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7). As a result, the particles prepared at pH 5 and pH 5.5 showed higher values ​​in a low pH environment than the particles prepared at pH 6, pH 6.5, or pH 7 (see Figure 33).

[0269] Example 11 Effect of Salt Concentration SS-OP and MC3 were used as ionic lipids. When SS-OP was used as the ionic lipid, the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000 = 52.5:7.5:40:1.5 in molar ratio, and when MC3 was used as the ionic lipid, the lipid composition was MC3:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5 in molar ratio.

[0270] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0271] The particle solution (total lipid 20 mM, 15 μL) was mixed with a solution containing luciferase-encoded mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 3 μg / 135 μL, buffer conditions NaCl 0 mM, 1 mM, 10 mM, 30 mM, 50 mM, 100 mM, 150 mM or 300 mM MES (pH 6), 135 μL) under stirring conditions with a vortex mixer while diluting, and the mixtures of each salt concentration and ionic lipid combination were incubated at 37 ° C. for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0272] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, the mRNA encapsulation rate tended to decrease as the salt concentration of the buffer solution increased (see Figures 34 and 35).

[0273] Example 12 Effect of Ionic Lipid Species LN-81 (Compound 34 in the Production Example described below) was used as the ionic lipid. The lipid compositions were, in molar ratios, LN-81:DEPC:Chol:DMG-PEG2000 = 52.5:17.5:30:1 and LN-81:DEPC:Chol:DMG-PEG2000 = 60:5:35:1.

[0274] 2400 μL of acidic malate buffer (20 mM, pH 5.0) and 800 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25 °C. After adding 3000 μL of MES buffer (pH 6.0) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6.0) and again concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). The entire concentrated mixture was transferred to an Amicon Ultra 4, and 3000 μL of MES buffer (pH 6.0) was added. Then, the mixture was ultrafiltered under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to form a particle solution.

[0275] The liquid formulation or frozen-thawed particles were mixed with a buffer solution of MES (pH 6) (nucleic acid concentration 11.1 μg / 180 μL, 180 μL) containing luciferase-encoding mRNA (CleanCap® FLuc mRNA − (L-7602)) while diluting under stirring conditions using a vortex mixer, and then incubated at 37°C for 5 minutes using an aluminum block incubator. 100 μL of the solution after incubation was dispensed, and 1010 μL of PBS was added to obtain nucleic acid-encapsulated nanoparticles.

[0276] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated (see Table 5).

[0277]

[0278] Comparative Example 1: Method for preparing a lyophilized product SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0279] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and concentrated to approximately 500 μL by ultrafiltration again under centrifugation conditions (25°C, 1000 g, 5 minutes). Sucrose solution was added to a final concentration of 160 mg / mL. 200 μL of the resulting solution was placed in a mighty vial and lyophilized to obtain a lyophilized product.

[0280] The lyophilized product was dissolved in a solution (nucleic acid concentration: 2 μg / 200 μL, dissolved in DDW) containing mRNA encoding luciferase (CleanCap® FLuc mRNA − (L-7602)). This mixture was incubated at 95°C for 5 minutes using an aluminum block incubator. After incubation, 200 μL of PBS was added to the solution to obtain nucleic acid-encapsulated nanoparticles.

[0281] Comparative Example 2: Method for preparing a lyophilized product SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0282] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and concentrated to approximately 500 μL by ultrafiltration again under centrifugation conditions (25°C, 1000 g, 5 minutes). Sucrose solution was added to a final concentration of 160 mg / mL. 200 μL of the resulting solution was placed in a mighty vial and lyophilized to obtain a lyophilized product.

[0283] The lyophilized product was dissolved in a solution (nucleic acid concentration 4 μg / 200 μL, dissolved in DDW) containing mRNA encoding luciferase (CleanCap® FLuc mRNA − (L-7602)). This mixture was incubated at 95°C for 5 minutes using an aluminum block incubator. 200 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0284] Comparative Example 3: Method for preparing a lyophilized product SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0285] 2100 μL of acidic malate buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 16 mL / min, and a syringe holder temperature of 25°C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and concentrated to approximately 500 μL by ultrafiltration again under centrifugation conditions (25°C, 1000 g, 5 minutes). Sucrose solution was added to a final concentration of 160 mg / mL. 200 μL of the resulting solution was placed in a mighty vial and lyophilized to obtain a lyophilized product.

[0286] The lyophilized product was dissolved in a solution (nucleic acid concentration 4 μg / 200 μL, dissolved in DDW) containing mRNA encoding luciferase (CleanCap® FLuc mRNA − (L-7602)). This mixture was incubated at 75°C for 5 minutes using an aluminum block incubator. 200 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0287] Test Example 1 In Vitro Activity SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0288] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0289] A particle solution (total lipid 20 mM, 15 μL) stored at 4°C (liquid) or a particle solution frozen at -80°C and then thawed at 4°C (frozen-thawed particles) was mixed with a solution containing luciferase-encoding mRNA (CleanCap (registered trademark) FLuc mRNA − (L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 5), 135 μL) while diluting under stirring conditions using a vortex mixer, and then incubated at 95°C for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0290] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles or the nucleic acid-encapsulated nanoparticles prepared by freeze-drying in Comparative Example 1 were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, the liquid formulation and the frozen-thawed formulations achieved an mRNA encapsulation rate equivalent to that of nucleic acid-encapsulated nanoparticles prepared by freeze-drying. Furthermore, the particle size of the nucleic acid-encapsulated nanoparticles obtained was smaller than that of nucleic acid-encapsulated nanoparticles prepared by freeze-drying (see Figures 14 and 15).

[0291] The resulting nucleic acid-encapsulated nanoparticles or the nucleic acid-encapsulated nanoparticles prepared by freeze-drying in Comparative Example 1 were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. HeLa cells were cultured at 5 x 10 in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution). ３The nucleic acid-encapsulated nanoparticles were seeded into a 96-well flat-bottom transparent plate at a concentration of 0.02 μg / 4 μL. The resulting nucleic acid-encapsulated particles were added to the medium so that the nucleic acid concentration in the medium was 0.02 μg / 4 μL. Luciferin was added to the 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 medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubator-type luminometer (Kronos), and the cumulative luminescence was measured every hour for 2 minutes. As a result, a higher nucleic acid introduction efficiency was obtained in the liquid formulation and the frozen-thawed formulation compared to nucleic acid-encapsulated nanoparticles prepared using freeze-drying technology (see Figures 16 and 17).

[0292] Test Example 2 In vivo activity SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0293] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0294] A particle solution (total lipid 20 mM, 15 μL) stored at 4°C (liquid) or a particle solution frozen at -80°C and then thawed at 4°C (frozen-thawed particles) was mixed with a solution containing luciferase-encoding mRNA (CleanCap (registered trademark) FLuc mRNA − (L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 5), 135 μL) while diluting under stirring conditions using a vortex mixer, and then incubated at 95°C for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0295] The resulting nucleic acid-encapsulated nanoparticles or the nucleic acid-encapsulated nanoparticles prepared by freeze-drying in Comparative Example 1 were administered to mice (Balb / c, 6 weeks old, female), and gene expression activity in the liver was evaluated using a GloMax 20 / 20 Luminometer. Each nucleic acid-encapsulated nanoparticle was administered to the mice via the tail vein at 0.05 mg / kg. Six hours after administration, the liver was removed and the expression level of luciferase protein was evaluated. As a result, the liquid formulation and the frozen-thawed formulations showed higher gene expression activity in the liver than the nucleic acid-encapsulated nanoparticles prepared by freeze-drying (see Figure 18).

[0296] Test Example 3 In vivo activity SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0297] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0298] The particle solution (total lipid 20 mM, 15 μL) (liquid) was mixed with a solution containing luciferase-encoding mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 3 μg / 135 μL, buffer condition MES (pH 6), 135 μL) while diluting under stirring conditions using a vortex mixer, and the mixture was incubated at 37°C for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0299] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles, or the nucleic acid-encapsulated nanoparticles prepared by freeze-drying in Comparative Examples 2 and 3, were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer®. The mRNA encapsulation rate was measured by the Ribogreen® assay. As a result, the liquid formulation achieved an mRNA encapsulation rate equivalent to that of nucleic acid-encapsulated nanoparticles prepared by freeze-drying. Furthermore, the particle size of the nucleic acid-encapsulated nanoparticles obtained was smaller than that of nucleic acid-encapsulated nanoparticles prepared by freeze-drying (see Figures 36 and 37). In Figures 36, 37, and 38, Comparative Example 2, in which the total flow rate during lipid nanoparticle preparation was 1 mL / min, is labeled "Freeze-dried (FR=1)," and Comparative Example 3, in which the total flow rate was 16 mL / min, is labeled "Freeze-dried (FR=16)."

[0300] The resulting nucleic acid-encapsulated nanoparticles or the nucleic acid-encapsulated nanoparticles prepared by freeze-drying in Comparative Examples 2 and 3 were administered to mice (Balb / c, 6 weeks old, female), and gene expression activity in the liver was evaluated using an IVIS Imaging System. Each nucleic acid-encapsulated nanoparticle was administered to the mice via the tail vein at 0.1 mg / kg. Six hours after administration, images were taken and the expression level of luciferase protein was evaluated. As a result, the liquid formulation demonstrated higher gene expression activity in the liver than the nucleic acid-encapsulated nanoparticles prepared by freeze-drying (see Figure 38).

[0301] Test Example 4 In vivo activity SS-OP was used as the ionic lipid, and DOPC, chol, and DMG-PEG 2000 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-PEG 2000 was used relative to the total of SS-OP, DOPC, and chol.

[0302] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were weighed into syringes. Using a NanoAssmblr® ultrafast nanomedicine production device (manufactured by Precision NanoSystems), LNPs were prepared under the following conditions: a flow ratio of acidic buffer solution to lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25 °C. After adding 9000 μL of MES buffer (pH 6) to the recovered material, the resulting mixture was transferred to an Amicon Ultra 15 and subjected to ultrafiltration under centrifugation conditions (25 °C, 1000 g, 5 minutes) to concentrate to approximately 1500 μL. The resulting concentrate was diluted to 15 mL with MES buffer (pH 6) and then concentrated to approximately 500 μL by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes). This procedure was repeated twice, and the resulting mixture was transferred to an Amicon Ultra 4. 3000 μL of MES buffer (pH 6) was added, followed by ultrafiltration under centrifugation conditions (25°C, 1000 g, 5 minutes) and concentrated to approximately 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0303] The particle solution (total lipid 20 mM, 15 μL) (liquid) was mixed with a solution containing luciferase-encoding mRNA (CleanCap® FLuc mRNA − (L-7602)) (nucleic acid concentration 3 μg / 135 μL, buffer condition MES (pH 6), 135 μL) while diluting under stirring conditions using a vortex mixer, and the mixture was incubated at 37°C for 5 minutes using an aluminum block incubator. 150 μL of PBS was added to the solution after incubation to obtain nucleic acid-encapsulated nanoparticles.

[0304] The particle size, polydispersity index (PdI), and mRNA encapsulation rate of the resulting nucleic acid-encapsulated nanoparticles (liquid formulation) or nucleic acid-encapsulated nanoparticles (MF) prepared using a microchannel (LNP prepared with the same lipid composition using an MES buffer (20 mM, pH 6.5) containing nucleic acid and an ethanolic lipid solution) were analyzed. Particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation rate was measured by the Ribogreen (registered trademark) assay. As a result, an mRNA encapsulation rate equivalent to that of nucleic acid-encapsulated nanoparticles prepared using a microchannel was obtained in the liquid formulation (see Figure 39).

[0305] The resulting nucleic acid-encapsulated nanoparticles (liquid formulation) or nucleic acid-encapsulated nanoparticles (MF) prepared using a microchannel were administered to mice (Balb / c, 6 weeks old, female), and gene expression activity in each organ (liver, heart, spleen, kidney, and lung) was evaluated using an IVIS Imaging System. Each nucleic acid-encapsulated nanoparticle was administered to the mice via the tail vein at 0.1 mg / kg. Six hours after administration, the mice were dissected and photographed to evaluate the expression level of luciferase protein. As a result, the liquid formulation showed a similar expression distribution to the nucleic acid-encapsulated nanoparticles prepared using a microchannel (see Figures 40 and 41).

[0306] Test Example 5: In vitro activity of particles of Example 12 The nucleic acid-encapsulated nanoparticles obtained in Example 12 or the nucleic acid-encapsulated nanoparticles prepared by freeze-drying in Comparative Example 1 were allowed to act on HeLa cells to evaluate the nucleic acid transfer efficiency. HeLa cells were cultured at 3 x 10 in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution). ４The cells were seeded into a 48-well flat-bottom transparent plate at a volume of 600 μL. The next day, after adding potassium luciferin to the medium, the resulting nucleic acid-encapsulated particles were added to the medium at a nucleic acid concentration of 0.12 μg / 60 μL, and the amount of luciferase protein introduced was evaluated using a Kronos HT for 24 hours. As a result, the solution and freeze-thawed particles exhibited higher nucleic acid introduction efficiency than nucleic acid-encapsulated nanoparticles prepared using freeze-drying technology. Furthermore, when comparing the solution and freeze-thawed particles, the freeze-thawed particles exhibited higher nucleic acid introduction efficiency. Meanwhile, the solution prepared with a composition of LN-81:DEPC:Chol:DMG-PEG2000 = 52.5:17.5:30:1 exhibited the highest nucleic acid introduction efficiency (see Figure 42).

[0307] Preparation of Ionic Lipid (2) Table 6 shows the names and structures of the ionic lipids prepared in the following preparation examples.

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] [Production Example 1] Synthesis of Compound 1 Compound 1 was produced by the following synthetic route, but the present invention is not limited to this synthetic route.

[0322]

[0323] <Synthesis of Intermediate 1> 4-Hydroxyphenylacetic acid (75.0 g) and PPTS (12.4 g) were dissolved in 400 g of dichloromethane at room temperature, and the resulting solution was cooled to 10-20°C. A solution obtained by dissolving DHP (207 g) in 100 g of dichloromethane was added dropwise thereto, and the reaction was carried out at 25°C for 2 hours. The reaction solution was cooled to 10-20°C, and DMAP (30.1 g) was added thereto to quench the reaction. 590 g of 2-propanol was added to the quenched solution, and the mixture was cooled to 10-20°C. A solution of 98.8 g of 400 g / L aqueous sodium hydroxide solution and 307 g of ion-exchanged water was added dropwise to the cooled solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator, and the dichloromethane and 2-propanol were distilled off. The resulting concentrate was washed twice with 750 g of chloroform, and then 6N hydrochloric acid was added to obtain a solution of pH 5.0. The resulting solution was extracted twice with 750 g of chloroform, and then 75.0 g of sodium sulfate was added to the organic layer to dehydrate it. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 98.2 g of Intermediate 1.

[0324] <Synthesis of Intermediate 2> Intermediate 1 (10.2 g), bis(2-hydroxyethyl) disulfide (9.99 g), and DMAP (1.06 g) were dissolved in 153 g of chloroform at room temperature. EDC (12.4 g) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 102 g of 20 wt% saline, and then 5.1 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, and the resulting crude product was purified using a column to obtain 9.01 g of Intermediate 2.

[0325] <Synthesis of Intermediate 3-a> Intermediate 2 (350 mg), dimethylglycine hydrochloride (144 mg), triethylamine (143 mg), and DMAP (23.0 mg) were dissolved in 5.25 mL of chloroform at room temperature. EDC (270 mg) was added to the resulting solution and the reaction was allowed to proceed at room temperature for 2 hours. The reaction solution was washed with 3.5 g of 0.5 M phosphate buffer (pH 4.0), 3.5 g of 7 wt % sodium bicarbonate water, and 3.5 g of 20 wt % saline, in that order, and then dehydrated by adding 175 mg of sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 378 mg of Intermediate 3-a.

[0326] <Synthesis of Intermediate 4-a> Intermediate 3-a (363 mg) was dissolved in 3.27 g of THF, and then 13.1 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at room temperature for 2 hours. 5.45 g of chloroform was added to the reaction solution for washing. After washing, a 1 N aqueous sodium hydroxide solution was added to the aqueous layer to adjust the pH to 6.0, and the pH-adjusted aqueous layer was extracted with 5.45 g of chloroform. 180 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 187 mg of Intermediate 4-a.

[0327] <Synthesis of Compound 1> Intermediate 4-a (187 mg), 2-hexyldecanoic acid (128 mg), and DMAP (12.0 mg) were dissolved in 2.81 g of chloroform at room temperature. EDC (144 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 1.87 g of 20 wt% saline, and then 93.0 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, and the resulting crude product was purified using a column to obtain 159 mg of Compound 1. <Synthesis of Compound 1> １ H-NMR (600MHz, CDCl ３)> δ: 0.86-0.90 (m, 6H), 1.20-1.45 (m, 22H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.8 8-2.97 (m, 4H), 3.19 (s, 2H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0328] [Production Example 2] Synthesis of Compound 2 <Synthesis of Compound 2> Compound 2 was synthesized using intermediate 4-a and 2-decyldodecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 34H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2 .97 (m, 4H), 3.19 (s, 2H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0329] [Production Example 3] Synthesis of Compound 3 <Synthesis of Compound 3> Compound 3 was synthesized using intermediate 4-a and 2-tetradecylhexadecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 50H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2 .97 (m, 4H), 3.19 (s, 2H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0330] [Production Example 4] Synthesis of Compound 4 <Synthesis of Intermediate 3-b> Intermediate 3-b represented by the following formula was synthesized in the same manner as in Production Example 1, except that 4-(dimethylamino)butanoic acid hydrochloride was used.

[0331]

[0332] <Synthesis of Intermediate 4-b> Intermediate 4-b represented by the following formula was synthesized in the same manner as in Production Example 1, except that intermediate 3-b was used.

[0333]

[0334] <Synthesis of Compound 4> Compound 4 was synthesized in the same manner as in Production Example 1, except that intermediate 4-b was used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.86-0.90 (m, 6H), 1.20-1.45 (m, 22H), 1.69-1.82 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.5 2-2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0335] [Production Example 5] Synthesis of Compound 5 <Synthesis of Compound 5> Compound 5 was synthesized using intermediate 4-b and 2-decyldodecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 34H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0336] [Production Example 6] Synthesis of Compound 6 <Synthesis of Compound 6> Compound 6 was synthesized using intermediate 4-b and 2-dodecyltetradecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 42H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0337] [Production Example 7] Synthesis of Compound 7 <Synthesis of Compound 7> Compound 7 was synthesized using intermediate 4-b and 2-tetradecylhexadecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 50H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0338] [Production Example 8] Synthesis of Compound 8 <Synthesis of Compound 8> Compound 8 was synthesized using intermediate 4-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0339] [Production Example 9] Synthesis of Compound 9 <Synthesis of Compound 9> Compound 9 was synthesized using intermediate 4-b and 2-octadecyleicosanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 66H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0340]

[0341] [Production Example 10] Synthesis of Compound 10 <Synthesis of Intermediate 5> Intermediate 2 (5.10 g), 4-bromobutyric acid (2.52 g), and DMAP (335 mg) were dissolved in 76.5 g of chloroform at room temperature. EDC (3.94 g) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 51.0 g of 20 wt% saline, and then 2.55 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 6.07 g of Intermediate 5.

[0342] <Synthesis of Intermediate 6-a> Diethylamine (258 mg) was dissolved in 1.04 g of THF at room temperature. A solution obtained by dissolving Intermediate 5 (230 mg) in 780 mg of THF was added dropwise thereto, and the mixture was allowed to react at 50°C for 15 hours. 3.45 g of chloroform was added to the reaction solution, and the mixture was washed successively with 2.30 g of 0.5 M acetate buffer (pH 4.0), 2.30 g of 7 wt% sodium bicarbonate water, and 2.30 g of 20 wt% saline. 130 mg of sodium sulfate was added and the mixture was dehydrated. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 130 mg of Intermediate 6-a.

[0343] <Synthesis of Intermediate 7-a> Intermediate 7-a was synthesized using intermediate 6-a in the same manner as in the synthesis of intermediate 4-a in Production Example 1.

[0344] <Synthesis of Compound 10> Compound 10 was synthesized using intermediate 7-a and 2-hexadecyl octadecanoic acid in the same manner as in the synthesis of Compound 1 in Production Example 1. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.28-2.60 (m, 9H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0345] Preparation Example 11 Synthesis of Compound 11 <Synthesis of Intermediate 6-b> Intermediate 6-b represented by the following formula was synthesized in the same manner as in Preparation Example 10, except that piperidine was used.

[0346]

[0347] <Synthesis of Intermediate 7-b> Intermediate 7-b represented by the following formula was synthesized in the same manner as in Production Example 10, except that intermediate 6-b was used.

[0348]

[0349] <Synthesis of Compound 11> Compound 11 was synthesized using intermediate 7-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 10. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 60H), 1.45-1.60 (m, 4H), 1.69-1.84 (m, 4H), 2.20-2.50 (m, 8H), 2.52-2 .59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0350] [Production Example 12] Synthesis of Compound 12 <Synthesis of Intermediate 6-c> Intermediate 6-c represented by the following formula was synthesized in the same manner as in Production Example 10, except that dipropylamine was used.

[0351]

[0352] <Synthesis of Intermediate 7-c> Intermediate 7-c represented by the following formula was synthesized in the same manner as in Production Example 10, except that intermediate 6-c was used.

[0353]

[0354] <Synthesis of Compound 12> Compound 12 was synthesized using intermediate 7-c and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 10. １ H-NMR (600MHz, CDCl ３ )> δ: 0.86-0.92 (m, 12H), 1.20-1.45 (m, 62H), 1.69-1.84 (m, 4H), 2.28-2.60 (m, 9H), 2.8 8-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0355]

[0356] [Production Example 13] Synthesis of Compound 13 <Synthesis of Compound 13> Compound 13 was synthesized by the same synthetic route as in Production Example 8, except that Intermediate 2 and 3-(dimethylamino)-2-methylpropionic acid hydrochloride were used as starting materials. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.13 (d, 3H), 1.20-1.45 (m, 58H), 1.68-1.78 (m, 2H), 2.18-2.22 (m, 7H), 2.52-2.7 0 (m, 3H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0357]

[0358] [Production Example 14] Synthesis of Compound 14 <Synthesis of Intermediate 8> 3,4-Dihydroxyphenylacetic acid (3.00 g) and PPTS (448 mg) were dissolved in 40.0 g of dichloromethane at room temperature, and the resulting solution was cooled to 20°C. DHP (7.50 g) was added dropwise thereto, and the reaction was carried out at room temperature for 2 hours. The reaction solution was cooled to 20°C, and DMAP (1.09 g) was added thereto to quench the reaction. 47.0 g of 2-propanol was added to the quenched solution, and the mixture was cooled to 10-20°C. 30.0 g of 1N aqueous sodium hydroxide solution was added dropwise to the cooled solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator, and dichloromethane and 2-propanol were distilled off. The resulting concentrate was washed with 30.0 g of chloroform, and 6N hydrochloric acid was added to obtain a solution with a pH of 5.0. The resulting solution was extracted with 30.0 g of chloroform, and then the organic layer was dehydrated by adding 1.50 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 4.98 g of Intermediate 8.

[0359] <Synthesis of Intermediate 9> Intermediate 8 (800 mg), bis(2-hydroxyethyl)disulfide (550 mg), and DMAP (58.0 mg) were dissolved in 12.0 g of chloroform at room temperature. EDC (684 mg) was added to the resulting solution, and the mixture was allowed to react at 25°C for 2 hours. The reaction solution was washed with 8.00 g of 20 wt% saline, and then 400 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, and the resulting crude product was purified using a column to obtain 530 mg of Intermediate 9.

[0360] <Synthesis of Intermediate 10-a> Intermediate 9 (525 mg), dimethylglycine hydrochloride (171 mg), triethylamine (169 mg), and DMAP (27.0 mg) were dissolved in 7.90 g of chloroform at room temperature. EDC (319 mg) was added to the resulting solution and the reaction was allowed to proceed at room temperature for 2 hours. The reaction solution was washed with 5.25 g of 0.5 M phosphate buffer (pH 4.0), 5.25 g of 7 wt % sodium bicarbonate water, and 5.25 g of 20 wt % saline, in that order, and then 250 mg of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 558 mg of Intermediate 10-a.

[0361] <Synthesis of Intermediate 11-a> Intermediate 10-a (550 mg) was dissolved in 4.95 g of THF, and then 19.8 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at room temperature for 2 hours. 8.25 g of chloroform was added to the reaction solution for washing. After washing, a 1 N aqueous sodium hydroxide solution was added to the aqueous layer to adjust the pH to 6.5, and the pH-adjusted aqueous layer was extracted with 8.25 g of chloroform. 300 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 503 mg of Intermediate 11-a.

[0362] <Synthesis of Compound 14> Intermediate 11-a (190 mg), oleic acid (289 mg), and DMAP (24.0 mg) were dissolved in 2.85 g of chloroform at room temperature. EDC (234 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 1.90 g of 20 wt% saline, and then dehydrated by adding 100 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 291 mg of Compound 14. <Synthesis of Compound 14> １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.22-1.42 (m, 40H), 1.69-1.83 (m, 6H), 1.99-2.05 (m, 8H), 2.36 (s, 6H), 2.50-2.5 3 (m, 4H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 5.32-5.38 (m, 4H), 7.10-7.17 (m, 3H)

[0363] [Production Example 15] Synthesis of Compound 15 <Synthesis of Intermediate 10-b> Intermediate 10-b represented by the following formula was synthesized in the same manner as in Production Example 14, except that 4-(dimethylamino)butanoic acid hydrochloride was used.

[0364]

[0365] <Synthesis of Intermediate 11-b> Intermediate 11-b represented by the following formula was synthesized in the same manner as in Production Example 14, except that intermediate 10-b was used.

[0366]

[0367] <Synthesis of Compound 15> Compound 15 was synthesized using intermediate 11-b and oleic acid according to the same synthetic route as in Production Example 14. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.22-1.42 (m, 40H), 1.69-1.83 (m, 6H), 1.99-2.05 (m, 8H), 2.26 (s, 6H), 2.33-2.54 (m, 4H), 2.50-2.53 (m, 4H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 5.32-5.38 (m, 4H), 7.10-7.17 (m, 3H)

[0368]

[0369] [Production Example 16] Synthesis of Compound 16 <Synthesis of Intermediate 12> Intermediate 2 (5.00 g) and triethylamine (2.72 g) were dissolved in 125 g of chloroform at room temperature. Methanesulfonyl chloride (2.31 g) was added to the resulting solution and reacted at 25°C for 3 hours. The reaction solution was washed with 75.0 g of 7 wt% aqueous sodium bicarbonate solution, and 5.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 4.42 g of Intermediate 12.

[0370] <Synthesis of Intermediate 13-a> Intermediate 12 (600 mg) was dissolved in 5.40 g of dimethylamine (2.0 mol / L in THF), and TBAI (49.0 mg) was added, followed by a reaction at 25°C for 20 hours. 9.00 g of chloroform was added to the reaction solution, which was then washed with 6.00 g of 0.5 M acetate buffer (pH 4.0), and 300 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 470 mg of Intermediate 13-a.

[0371] <Synthesis of Intermediate 14-a> Intermediate 14-a was synthesized in the same manner as for intermediate 4-a in Production Example 1, except that intermediate 13-a was used.

[0372] <Synthesis of Compound 16> Compound 16 was synthesized using intermediate 14-a and 2-hexadecyl octadecanoic acid in the same manner as in the synthesis of Compound 1 in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.25 (s, 6H), 2.52-2.60 (m, 3H), 2 .80 (t, 2H), 2.90 (t, 2H), 3.63 (s, 2H), 4.35 (t, 2H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0373] [Production Example 17] Synthesis of Compound 17 <Synthesis of Intermediate 13-b> 4.20 g of THF was added to intermediate 12 (600 mg) and diethylamine (779 mg), and the mixture was dissolved at room temperature. TBAI (49.0 mg) was added to the resulting solution, and the mixture was reacted at 25°C for 3 hours. 9.00 g of chloroform was added to the reaction solution, and the mixture was washed with 6.00 g of 0.5 M acetate buffer (pH 4.0), and 300 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 473 mg of intermediate 13-b represented by the following formula:

[0374]

[0375] <Synthesis of Intermediate 14-b> Intermediate 14-b represented by the following formula was synthesized in the same manner as in Production Example 16, except that intermediate 13-b was used.

[0376]

[0377] <Synthesis of Compound 17> Compound 17 was synthesized using intermediate 14-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 16. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.03 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.53-2.57 (m, 5H), 2.7 5-2.80 (m, 4H), 2.91 (t, 2H), 3.63 (s, 2H), 4.35 (t, 2H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0378]

[0379] [Production Example 18] Synthesis of Compound 18 <Synthesis of Intermediate 15-a> Cystamine dihydrochloride (227 mg), DIPEA (391 mg), and dimethylglycine hydrochloride (141 mg) were dissolved in 4.54 g of methanol at room temperature. DMT-MM (837 mg) was added to the resulting solution, and the reaction was allowed to proceed at 25°C for 2 hours. Subsequently, Intermediate 1 (239 mg) was added to the reaction solution, and the reaction was allowed to proceed at 25°C for 2 hours. After concentrating the reaction solution, 3.40 g of chloroform was added, and the mixture was washed sequentially with 2.27 g of 0.5 M phosphate buffer (pH 6.0), 2.27 g of 7 wt% aqueous sodium bicarbonate, and 2.27 g of 20 wt% saline. 100 mg of sodium sulfate was added to the resulting solution to dehydrate it. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 748 mg of Intermediate 15-a as a crude product.

[0380] <Synthesis of Intermediate 16-a> Intermediate 16-a was synthesized in the same manner as for intermediate 4-a in Production Example 1, except that a crude product of intermediate 15-a was used.

[0381] <Synthesis of Compound 18> Compound 18 was synthesized using intermediate 16-a and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.30 (s, 6H), 2.52-2.59 (m, 1H), 2.76-2.82 (m , 4H), 2.95 (s, 2H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0382] [Preparation Example 19] Synthesis of Compound 19 <Synthesis of Intermediate 15-b> Intermediate 15-b represented by the following formula was synthesized in the same manner as in Preparation Example 18, except that 3-(dimethylamino)propionic acid hydrochloride was used.

[0383]

[0384] <Synthesis of Intermediate 16-b> Intermediate 16-b represented by the following formula was synthesized in the same manner as in Production Example 18, except that intermediate 15-b was used.

[0385]

[0386] <Synthesis of Compound 19> Compound 19 was synthesized using intermediate 16-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 18. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.28 (s, 6H), 2.33-2.38 (m, 2H), 2.52-2.59 (m, 3H) ), 2.76-2.82 (m, 4H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0387] Preparation Example 20 Synthesis of Compound 20 <Synthesis of Intermediate 15-c> Intermediate 15-c represented by the following formula was synthesized in the same manner as in Preparation Example 18, except that 4-(dimethylamino)butanoic acid hydrochloride was used.

[0388]

[0389] <Synthesis of Intermediate 16-c> Intermediate 16-c represented by the following formula was synthesized in the same manner as in Production Example 18, except that intermediate 15-c was used.

[0390]

[0391] <Synthesis of Compound 20> Compound 20 was synthesized using intermediate 16-c and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 18. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.80 (m, 4H), 2.25-2.38 (m, 8H), 2.52-2.59 (m, 1H), 2.7 6-2.82 (m, 4H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0392]

[0393] [Production Example 21] Synthesis of Compound 21 <Synthesis of Intermediate 17-a> 7.10 g of dichloromethane was added to intermediate 2 (393 mg) and triethylamine (235 mg), and the mixture was dissolved at room temperature. DSC (541 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. After the DSC remaining in the reaction solution was removed by filtration, N,N-dimethylethylenediamine (112 mg) was added to the filtrate, and the reaction was carried out at 25°C for 1 hour. The reaction solution was washed with 5.90 g of 0.5 M acetate buffer (pH 4.0), 5.90 g of 7 wt% sodium bicarbonate water, and 5.90 g of 20 wt% saline, in that order, and then 200 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 472 mg of intermediate 17-a.

[0394] <Synthesis of Intermediate 18-a> Intermediate 18-a was synthesized in the same manner as for intermediate 4-a in Production Example 1, except that intermediate 17-a was used.

[0395] <Synthesis of Compound 21> Compound 21 was synthesized using intermediate 18-a and 2-hexadecyl octadecanoic acid in the same manner as in the synthesis of Compound 1 in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.22 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2 97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.26 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0396] Preparation Example 22 Synthesis of Compound 22 <Synthesis of Intermediate 17-b> Intermediate 17-b represented by the following formula was synthesized in the same manner as in Preparation Example 21, except that N,N-diethylethylenediamine was used.

[0397]

[0398] <Synthesis of Intermediate 18-b> Intermediate 18-b represented by the following formula was synthesized in the same manner as in Production Example 21, except that intermediate 17-b was used.

[0399]

[0400] <Synthesis of Compound 22> Compound 22 was synthesized using intermediate 18-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.47-2.57 (m, 7H), 2.88-2.97 (m, 4H) ), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0401] [Production Example 23] Synthesis of Compound 23 <Synthesis of Intermediate 17-c> Intermediate 17-c represented by the following formula was synthesized in the same manner as in Production Example 21, except that N,N-dimethyl-1,3-propanediamine was used.

[0402]

[0403] <Synthesis of Intermediate 18-c> Intermediate 18-c represented by the following formula was synthesized in the same manner as in Production Example 21, except that Intermediate 17-c was used.

[0404]

[0405] <Synthesis of Compound 23> Compound 23 was synthesized using intermediate 18-c and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.24 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2 97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.70 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0406] [Production Example 24] Synthesis of Compound 24 <Synthesis of Intermediate 17-d> Intermediate 17-d represented by the following formula was synthesized in the same manner as in Production Example 21, except that N,N,N'-trimethyl-1,3-propanediamine was used.

[0407]

[0408] <Synthesis of Intermediate 18-d> Intermediate 18-d represented by the following formula was synthesized in the same manner as in Production Example 21, except that Intermediate 17-d was used.

[0409]

[0410] <Synthesis of Compound 24> Compound 24 was synthesized using intermediate 18-d and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.24 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2.97 (m, 7H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0411] [Production Example 25] Synthesis of Compound 25 <Synthesis of Intermediate 17-e> Intermediate 17-e represented by the following formula was synthesized in the same manner as in Production Example 21, except that N,N-dimethyl-1,4-butanediamine was used.

[0412]

[0413] <Synthesis of Intermediate 18-e> Intermediate 18-e represented by the following formula was synthesized in the same manner as in Production Example 21, except that intermediate 17-e was used.

[0414]

[0415] <Synthesis of Compound 25> Compound 25 was synthesized using intermediate 18-e and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.50-1.62 (m, 4H), 1.69-1.84 (m, 2H), 2.20-2.42 (m, 8H), 2.52-2.59 (m, 1H), 2.8 8-2.97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.70 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0416] [Preparation Example 26] Synthesis of Compound 26 <Synthesis of Intermediate 17-f> Intermediate 17-f represented by the following formula was synthesized in the same manner as in Preparation Example 21, except for using 4-(2-aminoethyl)morpholine.

[0417]

[0418] <Synthesis of Intermediate 18-f> Intermediate 18-f represented by the following formula was synthesized in the same manner as in Production Example 21, except that intermediate 17-f was used.

[0419]

[0420] <Synthesis of Compound 26> Compound 26 was synthesized using intermediate 18-f and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.40-2.46 (m, 6H), 2.52-2.59 (m, 1H), 2.88-2.97 (m , 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.26 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0421] [Preparation Example 27] Synthesis of Compound 27 <Synthesis of Intermediate 17-g> Intermediate 17-g represented by the following formula was synthesized in the same manner as in Preparation Example 21, except that N,N-dibutylethylenediamine was used.

[0422]

[0423] <Synthesis of Intermediate 18-g> Intermediate 18-g represented by the following formula was synthesized in the same manner as in Production Example 21, except that Intermediate 17-g was used.

[0424]

[0425] <Synthesis of Compound 27> Compound 27 was synthesized using intermediate 18-g and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.87-0.93 (m, 12H), 1.20-1.45 (m, 66H), 1.69-1.84 (m, 2H), 2.39-2.57 (m, 7H), 2.88-2.97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0426] Preparation Example 28 Synthesis of Compound 28 <Synthesis of Intermediate 17-h> Intermediate 17-h represented by the following formula was synthesized in the same manner as in Preparation Example 21, except that N,N-diisopropylethylenediamine was used.

[0427]

[0428] <Synthesis of Intermediate 18-h> Intermediate 18-h represented by the following formula was synthesized in the same manner as in Production Example 21, except that Intermediate 17-h was used.

[0429]

[0430] <Synthesis of Compound 28> Compound 28 was synthesized using intermediate 18-h and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 0.95-1.03 (d, 12H), 1.20-1.45 (m, 58H), 1.60-1.84 (m, 6H), 2.52-2.59 (m, 1H), 2.88-3.00 (m , 6H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0431] Preparation Example 29 Synthesis of Compound 29 <Synthesis of Intermediate 17-i> Intermediate 17-i represented by the following formula was synthesized in the same manner as in Preparation Example 21, except that N,N-diethyl-N'-methylethylenediamine was used.

[0432]

[0433] <Synthesis of Intermediate 18-i> Intermediate 18-i represented by the following formula was synthesized in the same manner as in Production Example 21, except that intermediate 17-i was used.

[0434] <Synthesis of Compound 29> Compound 29 was synthesized using intermediate 18-i and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Production Example 21. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.47-2.57 (m, 7H), 2.88-2.97 (m, 7H) ), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0435]

[0436] [Production Example 30] Synthesis of Compound 30 <Synthesis of Intermediate 19> 5.10 g of dichloromethane was added to intermediate 2 (510 mg) and NPM (670 mg) and dissolved at room temperature. pNPCl (690 mg) was added to the resulting solution and allowed to react at 25°C for 2 hours. DMAP (33.0 mg) and 2-(dimethylamino)ethanol (976 mg) were added to this reaction solution and allowed to react at 25°C for 6 hours. The solution after the reaction was washed with 5.10 g of 0.5 M acetate buffer (pH 4.0), 5.10 g of 7 wt% sodium bicarbonate water, and 5.10 g of 20 wt% saline, in that order, and then dehydrated by adding 250 mg of sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 287 mg of intermediate 19.

[0437] <Synthesis of Intermediate 20> Intermediate 20 was synthesized in the same manner as for intermediate 4-a in Production Example 1, except that intermediate 19 was used.

[0438] <Synthesis of Compound 30> Compound 30 was synthesized using intermediate 20 and 2-hexadecyl octadecanoic acid in the same manner as in the synthesis of Compound 1 in Production Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.28 (s, 6H), 2.58-2.61 (m, 3H), 2.91-2 .94 (m, 4H), 3.63 (s, 2H), 4.23 (t, 2H), 4.32-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0439]

[0440] [Production Example 31] Synthesis of Compound 31 <Synthesis of Intermediate 21> Intermediate 18-b (1.00 g), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (445 mg), and DMAP (57.0 mg) were dissolved in 15.0 g of chloroform at room temperature. EDC (668 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 10.0 g of 20 wt% saline, and then 500 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 1.25 g of Intermediate 21.

[0441] <Synthesis of Intermediate 22> Intermediate 21 (1.25 g) was dissolved in 11.3 g of THF, and then 45.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 22 hours. 18.8 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 6.0. The aqueous layer was then extracted with 18.8 g of chloroform. 600 mg of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 908 mg of Intermediate 22.

[0442] <Synthesis of Compound 31> Intermediate 22 (200 mg), myristic acid (175 mg), and DMAP (18.0 mg) were dissolved in 3.00 g of chloroform at room temperature. EDC (175 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator and purified using a column to obtain 106 mg of Compound 31. <Synthesis of Compound 31> １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0443] [Production Example 32] Synthesis of Compound 32 <Synthesis of Compound 32> Compound 32 was synthesized using intermediate 22 and palmitic acid according to the same synthetic route as in Production Example 31. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 48H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0444] [Production Example 33] Synthesis of Compound 33 <Synthesis of Compound 33> Compound 33 was synthesized using intermediate 22 and stearic acid according to the same synthetic route as in Production Example 31. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 56H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0445] [Production Example 34] Synthesis of Compound 34 <Synthesis of Compound 34> Compound 34 was synthesized using intermediate 22 and oleic acid according to the same synthetic route as in Production Example 31. １ H-NMR (600MHz, CDCl ３)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6) H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0446] [Production Example 35] Synthesis of Compound 35 <Synthesis of Compound 35> Compound 35 was synthesized using intermediate 22 and linoleic acid according to the same synthetic route as in Production Example 31. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 28H), 1.42 (s, 3H), 1.55-1 .65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.75- 2.78 (m, 4H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30 -4.40 (m, 8H), 5.32-5.38 (m, 9H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0447]

[0448] [Production Example 36] Synthesis of Compound 36 <Synthesis of Intermediate 23> 36.0 g of dichloromethane was added to Intermediate 2 (2.0 g) and triethylamine (1.20 g) and dissolved at room temperature. DSC (2.75 g) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. After the DSC remaining in the reaction solution was removed by filtration, ethanolamine (394 mg) was added to the filtrate, and the reaction was carried out at 25°C for 1 hour. After the reaction, the solution was washed with 30.0 g of 0.5 M acetate buffer (pH 4.0), and then 1.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 1.41 g of Intermediate 23.

[0449] <Synthesis of Intermediate 24> Intermediate 23 (1.35 g) and triethylamine (594 mg) were dissolved in 20.0 g of chloroform at room temperature. Methanesulfonyl chloride (505 mg) was added to the resulting solution and reacted at room temperature for 3 hours. The reaction solution was washed with 15.0 g of 7 wt % aqueous sodium bicarbonate solution, and 500 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 1.34 g of Intermediate 24.

[0450] <Synthesis of Intermediate 25> Intermediate 2 (1.31 g) was dissolved in a mixed solvent of 11.8 g of THF and 13.1 g of IPA, and then 47.2 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 2 hours. 19.7 g of chloroform was added to the reaction solution for washing. After washing, a 400 g / L aqueous sodium hydroxide solution was added to the aqueous layer for neutralization, and then 19.7 g of chloroform was added to the aqueous layer for extraction. 3.0 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 939 mg of Intermediate 25.

[0451] <Synthesis of Intermediate 26> Intermediate 25 (930 mg), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (393 mg), and DMAP (50.0 mg) were dissolved in 15 g of chloroform at room temperature. EDC (590 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 9.30 g of 20 wt% saline, and then 500 mg of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator and purified using a column to obtain 1.06 g of Intermediate 26.

[0452] <Synthesis of Intermediate 27> Intermediate 26 (1.00 g) was dissolved in a mixed solvent of 9.00 g of THF and 9.00 g of IPA, and then 40.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 2 hours. 15.0 g of chloroform was added to the reaction solution for washing. After washing, a 400 g / L aqueous sodium hydroxide solution was added to the aqueous layer for neutralization, and then 15.0 g of chloroform was added to the aqueous layer for extraction. 500 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 785 mg of Intermediate 27.

[0453] <Synthesis of Intermediate 28> Intermediate 27 (770 mg), oleic acid (802 mg), and DMAP (66.0 mg) were dissolved in 11.6 g of chloroform at room temperature. EDC (648 mg) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 8.00 g of 20 wt% saline, and then 2.0 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 1.15 g of Intermediate 28.

[0454] <Synthesis of Compound 36> Intermediate 28 (250 mg) was dissolved in 2.50 g of THF, and 2-(methylamino)ethanol (137 mg) and TBAI (17.0 mg) were added, followed by a reaction at 40°C for 8 hours. 3.75 g of chloroform was added to the reaction solution, and the mixture was washed with 2.50 g of 0.5 M acetate buffer (pH 4.0), 2.50 g of 7 wt% sodium bicarbonate water, and 2.50 g of 20 wt% saline, in that order. The filtrate was concentrated using an evaporator and purified using a column to obtain 79 mg of Compound 36. <Synthesis of Compound 36> １ H-NMR (600MHz, CDCl ３)> δ: 0.89 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.30-2.40 (m, 7H), 2.52-2.58 (m, 4H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.58-3.63 (m, 4H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0455] [Production Example 37] Synthesis of Compound 37 <Synthesis of Compound 37> Compound 37 was synthesized using intermediate 28 and 3-(methylamino)-1-propanol according to the same synthetic route as in Production Example 36. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.72 (m, 6H), 1.99-2.05 (m, 8H), 2.30-2.40 (m, 7H), 2.52-2.58 (m, 4H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.58-3.63 (m, 4H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0456]

[0457] [Production Example 38] Synthesis of Compound 38 <Synthesis of Intermediate 29> Intermediate 2 (2.00 g), (E)-4-bromocrotonic acid (974 mg), and DMAP (131 mg) were dissolved in 30.0 g of chloroform at room temperature. EDC (1.54 g) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 9.0 mL of 20 wt% saline, and then 2.0 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 2.26 g of Intermediate 29.

[0458] <Synthesis of Intermediate 30> Intermediate 29 (2.25 g) was dissolved in a mixed solvent of 20.23 g of THF and 22.5 g of IPA, and then 81.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 2 hours. 34.0 g of chloroform was added to the reaction solution for washing. After washing, a 400 g / L aqueous sodium hydroxide solution was added to the aqueous layer for neutralization, and then 34.0 g of chloroform was added to the aqueous layer for extraction. 1.00 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 1.51 g of Intermediate 30.

[0459] <Synthesis of Intermediate 31> Intermediate 30 (1.40 g), 2-hexadecyl octadecanoic acid (1.80 g), and DMAP (79.0 mg) were dissolved in 21.0 g of chloroform at room temperature. EDC (925 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator, yielding 2.98 g of crude product Intermediate 31.

[0460] <Synthesis of Compound 38> 500 mg of the crude product of Intermediate 31 and 2.30 g of dimethylamine (2.0 mol / L in THF) were dissolved in 5.00 g of THF, and potassium iodide (0.12 mmol) was added and reacted at 25°C for 10 hours. After removing insoluble matter by filtration, 7.50 g of chloroform was added to the filtrate, which was then washed with 5.00 g of 0.5 M acetate buffer (pH 4.0), 5.00 g of 7 wt% sodium bicarbonate water, and 5.00 g of 20 wt% saline, in that order. 250 mg of sodium sulfate was added and dehydrated, after which the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator and purified using a column to obtain 270 mg of Compound 38. <Synthesis of Compound 38> １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.25 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2.97 (m, 4H) ), 3.07 (t, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.97-6.00 (m, 1H), 6.95-7.05 (m, 3H), 7.27-7.30 (m, 2H)

[0461] [Production Example 39] Synthesis of Compound 39 <Synthesis of Compound 39> Compound 39 was synthesized in the same manner as in Production Example 38, except that diethylamine was used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.03 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.50-2.59 (m, 5H), 2.88-2.97 (m, 4H) ), 3.23 (t, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.97-6.00 (m, 1H), 6.95-7.05 (m, 3H), 7.27-7.30 (m, 2H)

[0462] [Production Example 40] Synthesis of Compound 40 <Synthesis of Compound 40> Compound 40 was synthesized in the same manner as in Production Example 38, except that 2-(ethylamino)ethanol was used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.03 (t, 3H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.50-2.59 (m, 5H), 2.88-2.97 (m, 4H), 3.23 (t , 2H), 3.55-3.58 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.97-6.00 (m, 1H), 6.95-7.05 (m, 3H), 7.27-7.30 (m, 2H)

[0463]

[0464] [Production Example 41] Synthesis of Compound 41 <Synthesis of Compound 41> Compound 41 was synthesized using the same synthetic route as in Production Example 1, except that Intermediate 1, 4,4'-dithiobisbutan-1-ol, and 4-(dimethylamino)butanoic acid hydrochloride were used as starting materials. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.50 (m, 64H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.59-2.70 (m, 4H), 3.63 (s, 2H), 4.22-4.33 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0465]

[0466] [Production Example 42] Synthesis of Compound 42 <Synthesis of Intermediate 32> Bis(2-hydroxyethyl)disulfide (33.2 g), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (25.0 g), and DMAP (3.50 g) were dissolved in 250 g of chloroform at room temperature. EDC (41.0 g) was added to the resulting solution and reacted at 25°C for 2 hours. The reaction solution was washed with 9.0 mL of 20 wt% saline, and then 25.0 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 21.8 g of Intermediate 32.

[0467] <Synthesis of Intermediate 33> 180 g of dichloromethane was added to Intermediate 32 (10.0 g) and triethylamine (7.17 g) and dissolved at room temperature. DSC (16.5 g) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. After the DSC remaining in the reaction solution was removed by filtration, N,N-diethylethylenediamine (5.00 g) was added to the filtrate, and the reaction was carried out at 25°C for 1 hour. After the reaction, the solution was washed with 150 g of 0.5 M acetate buffer (pH 4.0), 150 g of 7 wt% sodium bicarbonate water, and 150 g of 20 wt% saline, in that order, and then 5.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 13.1 g of Intermediate 33.

[0468] <Synthesis of Intermediate 34> Intermediate 33 (13.0 g) was dissolved in 49.0 g of THF, and then 195 g of 0.5 M phosphate buffer (pH 2.0) was added and the mixture was reacted at 40°C for 3 hours. 120 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 7.0. The aqueous layer was then extracted with 195 g of chloroform. 6.50 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 5.01 g of Intermediate 34.

[0469] <Synthesis of Intermediate 35> Intermediate 34 (5.00 g), Intermediate 1 (6.01 g), and DMAP (592 mg) were dissolved in 75.0 g of chloroform at room temperature. EDC (5.81 g) was added to the resulting solution and the mixture was allowed to react at 25°C for 2 hours. The reaction solution was washed with 50.0 g of 20 wt% saline, and then 2.50 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 6.25 g of crude Intermediate 35.

[0470] <Synthesis of Intermediate 36> 6.25 g of the crude product of Intermediate 35 was dissolved in 56.3 g of THF, and then 225 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 25°C for 2 hours. 94.0 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 6.0. The aqueous layer was then extracted with 94.0 g of chloroform. 3.00 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 3.7 g of Intermediate 36.

[0471] <Synthesis of Compound 42> Intermediate 36 (500 mg), oleic acid (436 mg), and DMAP (36.0 mg) were dissolved in 7.50 g of chloroform at room temperature. EDC (352 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 5.00 g of 20 wt% saline, and then dehydrated by adding 250 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 453 mg of compound 42. <Synthesis of Compound 42> １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6) H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0472] [Production Example 43] Synthesis of Compound 43 <Synthesis of Compound 43> Compound 43 was synthesized using intermediate 36 and myristic acid according to the same synthetic route as in Production Example 42. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32 (s, 1H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0473]

[0474] [Production Example 44] Synthesis of Compound 44 <Synthesis of Intermediate 37> Intermediate 34 (1.60 g), Intermediate 1 (4.19 g), and DMAP (165 mg) were dissolved in 24.0 g of chloroform at room temperature. EDC (1.95 g) was added to the resulting solution and reacted at 25°C for 2 hours. The reaction solution was washed with 16.0 g of 20 wt% saline, and then dehydrated by adding 500 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 1.85 g of Intermediate 37.

[0475] <Synthesis of Intermediate 38> Intermediate 37 (450 mg), oleic acid (241 mg), and DMAP (19.0 mg) were dissolved in 6.75 g of chloroform at room temperature. EDC (223 mg) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 4.5 g of 20 wt% saline, and then 2.0 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 670 mg of crude product, Intermediate 38.

[0476] Synthesis of Intermediate 39: The crude product of Intermediate 38 (670 mg) was dissolved in a mixed solvent of 4.00 g of THF and 4.00 g of IPA, and then 16.2 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 3 hours. 13.5 g of chloroform was added to the reaction solution for extraction, and then 9.00 g of 0.5 M phosphate buffer (pH 6.5) was added to the organic layer for washing. 300 mg of sodium sulfate was added to the washed organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator and purified using a column to obtain 240 mg of Intermediate 39.

[0477] <Synthesis of Compound 44> Intermediate 39 (150 mg), lauric acid (41.0 mg), and DMAP (5.0 mg) were dissolved in 2.25 g of chloroform at room temperature. EDC (53.0 mg) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 1.50 g of 20 wt% saline, and then 50.0 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 116 mg of Compound 44. <Synthesis of Compound 44>１ H-NMR (600MHz, CDCl ３ )> δ: 0.80-0.92 (m, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 36H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 4H), 2.36 (t, 2H), 2.52-2.58 (m , 6H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32-5.36 (m, 3H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0478]

[0479] [Production Example 45] Synthesis of Compound 45 <Synthesis of Intermediate 40> 30.0 g of dichloromethane was added to intermediate 18-b (3.00 g) and NPM (3.41 g) and dissolved at room temperature. pNPCl (3.51 g) was added to the resulting solution and reacted at 25°C for 10 hours. DMAP (170 mg) and 1,2-isopropylidene glycol (7.37 g) were added to this reaction solution and reacted at 25°C for 8 hours. The reaction solution was washed with 30.0 g of 7 wt% sodium bicarbonate water and 30.0 g of 20 wt% saline, in that order, and then dehydrated by adding 9.00 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 2.16 g of intermediate 40.

[0480] <Synthesis of Intermediate 41> Intermediate 40 (2.16 g) was dissolved in 11.3 g of THF, and then 45.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the mixture was reacted at 40°C for 2 hours. 19.0 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 6.0. The aqueous layer was then extracted with 19.0 g of chloroform. 1.20 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 850 mg of Intermediate 41.

[0481] <Synthesis of Compound 45> Compound 45 was synthesized using the same synthetic route as in Production Example 42, except that intermediate 41 and oleic acid were used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6) H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.50 (m, 8H), 5.28-5.38 (m, 6H), 6.97-7.00 (m, 2H), 7.27-7.30 (m, 2H)

[0482] [Production Example 46] Synthesis of Compound 46 <Synthesis of Compound 46> Compound 46 was synthesized using the same synthetic route as in Production Example 42, except that intermediate 41 and myristic acid were used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 32H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.92- 2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.50 (m, 8H), 5.28-5.31 (m, 2H), 6.97-7.00 (m, 2H), 7.27-7.30 (m, 2H)

[0483] [Comparative Example] Synthesis of O-Ph-P4C2 O-Ph-P4C2 was synthesized according to the synthetic route described in Production Example 3 of Patent Document 3. １ H-NMR (600MHz, CDCl ３ )> δ0.88 (t, 6H), 1.22-1.42 (m, 46H), 1.54-1.76 (m, 12H), 1.94-2.03 (m, 12H), 2.52-2.56 (m, 4H), 2.62-2.66 (m , 4H), 2.80-2.89 (m, 8H), 3.59 (s, 4H), 4.13 (t, 4H), 5.34-5.37 (m, 4H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0484] According to the present invention, nucleic acids can be introduced into cells with high efficiency, and therefore, the present invention is useful for nucleic acid medicine, gene therapy, and biochemical experiments.

[0485] This application is based on patent application No. 2022-051913 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing nucleic acid-encapsulated lipid nanoparticles, comprising the following steps: a) A step of preparing a suspension of nucleic acid-free lipid nanoparticles by mixing an alcohol solution containing ionic lipids, sterols, and PEG lipids with an acidic buffer having a buffering effect at pH 1 to 6.5, and b) A step to obtain nucleic acid-encapsulated lipid nanoparticles by mixing the suspension of lipid nanoparticles obtained in step a with an aqueous solution containing nucleic acid and optionally containing 0 to 25 v / v% alcohol, without freeze-drying the suspension, and optionally incubating the mixture at 0 to 95°C for 0 to 60 minutes.

2. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 1, comprising the following step c after step b: c) A step of replacing the outer aqueous phase of the obtained nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration, or dilution.

3. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 1 or 2, further comprising the step of freezing nucleic acid-free lipid nanoparticles at -80 to 0°C and then thawing them at 0 to 95°C in step a.

4. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 1 or 2, further comprising the step of replacing the outer aqueous phase with another acidic buffer having a buffering effect at pH 1 to 6.5 by dialysis, ultrafiltration, or dilution after the preparation of a suspension of lipid nanoparticles in step a.

5. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 1 or 2, wherein in step a, the alcohol solution further contains phospholipids.

6. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 1 or 2, wherein the ionic lipid is a compound represented by formula (1): 【Chemistry 1】 (In formula (1), R １ａ and R １ｂ Each of these independently represents an alkylene group with 1 to 6 carbon atoms. X ａ and X ｂ Each of these independently represents 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 of these independently represents an alkylene group or oxydialkylene group having 8 or fewer carbon atoms. Y ａ and Y ｂ Each of these independently represents an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z ａ and Z ｂ each independently represents 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. n ａ and n ｂ Each of these is independently either 0 or 1, R ３ａ and R ３ｂ Each of these is independently a residue derived from a reaction product of a lipid-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms having a cyclopropane ring, or formula (3): R ９ -O-CO-(CH ２ )a- (3) (In formula (3), R ９ This represents an aliphatic hydrocarbon group with 2 to 20 carbon atoms. (where 'a' represents an integer between 2 and 10.) (This represents the base represented by [the symbol].)

7. A method for producing nucleic acid-encapsulated lipid nanoparticles according to claim 1 or 2, wherein the ionic lipid is a compound represented by formula (2): 【Chemistry 2】 (In the formula, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen atoms. R １ This represents an aliphatic hydrocarbon group with 8 or fewer carbon atoms. L １ This represents an ester bond, amide bond, carbamate bond, N-alkylcarbamate bond, carbonate bond, or urea bond. k represents 0 or 1, R ｘ and R ｙ Each of these independently represents an alkylene group with 2 to 5 carbon atoms. L ２ This represents an ester bond, amide bond, carbamate bond, carbonate bond, ether bond, or urea bond. R ２ This represents an alkylene group with 8 or fewer carbon atoms, or it does not exist. Y represents a group that (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, (ii) has at least one selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) contains at least one selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a lipid-soluble vitamin residue, and a sterol derivative residue.

8. A method for introducing nucleic acids into cells, comprising the step of bringing nucleic acid-encapsulated lipid nanoparticles produced by the method of claim 1 or 2 into contact with cells in vitro.

9. A method for introducing nucleic acids into target cells, comprising the step of administering nucleic acid-encapsulated lipid nanoparticles produced by the method described in claim 1 or 2 to a living organism (excluding humans).

10. A method for producing a pharmaceutical composition, comprising the method according to claim 1 or 2.

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