Method for producing nucleic acid-encapsulated ligand-modified lipid nanoparticles

Abstract

The present invention provides a method for producing nucleic acid-encapsulated ligand-modified lipid nanoparticles, the method comprising the following steps a)-c), etc.: a step a) in which an alcoholic solution containing an ionic lipid, a sterol, and a PEG lipid is mixed with an acidic buffer solution having a pH of 1-6.5 to obtain a suspension of lipid nanoparticles not containing nucleic acid; the method comprises a step b) of mixing nucleic acid-free lipid nanoparticles with a nucleic acid solution to obtain a suspension of nucleic acid-encapsulated lipid nanoparticles, and a step c) of mixing the suspension of nucleic acid-encapsulated lipid nanoparticles with a ligand-bound lipid to obtain a suspension of nucleic acid-encapsulated ligand-modified lipid nanoparticles.

Description

Technical Field

[0001] This invention relates to a method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids. Background Technology

[0002] To realize the practical application of nucleic acid therapy using oligonucleotides such as siRNA and gene therapy using mRNA, pDNA, etc., efficient and safe nucleic acid delivery vectors are needed. Viral vectors are nucleic acid delivery vectors with high expression efficiency, but non-viral nucleic acid delivery vectors that can be used more safely are being developed. Among them, vectors using ionic lipids, namely lipid nanoparticles, are currently the most widely used non-viral nucleic acid delivery vectors.

[0003] Ionic lipids are generally composed of amine sites and lipid sites. Under acidic conditions, the amine sites, which are protonated, interact electrostatically with nucleic acids, which are polyanions, to form lipid nanoparticles, thereby promoting their absorption by lipids and delivering nucleic acids into cells.

[0004] 1,2-Dioleoyl-3-dimethylammonium propane (DODAP) is a well-known and widely used ionic lipid. It is known that by combining well-known ionic lipids with phospholipids, cholesterol, and PEG lipids, lipid nanoparticles can be formed to deliver nucleic acids into cells (see, for example, Non-Patent Literature 1).

[0005] Furthermore, Patent Document 1 describes an ionic lipid having the following structure: a compound consisting of one or two amine sites and one lipid site linked together by disulfide bonds exhibiting biodegradability. This document indicates that the ionic lipid can improve pharmacokinetic properties such as blood stability and tumor targeting. Furthermore, by altering the structure surrounding the amine site, the pKa of the lipid membrane structure can be adjusted to a value favorable for endosome escape from the cell. Moreover, utilizing the property of intracellular disulfide bond cleavage, it has the effect of dissociating nucleic acids from the lipid membrane structure. In fact, since this ionic lipid exhibits higher nucleic acid delivery efficiency compared to the known ionic lipid DODAP, it indicates that this ionic lipid can improve intracellular kinetics, such as increasing the efficiency of nucleic acid delivery into the cytoplasm.

[0006] Furthermore, Patent Document 2 discloses a lipid membrane structure that, in addition to the tertiary amine site and disulfide bond, improves its fusion ability with the endosome membrane by using ionic lipids with aromatic rings introduced near the lipid site, thereby further improving the efficiency of nucleic acid delivery to the cytoplasm.

[0007] As mentioned above, lipid nanoparticles are being developed to improve intracellular dynamics by enhancing endosome escape efficiency and membrane fusion capabilities. On the other hand, in order for lipid nanoparticles composed of ionic lipids to exert a more practical effect as nucleic acid delivery carriers in vivo, they need to have targeting ability to target organs and cells.

[0008] As a method to improve targeting of target organs and cells, methods using ligand-modified lipid nanoparticles are known.

[0009] For example, Non-Patent Literature 2 illustrates lipid nanoparticles modified with GalNAc. In this literature, ligand-modified lipid nanoparticles encapsulated with nucleic acids are manufactured by mixing PEG lipids with ligands, which are used to impart cell targeting, with ionic lipids, phospholipids, and cholesterol. This literature shows that intravenous injection of these nucleic acid-encapsulated ligand-modified lipid nanoparticles improves gene silencing efficiency.

[0010] Furthermore, Non-Patent Literature 3 illustrates lipid nanoparticles modified with RGD peptides. In this literature, PEG lipids are pre-bound with ligands to synthesize ligand-bound PEG lipids, thereby creating nucleic acid-encapsulated lipid nanoparticles composed of other components. The nucleic acid-encapsulated lipid nanoparticles are then incubated with the ligand-bound PEG lipids to produce ligand-modified lipid nanoparticles encapsulated with nucleic acids. This literature demonstrates that intravenous injection of these nucleic acid-encapsulated ligand-modified lipid nanoparticles improves the efficiency of nucleic acid delivery to tumors.

[0011] Furthermore, Patent Document 3 illustrates lipid nanoparticles modified with an anti-CD3-IgG antibody. In this document, ligand-modified lipid nanoparticles encapsulated with nucleic acids are manufactured by adding a ligand to nucleic acid-encapsulated lipid nanoparticles produced using activated PEG lipids. This document demonstrates that intravenous injection of these nucleic acid-encapsulated ligand-modified lipid nanoparticles improves the efficiency of nucleic acid delivery to T cells in the spleen.

[0012] [Existing Technical Documents]

[0013] [Patent Documents]

[0014] [Patent Document 1] International Publication No. 2013 / 073480

[0015] [Patent Document 2] International Publication No. 2019 / 188867

[0016] [Patent Document 3] International Publication No. 2023 / 054243

[0017] [Non-patent literature]

[0018] [Non-Patent Literature 1] Biomaterials 29 (2008) 3477-3496

[0019] [Non-Patent Literature 2] Molecular Therapy vol. 18 no. 7, 1357-1364 July 2010

[0020] [Non-Patent Literature 3] Journal of Controlled Release 173 (2014) 110-118 Summary of the Invention

[0021] [The problem the invention aims to solve]

[0022] The method in Non-Patent Document 2 described above involves a one-time mixing of the constituent components of lipid nanoparticles, nucleic acids, and PEG lipids bound with ligands to obtain ligand-modified lipid nanoparticles encapsulated with nucleic acids. Furthermore, Non-Patent Document 3 and Patent Document 3 involve ligand modification after the manufacture of lipid nanoparticles encapsulated with nucleic acids. These methods all involve simultaneously forming lipid nanoparticles and encapsulating nucleic acids within them, thus producing lipid nanoparticles encapsulated with nucleic acids. There is still room for improvement in ensuring that nucleic acids are encapsulated within the lipid nanoparticles.

[0023] The present invention was made in view of the above situation, and its purpose is to provide a method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids, which encapsulates nucleic acids in lipid nanoparticles through steps different from the prior art.

[0024] [Methods for solving the problem]

[0025] Based on in-depth research into the aforementioned issues, the inventors discovered that, unlike existing technologies, by first preparing lipid nanoparticles that do not contain nucleic acids, and then encapsulating nucleic acids within these lipid nanoparticles, nucleic acids can be encapsulated within the lipid nanoparticles. The present invention, based on this discovery, is described below.

[0026] [1] A method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids, characterized in that it comprises:

[0027] Step a) involves mixing an alcoholic solution containing ionic lipids, sterols, and PEG lipids with an acidic buffer solution with a pH of 1–6.5 to obtain a suspension of lipid nanoparticles that do not contain nucleic acids; and includes

[0028] Step b) mixing lipid nanoparticles without nucleic acid with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulated with nucleic acid; and step c) mixing the suspension of lipid nanoparticles encapsulated with nucleic acid with lipids bound to ligands to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

[0029] Or include

[0030] Step c') involves mixing lipid nanoparticles without nucleic acids, water, and lipids bound with ligands to obtain a suspension of ligand-modified lipid nanoparticles without nucleic acids; and step b') involves mixing ligand-modified lipid nanoparticles without nucleic acids with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids.

[0031] Or include

[0032] Step d) involves mixing lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

[0033] [2] According to the method described in [1] above, wherein in step b), a suspension of lipid nanoparticles without nucleic acid is mixed with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulated with nucleic acid; or

[0034] In step c'), a suspension of nucleic acid-free lipid nanoparticles is mixed with lipids bound to ligands to obtain a suspension of nucleic acid-free ligand-modified lipid nanoparticles; and in step b'), the suspension of nucleic acid-free ligand-modified lipid nanoparticles is mixed with a nucleic acid solution to obtain a suspension of nucleic acid-encapsulated ligand-modified lipid nanoparticles; or

[0035] In step d), a suspension of lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

[0036] [3] According to the method described in [1] or [2] above, wherein it includes process a), process b) and process c), or it includes process a), process c') and process b').

[0037] [4] The method according to any one of [1] to [3] above includes the following steps: maintaining the mixture of the nucleic acid-free lipid nanoparticles of step b) and the nucleic acid solution, or the mixture of the nucleic acid-free ligand-modified lipid nanoparticles of step b') and the nucleic acid solution at 0 to 95°C for 1 minute to 2 hours.

[0038] [5] The method according to any one of [1] to [4] above includes the following steps: maintaining the suspension of nucleic acid-encapsulated lipid nanoparticles in step c) with a mixture of lipids bound with ligands, or the mixture of nucleic acid-free lipid nanoparticles, water and lipids bound with ligands in step c') at 0 to 95°C for 1 minute to 5 hours.

[0039] [6] The method according to any one of [1], [2] and [4] to [5] above, wherein the method includes the following step: maintaining the mixture of lipid nanoparticles without nucleic acid, nucleic acid solution and lipid bound with ligand in step d) at 0 to 95°C for 1 minute to 2 hours.

[0040] [7] According to the method described in [1] to [6] above, wherein, in step c), a suspension of lipid nanoparticles encapsulated with nucleic acids, lipids bound with ligands, and lipids not bound with ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids; or

[0041] In step c'), lipid nanoparticles without nucleic acids, water, lipids bound to ligands, and lipids without bound ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles without nucleic acids; or

[0042] In step d), lipid nanoparticles without nucleic acid, nucleic acid solution, lipids bound with ligands and lipids without ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

[0043] [8] According to any one of [1] to [7] above, wherein the alcohol solution in step a) further contains phospholipids.

[0044] [9] The method according to any one of [1] to [8] above, wherein the ionic lipid comprises a compound selected from the group consisting of: [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate); heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; and (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate.

[0045] Compounds represented by formula (1):

[0046] [Chemical Formula 1]

[0047]

[0048] (In formula (1),

[0049] R 1a and R 1b Each of the alkylene groups having 1 to 6 carbon atoms can be independently represented.

[0050] X a and X b Each of these can be used independently to represent a noncyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0051] R 2a and R 2b Each can be used independently to represent an alkylene group or an oxadiene group having 8 or fewer carbon atoms.

[0052] Y a and Y b Each can be used independently to represent an ester bond, amide bond, carbamate bond, ether bond, or urea bond.

[0053] Z a and Z b Each of these groups independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms.

[0054] n a and n b Each can be independently 0 or 1.

[0055] R 3a and R 3b Represented independently:

[0056] Derived from residues of fat-soluble vitamins with hydroxyl groups reacting with succinic anhydride or glutaric anhydride.

[0057] Residues derived from the reaction products of sterol derivatives with hydroxyl groups and succinic anhydride or glutaric anhydride.

[0058] Aliphatic hydrocarbon groups with 1 to 40 carbon atoms,

[0059] Alkyl groups having a cyclopropane ring with 3 to 40 carbon atoms,

[0060] The group represented by formula (3):

[0061] R 9 -O-CO-(CH2) a -* (3)

[0062] (In formula (3),

[0063] * indicates the bonding location.

[0064] R 9This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms.

[0065] 'a' represents an integer from 2 to 10.

[0066] Groups with 50 or fewer carbon atoms represented by formula (4):

[0067]

Chemical Formula 2

[0068]

[0069] (In formula (4),

[0070] * indicates the bonding location, and

[0071] R 10 It represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon cyclic group having 3 to 12 carbon atoms, and R 10 It can be substituted by substituents selected from the group consisting of heterocyclic groups with 3 to 14 members and hydrocarbon cyclic groups with 3 to 12 carbon atoms.

[0072] or

[0073] Groups with 50 or fewer carbon atoms represented by formula (5):

[0074]

Chemical Formula 3

[0075]

[0076] (in formula (5),

[0077] * indicates the bonding location.

[0078] R 11 It represents an alkylene group with 2 to 9 carbon atoms, an alkenyl group with 2 to 9 carbon atoms, or an alkynyl group with 2 to 9 carbon atoms, and R 11 It can be substituted by substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups with 3 to 12 carbon atoms.

[0079] R 12 and R 13 R represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, respectively. 12 At least one ethylene or at least one trimethylene group can be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, R 13 At least one ethylene or at least one trimethylene group can be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R 12 and R 13They can be independently substituted by substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon cyclic groups with 3 to 12 carbon atoms, and

[0080] X 4 (This represents an oxygen atom, NH4+, or a sulfur atom.)

[0081]

[10] According to the method described in [9] above, wherein the ionic lipid comprises a compound represented by formula (1).

[0082]

[11] According to any one of [1] to

[10] above, wherein the ligand in the lipid bound to the ligand is a ligand that targets brain cells or a ligand that targets T cells.

[0083]

[12] A method for introducing nucleic acid into cells, characterized in that it includes the following steps: contacting the cells in vitro with ligand-modified lipid nanoparticles encapsulated with nucleic acid obtained by any one of the methods described in [1] to

[11] .

[0084]

[13] A method for introducing nucleic acid into target cells in a living organism, characterized by comprising the following steps: administering ligand-modified lipid nanoparticles encapsulated with nucleic acid obtained by any one of the methods described in [1] to

[11] to the living organism.

[0085]

[14] A method for manufacturing a pharmaceutical composition, characterized in that it comprises any one of the methods described in [1] to

[11] above.

[0086] [Invention Effects]

[0087] The method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids according to the present invention can encapsulate nucleic acids in lipid nanoparticles through steps different from those in the prior art. Detailed Implementation

[0088] The following describes embodiments of the present invention, but the invention is not limited thereto. Furthermore, except where explicitly stated that they are not combinable, the descriptions in this specification can be combined with each other.

[0089] <Method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids>

[0090] This invention provides a method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids (hereinafter sometimes referred to as "target lipid nanoparticles") (hereinafter sometimes referred to as "the manufacturing method of this invention"), comprising:

[0091] Step a) involves mixing an alcoholic solution containing ionic lipids, sterols, and PEG lipids (hereinafter sometimes referred to as "lipid solution") with an acidic buffer solution with a pH of 1–6.5 (hereinafter sometimes referred to as "acidic buffer solution") to obtain a suspension of lipid nanoparticles without nucleic acids; and

[0092] Step b) mixing lipid nanoparticles without nucleic acid with a nucleic acid solution (preferably mixing a suspension of lipid nanoparticles without nucleic acid with a nucleic acid solution) to obtain a suspension of lipid nanoparticles encapsulated with nucleic acid; and step c) mixing the suspension of lipid nanoparticles encapsulated with nucleic acid with lipids bound to ligands to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

[0093] Or include:

[0094] Step c') involves mixing nucleic acid-free lipid nanoparticles, water, and lipids bound with ligands (preferably, mixing a suspension of nucleic acid-free lipid nanoparticles with lipids bound with ligands) to obtain a suspension of nucleic acid-free ligand-modified lipid nanoparticles; and step b') involves mixing nucleic acid-free ligand-modified lipid nanoparticles with a nucleic acid solution (preferably, mixing a suspension of nucleic acid-free ligand-modified lipid nanoparticles with a nucleic acid solution) to obtain a suspension of nucleic acid-encapsulated ligand-modified lipid nanoparticles.

[0095] Or include:

[0096] Step d) involves mixing lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands (preferably, mixing a suspension of lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands) to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

[0097] The manufacturing method of the present invention includes "step a), step b), and step c), "step a), step c'), and step b'"), or "step a) and step d"). Other steps (e.g., exchange of dispersion media) may be performed between these steps as needed. The manufacturing method of the present invention preferably includes "step a), step b), and step c") or "step a), step c'), and step b'"), and more preferably includes "step a), step b), and step c").

[0098] In this specification, "lipid nanoparticles" refers to particles with a membrane structure in which the hydrophilic groups of amphiphilic lipids are arranged facing the aqueous phase side of the interface. "Amphiphilic lipids" refers to lipids that have both a hydrophilic group exhibiting hydrophilicity and a hydrophobic group exhibiting hydrophobicity. Examples of amphiphilic lipids include ionic lipids, phospholipids, and PEG lipids.

[0099] The target lipid nanoparticles obtained by the manufacturing method of the present invention contain ionic lipids, sterols, and PEG lipids as membrane components, and may further contain phospholipids. The particle size of the target lipid nanoparticles is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Particle size can be measured, for example, using a particle size distribution measuring device such as a Zetasizer Nano (Malvern). In this specification, "particle size" refers to the average particle size (Zeta average) measured by dynamic light scattering.

[0100] In this specification, "total lipids" refers to the total amount of lipids. Examples of lipids include, for example, ionic lipids, sterols, PEG lipids, and phospholipids.

[0101] In this specification, "ligand-modified lipid nanoparticles without nucleic acid" refers to (ligand-modified) lipid nanoparticles with nucleic acid content below the detection limit.

[0102] In this specification, "(ligand-modified) lipid nanoparticles encapsulated with nucleic acids" refers to (ligand-modified) lipid nanoparticles containing nucleic acids encapsulated inside (ligand-modified) lipid nanoparticles.

[0103] In this specification, "ligand" refers to a substance that has a specific affinity for molecules expressed on the surface of a target cell (e.g., a cell that exerts a therapeutic effect on a disease by introducing a drug (preferably a nucleic acid) encapsulated in the lipid nanoparticles) into the cell.

[0104] In this specification, "ligand-modified lipid nanoparticles" refers to lipid nanoparticles with ligands, wherein the ligands are presented on the surface of the lipid nanoparticles to interact with molecules with specific affinity expressed on target cells and be delivered to the target cells.

[0105] <Process a)>

[0106] In step a), a suspension of lipid nanoparticles without nucleic acids is obtained by mixing a lipid solution with an acidic buffer solution. The acidic buffer solution used in step a) contains water as a solvent, and the suspension of lipid nanoparticles without nucleic acids obtained in step a) contains water as a dispersion medium.

[0107] Alcohols used as lipid solutions include, for example, ethanol and tert-butanol. Only one type of alcohol may be used, or two or more may be used in combination. Ethanol is preferred.

[0108] In step a), the mixing of the lipid solution and the acidic buffer solution is preferably performed using a device containing a microfluidic path or a vortex mixer, and more preferably using a device containing a microfluidic path. Examples of devices containing a microfluidic path include "NanoAssemblr Ignite" (manufactured by Precision NanoSystems) and the nanoparticle manufacturing device "iLiNP" (manufactured by Lilac Pharma Co., Ltd.).

[0109] When mixing the lipid solution with the acidic buffer solution in step a) using a device incorporating a microfluidic path, the flow rates of the lipid solution and the acidic buffer solution, as well as their flow rate ratio, are not particularly limited as long as they enable the formation of lipid nanoparticles. They can be appropriately selected based on the microfluidic path used and the performance of the device. The flow rates of the lipid solution and the acidic buffer solution are preferably 100 to 200,000 μL / min, more preferably 1,000 to 200,000 μL / min. The flow rate ratio of the lipid solution to the acidic buffer solution (lipid solution flow rate / acidic buffer flow rate) is preferably 1 / 10 to 1 / 1, more preferably 1 / 8 to 1 / 2. Furthermore, the "flow rate ratio of the lipid solution to the acidic buffer solution (lipid solution flow rate / acidic buffer flow rate)" is equal to the "volume ratio of the lipid solution to the acidic buffer solution (lipid solution volume / acidic buffer volume)".

[0110] The temperature at which the lipid solution is mixed with the acidic buffer solution in step a) is not particularly limited as long as it is a temperature at which lipid nanoparticles can be formed. From the viewpoint that it is easy to form lipid nanoparticles, it is preferably 4~50°C, and more preferably 20~30°C.

[0111] The alcohol solution (i.e., lipid solution) in step a) contains ionic lipids.

[0112] In one embodiment of the present invention, ionic lipids

[0113] Preferably, it comprises at least one compound selected from the group consisting of a compound represented by the following formula (1) (sometimes referred to simply as "ionic lipid (1)" in this specification), [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate.

[0114] More preferably, it contains ionic lipids (1).

[0115] In another embodiment of the present invention, ionic lipids

[0116] Preferably, it is selected from at least one of the following groups: ionic lipid (1), [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate.

[0117] More preferably, ionic lipids (1), [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate,

[0118] Further preferred are ionic lipids (1).

[0119] Ionic lipids (1) can be used in combination with one type or two or more types.

[0120] [Chemical Formula 4]

[0121]

[0122] (In formula (1),

[0123] R 1a and R 1b Each of the alkylene groups having 1 to 6 carbon atoms can be independently represented.

[0124] X a and X b Each of these can be used independently to represent a noncyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0125] R 2a and R 2b Each can be used independently to represent an alkylene group or an oxadiene group having 8 or fewer carbon atoms.

[0126] Y a and Y b Each can be used independently to represent an ester bond, amide bond, carbamate bond, ether bond, or urea bond.

[0127] Z a and Z bEach of these groups independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms.

[0128] n a and n b Each can be independently 0 or 1.

[0129] R 3a and R 3b Represented independently:

[0130] Derived from residues of fat-soluble vitamins with hydroxyl groups reacting with succinic anhydride or glutaric anhydride.

[0131] Residues derived from the reaction products of sterol derivatives with hydroxyl groups and succinic anhydride or glutaric anhydride.

[0132] Aliphatic hydrocarbon groups with 1 to 40 carbon atoms,

[0133] Alkyl groups having a cyclopropane ring with 3 to 40 carbon atoms,

[0134] The group represented by formula (3):

[0135] R 9 -O-CO-(CH2) a -* (3)

[0136] (In formula (3),

[0137] * indicates the bonding location.

[0138] R 9 This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms.

[0139] 'a' represents an integer from 2 to 10.

[0140] Groups with 50 or fewer carbon atoms represented by formula (4):

[0141] [Chemical Formula 5]

[0142]

[0143] (In formula (4),

[0144] * indicates the bonding location, and

[0145] R 10 It represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon cyclic group having 3 to 12 carbon atoms, and R 10 It can be substituted by substituents selected from the group consisting of heterocyclic groups with 3 to 14 members and hydrocarbon cyclic groups with 3 to 12 carbon atoms.

[0146] or

[0147] Groups with 50 or fewer carbon atoms represented by formula (5):

[0148]

Chemical Formula 6

[0149]

[0150] (in formula (5),

[0151] * indicates the bonding location.

[0152] R 11 It represents an alkylene group with 2 to 9 carbon atoms, an alkenyl group with 2 to 9 carbon atoms, or an alkynyl group with 2 to 9 carbon atoms, and R 11 It can be substituted by substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups with 3 to 12 carbon atoms.

[0153] R 12 and R 13 R represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, respectively. 12 At least one ethylene or at least one trimethylene group can be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, R 13 At least one ethylene or at least one trimethylene group can be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R 12 and R 13 They can be independently substituted by substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon cyclic groups with 3 to 12 carbon atoms, and

[0154] X 4 This represents an oxygen atom, NH (nitrated oxygen), or a sulfur atom.

[0155] The definitions of each symbol in equation (1) will be explained in turn below.

[0156] R 1a and R 1b Each alkylene group, having 1 to 6 carbon atoms, can be linear or branched, preferably linear. The number of carbon atoms in this alkylene group is preferably 1 to 4, 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. 1a and R 1b Preferably, each of the following is independently methylene, ethylene, trimethylene, isopropylene, or tetramethylene, with ethylene being the most preferred.

[0157] R 1a With R 1b They can be the same or different, but R is preferred. 1a With R 1b They are the same group.

[0158] X a and X b Each of the following can be independently represented as 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, preferably a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0159] The alkyl group with 1 to 6 carbon atoms and 1 tertiary amino group in the non-cyclic alkyl tertiary amino group can be straight-chain, branched, or cyclic. The number of carbon atoms in this alkyl group is preferably 1 to 3. Specific examples of alkyl groups with 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl, with methyl, ethyl, propyl, or isopropyl being preferred, and methyl being the most preferred.

[0160] The preferred specific structure of a noncyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is given by X. 1 In addition, X 1 The two ends of the following formula represent the bonding positions, not carbon atoms.

[0161] [Chemical Formula 7]

[0162]

[0163] X 1 R 5 The alkyl group having 1 to 6 carbon atoms can be linear, branched, or cyclic. The preferred carbon number of the alkyl group is 1 to 3. Specific examples of alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl, with methyl, ethyl, propyl, or isopropyl being preferred, and methyl being the most preferred.

[0164] The cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups preferably has 4 to 5 carbon atoms. Specifically, cyclic alkylene tertiary amino groups having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups are aziridinidine dieryl, aziridine dieryl, pyrrolidine dieryl, piperidine dieryl, imidazolidinidine dieryl, and piperazine dieryl, preferably pyrrolidine dieryl, piperidine dieryl, and piperazine dieryl, with piperidine dieryl being the most preferred. Furthermore, in this specification, "compound name + dieryl (e.g., aziridinidine dieryl)" refers to a divalent group having a structure obtained by removing two hydrogen atoms from the compound (e.g., aziridinium).

[0165] The preferred specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group is given by X. 2 In addition, X 2 The two ends of the following formula represent the bonding positions, not carbon atoms.

[0166] [Chemical Formula 8]

[0167]

[0168] X 2 p is 1 or 2. When p is 1, X 2 It is a pyrrolidine dimethyl group, and when p is 2, X 2 It is piperidine dimethyl. Preferably, p is 2.

[0169] The preferred specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amine groups is given by X. 3 In addition, X 3 The two ends of the following formula represent the bonding positions, not carbon atoms.

[0170] [Chemical Formula 9]

[0171]

[0172] X 3 w is 1 or 2. When w is 1, X 3 It is an imidazolidinediyl group, and when w is 2, X 3 It is piperazine dimethyl.

[0173] X a With X b They can be the same or different, but X is preferred. a To be with X b Same group.

[0174] R 2a and R 2b Each alkylene group having 8 or fewer carbon atoms or an oxadiene group is independently represented, preferably each alkylene group having 8 or fewer carbon atoms.

[0175] The alkylene group having 8 or fewer carbon atoms can be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 6 or fewer, and most preferably 4 or fewer. Examples of alkylene groups having 8 or fewer carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, and octamethylene, with methylene, ethylene, trimethylene, and tetramethylene being preferred, and ethylene being most preferred.

[0176] In this specification, "oxadiene with 8 or fewer carbon atoms" refers to an alkylene group (alkylene-O-alkylene, in other words, "alkyleneoxyalkylene") formed by an ether bond, which is a group in which the sum of the carbon atoms of the two alkylene groups is 8 or fewer. The two alkylene groups may be the same or different, but are preferably the same. Specific examples of oxadiene with 8 or fewer carbon atoms include oxadimethylene, oxadiethylene, oxadi(trimethylene) (i.e., trimethyleneoxytrimethylene), and oxadi(tetramethylene) (i.e., tetramethyleneoxytetramethylene). Oxadiethylene, oxadiethylene, and oxadi(trimethylene) are preferred, with oxadiethylene being the most preferred.

[0177] R 2a With R 2b They can be the same or different, but R is preferred. 2a To be with R 2b Same group.

[0178] Y a and Y b Each bond is independently composed of an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, preferably independently composed of an ester bond, an amide bond, or a carbamate bond, more preferably independently composed of an ester bond or an amide bond, and most preferably composed of ester bonds. a and Y b The orientation of the Y bond is not restricted, but... a and Y b When the bond is an ester bond, it preferably presents as -Z. a -CO-OR 2a -and-Z b -CO-OR 2b - structure.

[0179] Y a With Y b They can be the same or different, but Y is preferred. a To be with Y b Same group.

[0180] Z a and Z bEach of these terms independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms. The aromatic compound preferably contains 6 to 12 carbon atoms, and most preferably 6 to 7. Furthermore, the aromatic compound preferably contains only one aromatic ring.

[0181] The types of aromatic rings included in aromatic compounds with 3 to 16 carbon atoms include, for aromatic hydrocarbon rings, benzene rings, naphthyl rings, and anthracene rings; for aromatic heterocycles, imidazole rings, pyrazole rings, oxazole rings, isoxazole rings, thiazole rings, isothiazole rings, triazine rings, pyrrole rings, furanothiophene rings, pyrimidine rings, pyridazine rings, pyrazine rings, pyridine rings, purine rings, pteridine rings, benzimidazole rings, indole rings, benzofuran rings, quinazoline rings, phthalazine rings, quinoline rings, isoquinoline rings, coumarin rings, chromone rings, benzodiazepine rings, phenoxazine rings, phenothiazine rings, and acridine rings, etc., with benzene rings, naphthyl rings, and anthracene rings being the most preferred.

[0182] Aromatic rings can have substituents. Examples of substituents include acyl groups with 2-4 carbon atoms, alkoxycarbonyl groups with 2-4 carbon atoms, carbamoyl groups with 2-4 carbon atoms, acyloxy groups with 2-4 carbon atoms, amide groups with 2-4 carbon atoms, alkoxycarbonylamino groups with 2-4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylthio groups with 1-4 carbon atoms, alkylsulfonyl groups with 1-4 carbon atoms, arylsulfonyl groups with 6-10 carbon atoms, nitro groups, trifluoromethyl groups, cyano groups, and alkyl groups with 1-4 carbon atoms. Ureidyl groups with 1 to 4 carbon atoms, alkoxy groups with 1 to 4 carbon atoms, aryl groups with 6 to 10 carbon atoms, and aryloxy groups with 6 to 10 carbon atoms are among the preferred examples. Examples include acetyl groups, methoxycarbonyl groups, methylcarbamoyl groups, acetoxy groups, acetamido groups, methoxycarbonylamino groups, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methylthio groups, benzenesulfonyl groups, nitro groups, trifluoromethyl groups, cyano groups, methyl groups, ethyl groups, propyl groups, isopropyl groups, tert-butyl groups, ureidyl groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups, phenyl groups, and phenoxy groups.

[0183] As Z a and Z b The preferred specific structures can be listed as Z 1 In addition, Z 1 The two ends of the following formula represent the bonding positions, not carbon atoms.

[0184]

Chemical Formula 10

[0185]

[0186] In the formula, s represents an integer from 0 to 3, t represents an integer from 0 to 3, u represents an integer from 0 to 4, and u represents R. 4 Substituents are represented independently.

[0187] Z 1 The s is preferably an integer from 0 to 1, and more preferably 0.

[0188] Z 1 The t is preferably an integer from 0 to 2, and more preferably 1.

[0189] Z 1 u is preferably an integer from 0 to 2, and more preferably an integer from 0 to 1.

[0190] Z 1 R 4 These are substituents on the aromatic ring (benzene ring) of aromatic compounds with 3 to 16 carbon atoms, which do not hinder the reaction in the synthesis of the ionic lipid. Examples of such substituents include acyl groups with 2 to 4 carbon atoms, alkoxycarbonyl groups with 2 to 4 carbon atoms, carbamoyl groups with 2 to 4 carbon atoms, acyloxy groups with 2 to 4 carbon atoms, amide groups with 2 to 4 carbon atoms, alkoxycarbonylamino groups with 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylthio groups with 1 to 4 carbon atoms, alkylsulfonyl groups with 1 to 4 carbon atoms, arylsulfonyl groups with 6 to 10 carbon atoms, nitro groups, trifluoromethyl groups, cyano groups, alkyl groups with 1 to 4 carbon atoms, and alkyl groups with 1 to 4 carbon atoms. Ureidyl groups with ~4 carbon atoms, alkoxy groups with 1 to 4 carbon atoms, aryl groups with 6 to 10 carbon atoms, and aryloxy groups with 6 to 10 carbon atoms are preferred examples. Examples include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylthio, benzenesulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, ureidyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, and phenoxy groups. Multiple R groups are present. 4 At that time, each R 4 They can be the same or different.

[0191] Z a With Z b They can be the same or different, but Z is preferred. a To be with Z b Same group.

[0192] n a and n b Each can be 0 or 1 independently.

[0193] n a With n b They can be the same or different, but n is preferred. a With nb same.

[0194] R 3a and R 3b Each of the following is independently a residue derived from the reaction of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from the reaction 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 having 3 to 40 carbon atoms, a group represented by the formula (3), a group represented by the formula (4) having 50 or fewer carbon atoms, or a group represented by the formula (5) having 50 or fewer carbon atoms.

[0195] In one embodiment of the present invention, R 3a and R 3b

[0196] Preferably, the residues are independently derived from the reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, residues derived from the reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, aliphatic hydrocarbon groups having 1 to 40 carbon atoms, alkyl groups having a cyclopropane ring having 3 to 40 carbon atoms, or groups represented by formula (3).

[0197] More preferably, the residues are independently derived from the reactants of fat-soluble vitamins with hydroxyl groups and succinic anhydride or glutaric anhydride, or aliphatic hydrocarbon groups having 12 to 22 carbon atoms.

[0198] Further preferred groups are aliphatic hydrocarbon groups with 12 to 22 carbon atoms each.

[0199] In another embodiment of the present invention, R 3a and R 3b Preferably, the residues are individually derived from the reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, aliphatic hydrocarbon groups with 12 to 22 carbon atoms, groups with 50 or fewer carbon atoms represented by formula (4), or groups with 50 or fewer carbon atoms represented by formula (5).

[0200] In another embodiment of the present invention, it is more preferably:

[0201] R 3a and R 3b Each is independently a residue derived from the reaction product of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, or

[0202] R 3a and R 3b Each is independently an aliphatic hydrocarbon group with 12 to 22 carbon atoms, or

[0203] R 3aand R 3b Each is independently a group with 50 or fewer carbon atoms represented by the formula (4), or

[0204] R 3a and R 3b One of them is an aliphatic hydrocarbon group with 12 to 22 carbon atoms, and the other is a group with 50 or fewer carbon atoms represented by the formula (4), or

[0205] R 3a and R 3b One of them is an aliphatic hydrocarbon group with 12 to 22 carbon atoms, and the other is a group with 50 or less carbon atoms represented by the formula (5).

[0206] In another embodiment of the present invention, it is further preferred that:

[0207] R 3a and R 3b Each is independently a residue derived from the reaction product of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, or

[0208] R 3a and R 3b Each is independently an aliphatic hydrocarbon group with 12 to 22 carbon atoms, or

[0209] R 3a and R 3b One of them is an aliphatic hydrocarbon group with 12 to 22 carbon atoms, and the other is a group with 50 or fewer carbon atoms represented by the formula (4), or

[0210] R 3a and R 3b One of them is an aliphatic hydrocarbon group with 12 to 22 carbon atoms, and the other is a group with 50 or less carbon atoms represented by the formula (5).

[0211] In another embodiment of the present invention, it is particularly preferred that:

[0212] R 3a and R 3b Each is independently a residue derived from the reaction product of a fat-soluble vitamin with a hydroxyl group and succinic anhydride or glutaric anhydride, or

[0213] R 3a and R 3b Each is an aliphatic hydrocarbon group with 12 to 22 carbon atoms.

[0214] In another embodiment of the present invention, it is preferred that:

[0215] R 3a and R 3bEach is independently an aliphatic hydrocarbon group with 12 to 22 carbon atoms, or

[0216] R 3a and R 3b Each is independently a group with 50 or fewer carbon atoms represented by the formula (4), or

[0217] R 3a and R 3b One of them is an aliphatic hydrocarbon group with 12 to 22 carbon atoms, and the other is a group with 50 or less carbon atoms represented by the formula (4).

[0218] In another embodiment of the present invention, R 3a and R 3b More preferably, each of the following is an aliphatic hydrocarbon group, which is independently composed of 12 to 22 carbon atoms.

[0219] "Residues derived from the reaction of fat-soluble vitamins with hydroxyl groups with succinic anhydride or glutaric anhydride" refers to groups in fat-soluble vitamins whose hydroxyl groups are replaced by *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-. * indicates the bonding position with the fat-soluble vitamin.

[0220] "Residues derived from the reaction of hydroxyl-containing sterol derivatives with succinic anhydride or glutaric anhydride" refers to groups in hydroxyl-containing sterol derivatives where the hydroxyl group is replaced by *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2-. * indicates the bonding position with the sterol derivative.

[0221] Examples of fat-soluble vitamins containing hydroxyl groups include retinol, ergosterol, 7-dehydrocholesterol, calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. Tocopherol is preferred among fat-soluble vitamins containing hydroxyl groups.

[0222] Examples of steroidal derivatives containing hydroxyl groups include cholesterol, cholesterol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, with cholesterol or cholesterol being preferred.

[0223] The aliphatic hydrocarbon group with 1 to 40 carbon atoms can be linear or branched. The aliphatic hydrocarbon group can be saturated or unsaturated. When it is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds it contains is typically 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, but carbon-carbon double bonds are preferred. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes alkyl, alkenyl, and alkynyl groups, but preferably includes alkyl or alkenyl groups. Examples of aliphatic hydrocarbon groups with 1 to 40 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, triadecyl, tetradecyl, ... Alkenyl, pentadecyl alkenyl, hexadecyl alkenyl, heptadecanyl alkenyl, octadecyl alkenyl, nonadecyl alkenyl, icosyl alkenyl, icosyl alkenyl, dodecanyl alkenyl, tridecyl alkenyl, tetradecyl alkenyl, pentadecyl alkenyl, hexadecyl alkenyl, heptadecanyl, octadecyl alkenyl, nonadecyl alkenyl, icosyl alkenyl, icosyl alkenyl, octadecyl trienyl, icosyl trienyl, icosyl tetraenyl, icosyl pentaenyl, icosyl hexaenyl, isostearyl, 1-hexylheptyl, 1-hexyl nonyl, 1-octyl nonyl, 1-octyl undecyl, 1-decyl undecyl, etc. The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably tridecyl, pentadecyl, heptadecanyl, nonadecanyl, heptadecanyl-enyl, heptadecanyl-dienyl, or 1-hexylnonyl, and particularly preferably tridecyl, heptadecanyl, heptadecanyl-enyl, or heptadecanyl-dienyl.

[0224] In one embodiment of the present invention, R 3a and R 3b The aliphatic hydrocarbon group with 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) is derived from fatty acids. In this case, the carbonyl carbon derived from the fatty acid is contained in -CO-O- in formula (1). As specific examples of aliphatic hydrocarbon groups, it is heptadecanediol when linoleic acid is used as the fatty acid and heptadecanediol when oleic acid is used as the fatty acid.

[0225] R 3a and R 3bThe phrase "alkyl group having 3 to 40 carbon atoms with a cyclopropane ring" refers to an alkyl group having 3 to 40 carbon atoms in an alkyl chain containing at least one cyclopropane ring. The 3 to 40 carbon atoms in the alkyl group do not include the carbon atoms of the cyclopropane ring. Preferably, the alkyl group has one cyclopropane ring. 3a and R 3b The alkyl group having 3 to 40 carbon atoms in the cyclopropane ring is preferably a group represented by formula (6):

[0226]

Chemical Formula 11

[0227]

[0228] (In equation (6), * represents the bonding position, b and c represent integers independently, and the sum of b and c is 2~39.)

[0229] Preferably, b is an integer from 1 to 20, and c is an integer from 1 to 19. More preferably, b is an integer from 2 to 18, even more preferably, an integer from 3 to 17, and even more preferably, an integer from 4 to 12. More preferably, c is an integer from 3 to 15, even more preferably, an integer from 3 to 11, and even more preferably, an integer from 3 to 9. As a group represented by formula (6), for example, 7-(2-octylcyclopropyl)heptyl, etc., can be listed.

[0230] In equation (3), * represents the bonding position as described above, rather than a carbon atom.

[0231] In equation (3), R 9 Aliphatic hydrocarbon groups with 2 to 20 carbon atoms can be either straight-chain or branched.

[0232] The aforementioned aliphatic hydrocarbon groups can be saturated or unsaturated. When the aliphatic hydrocarbon group is unsaturated, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is typically 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, but carbon-carbon double bonds are preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, further preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes alkyl, alkenyl, alkynyl, etc., but preferably includes alkyl or alkenyl groups, more preferably alkyl. Examples of aliphatic hydrocarbon groups with 2 to 20 carbon atoms include ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecanyl, and octadecenyl. The aliphatic hydrocarbon groups having 2 to 20 carbon atoms are preferably tridecyl, pentadecyl, hexadecyl, docosadenyl, dodecadienyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, icosadecanyl, icosadecanyl-trienyl, icosadecanyl-tetraenyl, icosadecanyl-pentaenoyl, isostearyl, 1-hexylheptyl, 1-ethylnonyl, 1-butylnonyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 3-octylundecyl, etc. The preferred aliphatic hydrocarbon groups are tridecyl, pentadecyl, heptadecanyl, nonadecanyl, heptadecanyl, heptadecanyl-dienyl, and 1-hexylnonyl, with particular preference for tridecyl, heptadecanyl, heptadecanyl, and heptadecanyl-dienyl.

[0233] In formula (3), a is preferably an integer from 3 to 9, more preferably an integer from 3 to 7, even more preferably an integer from 5 to 7, and most preferably 5 or 7.

[0234] In equation (4), * represents the bonding position as described above, rather than a carbon atom.

[0235] In equation (4), R 10 It represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon cyclic group having 3 to 12 carbon atoms, and R 10 It can be substituted by substituents selected from the group consisting of heterocyclic groups with 3 to 14 members and hydrocarbon cyclic groups with 3 to 12 carbon atoms.

[0236] In this instruction manual, “R” 10"Substituents that can be selected from the group consisting of heterocyclic groups of 3 to 14 members and hydrocarbon cyclic groups of 3 to 12 carbon atoms" (hereinafter referred to as "the Statement") means: R 10 The alkyl, alkenyl, alkynyl, or cycloalkyl group can each be independently substituted by a substituent selected from the group consisting of a 3- to 14-membered heterocyclic group and a cycloalkyl group having 3 to 12 carbon atoms. Other expressions similar to those described herein also mean the same as those described herein. Furthermore, in this specification, unless otherwise specified, "alkyl," "alkenyl," "alkynyl," "alkylene," "alkenidyl," and "alkynidyl" refer to unsubstituted groups.

[0237] In this specification, alkyl groups can be any type, whether straight-chain or branched. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, triadecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl, tridecyl.

[0238] In this specification, the alkenyl group can be any type, either straight-chain or branched. Furthermore, the alkenyl group may contain only one olefinic carbon-carbon double bond or more than two. Examples of alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecanyl, octadecenyl, nonadecanyl, icosyl, icosyl, icosyl-2-carbon, icosyl-3-carbon, icosyl-2 ...

[0239] In this specification, the alkynyl group can be any type, either straight-chain or branched. Furthermore, the number of carbon-carbon triple bonds in the alkynyl group can be either one or two or more. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, hepynyl, octyynyl, nonynyl, decyynyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptynyl, octadecynyl, nonadecynyl, icosyl, icosyl, icosyl, icosyl, icosyl, tridecynyl, icosyl, icosyl, pentadecynyl, hexadecynyl, heptynyl, octadecynyl, nonadecynyl, icosyl, tridecynyl, tridecynyl, and trididecynyl.

[0240] In this specification, "hydrocycloalkyl group with 3 to 12 carbon atoms" refers to a cyclic group, which is a group whose ring consists of 3 to 12 carbon atoms. Examples of hydrocarbon cycloalkyl groups with 3 to 12 carbon atoms include cycloalkyl, phenyl, naphthyl, and adamantyl groups with 3 to 8 carbon atoms. Examples of cycloalkyl groups with 3 to 8 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Hydrocycloalkyl groups with 3 to 12 carbon atoms are preferably non-aromatic hydrocarbon cycloalkyl groups with 3 to 12 carbon atoms, more preferably cycloalkyl or adamantyl groups with 3 to 8 carbon atoms, and even more preferably cyclohexyl or adamantyl groups.

[0241] In this specification, "3- to 14-membered heterocyclic group" refers to a heterocyclic group with 3 to 14 ring atoms. Examples of 3- to 14-membered heterocyclic groups include 5- to 14-membered aromatic heterocyclic groups and 3- to 14-membered non-aromatic heterocyclic groups. 3- to 14-membered heterocyclic groups are preferably 3- to 14-membered non-aromatic heterocyclic groups.

[0242] In this specification, the following groups can be listed as 5- to 14-membered aromatic heterocyclic groups:

[0243] (i) 5- to 6-membered monocyclic aromatic heterocyclic groups such as thiophene, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl, etc.

[0244] (ii) benzothiophene, benzofuran, benzoimidazolyl, benzooxazolyl, benzoisooxazolyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridyl, thiophene-pyridyl, furan-pyridyl, pyrrolopyridyl, pyrazololopyridyl, oxazololopyridyl, thiazopyridyl, imidazopyrazinyl, imidazopyrimidyl, thiophene-pyrimidyl, furan-pyrimidyl, pyrrolopyrimidyl, pyrazololopyrimidyl It includes 8- to 14-membered fused polycyclic aromatic heterocyclic groups such as oxazolopyrimidinyl, thiazopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thiopheneyl, phenoxthianinyl, indolyl, isoindolyl, 1H-inzolyl, purineyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthidyl, quinoxolinyl, quinoxolinyl, cyclophosphinyl, carbazoyl, β-carolinyl, phenanthidyl, acridineyl, phenazinyl, phenothiazinyl, and pheoxazinyl.

[0245] As a non-aromatic heterocyclic group with 3 to 14 members in this specification, for example, the following groups can be listed:

[0246] (i) Aziridinyl, ethylene oxide, thiocyclopropane, azacyclobutane, oxacyclobutane, thiocyclobutane, tetrahydrothiopheneyl, tetrahydrofuranyl, pyrrolinyl, pyrrolylyl, imidazolinyl, imidazolinyl, oxazolinyl, oxazolinyl, pyrazolinyl, pyrazolinyl, thiazolinyl, thiazolinyl, tetrahydroisothiazolinyl, dithiopentaneyl (e.g., 1,2-dithiopentane-3-yl), tetrahydrooxazolinyl Azolyl, tetrahydroisoxazolyl, piperidinyl, piperazine, tetrahydropyridinyl, dihydropyridinyl, dihydrothiaranyl, tetrahydropyrimidinyl, tetrahydropyridinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, thiomorpholinyl, azirheptanyl, diazaheptanyl, azirheptantrienyl, oxaheptanyl, azirheptanyl, diazaheptanyl, diazaheptanyl, etc., are 3- to 8-membered monocyclic non-aromatic heterocyclic groups.

[0247] (ii) Dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronaphtho[2,3-b]thiophenyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 4H-quinazinyl, indololinyl, isoindololinyl, tetrahydrothienzo[2,3-c]pyridyl, tetrahydrobenzozazolyl, tetrahydroquinoxalinyl, tetrahydrophenanthridine, hexahydrophenothiazinyl, hexahydrophenothiazinyl, tetrahydrophthalazinyl, tetrahydronaphridinyl, tetrahydroquinazolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbazolyl, tetrahydroacridinyl, tetrahydrophenothiazinyl, tetrahydrothiazolyl, octahydroisoquinolinyl and other 9- to 14-membered fused polycyclic nonaromatic heterocyclic groups.

[0248] In one embodiment of the present invention, R 10Preferably, the alkyl group has 1 to 20 carbon atoms, the alkenyl group has 2 to 20 carbon atoms, the alkynyl group has 2 to 20 carbon atoms, or the hydrocarbon cycloyl group has 3 to 12 carbon atoms. The alkyl group can be replaced by the hydrocarbon cycloyl group with 3 to 12 carbon atoms.

[0249] In one embodiment of the present invention, R 10 More preferably, it is an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a cyclohexyl group, wherein the alkyl group may be replaced by an adamantyl group.

[0250] In one embodiment of the present invention, R 10 Further preferred are alkyl groups having 1 to 20 carbon atoms, particularly preferred are alkyl groups having 1 to 10 carbon atoms, and most preferred are heptyl groups.

[0251] In another embodiment of the present invention, R 10 Preferably, the alkyl group has 1 to 20 carbon atoms, the alkenyl group has 2 to 20 carbon atoms, the alkynyl group has 2 to 20 carbon atoms, or the hydrocarbon cyclic group has 3 to 12 carbon atoms. The alkyl group can be replaced by a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentyl).

[0252] In another embodiment of the present invention, R 10 More preferably, it is an alkyl group with 1 to 20 carbon atoms or an alkynyl group with 2 to 20 carbon atoms, wherein the alkyl group may be substituted with a non-aromatic heterocyclic group of 3 to 14 members (e.g., dithiopentyl).

[0253] In one embodiment of the present invention, R 10 More preferably, it is an alkyl group with 1 to 10 carbon atoms or an alkynyl group with 1 to 10 carbon atoms, wherein the alkyl group may be replaced by a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentyl).

[0254] In equation (5), * represents the bonding position as described above, rather than a carbon atom.

[0255] In equation (5), R 11 It represents an alkylene group with 2 to 9 carbon atoms, an alkenyl group with 2 to 9 carbon atoms, or an alkynyl group with 2 to 9 carbon atoms, and R 11 It can be substituted by substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon cyclic groups with 3 to 12 carbon atoms.

[0256] In this specification, alkylene groups can be any type, whether straight-chain or branched. Examples of alkylene groups include methylene, ethylene, trimethylene (-(CH2)3-), propylene (-CH(CH3)CH2-, -CH2CH(CH3)-), tetramethylene (-(CH2)4-), butylene (-CH(C2H5)CH2-, -CH2CH(C2H5)-), pentamethylene (-(CH2)5-), hexamethylene (-(CH2)6-), heptamethylene (-(CH2)7-), octamethylene (-(CH2)8-), nonamethylene (-(CH2)9-), and decamethylene (-(CH2)... 10 - (In the above formulas, "-" indicates a single bond.)

[0257] In this specification, "alkendiyl" refers to a divalent group having a structure obtained by removing two hydrogen atoms from an alkene. In this specification, the alkendiyl group can be of any type, either linear or branched. Furthermore, in this specification, the number of alkene-type carbon-carbon double bonds in the alkene or alkendiyl group can be either only one or more. Examples of alkendiyl groups include ethylenediyl, propenediyl, butenediyl, pentenediyl, hexenediyl, heptenediyl, octenediyl, nonenediyl, and decenediyl. Furthermore, in this specification, "compound name + diyl (e.g., ethylenediyl)" refers to a divalent group having a structure obtained by removing two hydrogen atoms from the compound.

[0258] In this specification, "acetylenic diol" refers to a divalent group having a structure obtained by removing two hydrogen atoms from an alkyne. In this specification, the acetylenic diol can be of any type, either straight-chain or branched. Furthermore, in this specification, the number of carbon-carbon triple bonds in the alkyne or the acetylenic diol can be either only one or two or more. Examples of acetylenic diols include, for example, acetylenic diol, propylenic diol, butylenic diol, pentylenic diol, hexylenic diol, heptylenic diol, octylenic diol, nonylenic diol, and decylenic diol.

[0259] In equation (5), R 11 Preferably, it is an alkylene group with 2 to 9 carbon atoms, an alkenyl group with 2 to 9 carbon atoms, or an alkynyl group with 2 to 9 carbon atoms; more preferably, it is an alkylene group with 2 to 9 carbon atoms or an alkenyl group with 2 to 9 carbon atoms; even more preferably, it is an alkylene group with 2 to 9 carbon atoms; particularly preferably, it is an alkylene group with 2 or 3 carbon atoms; and most preferably, it is a trimethylene group.

[0260] In equation (5), R 12 and R 13 R represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, respectively. 12At least one ethylene or at least one trimethylene group can be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, R 13 At least one ethylene or at least one trimethylene group can be replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R 12 and R 13 They can be independently substituted by substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon cyclic groups with 3 to 12 carbon atoms.

[0261] R 12 and R 13 Preferably, each is an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, more preferably an alkyl group having 1 to 17 carbon atoms, even more preferably an alkyl group having 1 to 10 carbon atoms, and particularly preferably a hexyl group.

[0262] In equation (5), X 4 Represents an oxygen atom, NH, or a sulfur atom. X 4 Preferably, it contains oxygen atoms or NH, and more preferably oxygen atoms.

[0263] In equation (5), the optimal choice is R. 11 It is trimethylene, R 12 and R 13 All are hexadi, and X 4 It is an oxygen atom.

[0264] R 3a With R 3b They can be the same or different, but R is preferred. 3a To be with R 3b Same group.

[0265] In one embodiment of the present invention, R 1a With R 1b Same, X a With X b Same, R 2a With R 2b Same, Y a With Y b Same, Z a With Z b Same, R 3a With R 3b same.

[0266] As preferred examples of ionic lipids (1), the following ionic lipids can be listed.

[0267] [Ionic lipids (1-1a)]

[0268] An ionic lipid (1), wherein,

[0269] R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene);

[0270] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0271] R 2a and R 2b Each is independently an alkylene group having 8 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0272] Y a and Y b Each is an ester bond or an amide bond, independently;

[0273] Z a and Z b Each of these can be a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-) derived independently from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms.

[0274] n a and n b Each is independently 0 or 1; and

[0275] R 3a and R 3b Each of the following is an aliphatic hydrocarbon group with 12 to 22 carbon atoms (e.g., heptadecanyl, heptadecanediyl, 1-hexylnonyl) derived independently from the reactants of fat-soluble vitamins (e.g., tocopherol) with succinic anhydride or glutaric anhydride, and a group with 50 or fewer carbon atoms represented by the formula (4) (R in formula (4)). 10 It is an alkyl group with 1 to 20 carbon atoms or an alkynyl group with 2 to 20 carbon atoms, wherein the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentane). Alternatively, it may be a group with 50 or fewer carbon atoms represented by the formula (R in formula (5)). 11 R is an alkylene group having 2 to 9 carbon atoms. 12 and R 13 Each is independently an alkyl group having 1 to 17 carbon atoms, and X 4 (It is an oxygen atom or NH.)

[0276] [Ionic lipids (1-1b)]

[0277] An ionic lipid (1), wherein,

[0278] R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene);

[0279] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0280] R 2a and R 2b Each is independently an alkylene group having 8 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0281] Y a and Y b Each is an ester bond or an amide bond, independently;

[0282] Z a and Z b Each of these can be a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-) derived independently from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms.

[0283] n a and n b Each is independently 0 or 1; and

[0284] R 3a and R 3b Each of the following is an aliphatic hydrocarbon group with 12 to 22 carbon atoms (e.g., heptadecanyl, heptadecanediyl, 1-hexylnonyl) derived independently from the reactants of fat-soluble vitamins (e.g., tocopherol) with succinic anhydride or glutaric anhydride, and a group with 50 or fewer carbon atoms represented by the formula (4) (R in formula (4)). 10 It is an alkyl group with 1 to 20 carbon atoms, or a group with 50 or fewer carbon atoms represented by the formula (5) (R in formula (5)). 11 R is an alkylene group having 2 to 9 carbon atoms. 12 and R 13 Each is independently an alkyl group having 1 to 17 carbon atoms, and X 4 (It is an oxygen atom or NH.)

[0285] [Ionic lipids (1-1c)]

[0286] An ionic lipid (1), wherein,

[0287] R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene);

[0288] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0289] R 2a and R 2b Each is independently an alkylene group having 8 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0290] Y a and Y b Each is an ester bond or an amide bond, independently;

[0291] Z a and Z b Each of these can be a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-) derived independently from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms.

[0292] n a and n b Each is independently 0 or 1; and

[0293] R 3a and R 3b Each residue is independently derived from a reaction product of a fat-soluble vitamin with a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group with 12 to 22 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl).

[0294] [Ionic lipids (1-1d)]

[0295] An ionic lipid (1), wherein,

[0296] R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene);

[0297] X a and X bEach is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0298] R 2a and R 2b Each is independently an alkylene group having 8 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0299] Y a and Y b Each is an ester bond or an amide bond, independently;

[0300] Z a and Z b Each of these can be a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-) derived independently from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms.

[0301] n a and n b Each is independently 0 or 1; and

[0302] R 3a and R 3b Each is independently an aliphatic hydrocarbon group with 12 to 22 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl), or a group with 50 or fewer carbon atoms represented by formula (4) (R in formula (4)). 10 It is an alkyl group with 1 to 20 carbon atoms or an alkynyl group with 2 to 20 carbon atoms, wherein the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentane).

[0303] [Ionic lipids (1-2a)]

[0304] An ionic lipid (1), wherein,

[0305] R 1a and R 1b Each is an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene);

[0306] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 3 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 tertiary amino group.

[0307] R 2a and R 2bEach is independently an alkylene group having 6 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0308] Y a and Y b Each is an ester bond or an amide bond, independently;

[0309] Z a and Z b Each of these can be independently derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and possibly heteroatoms, and is a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-).

[0310] n a and n b Each is independently 0 or 1; and

[0311] R 3a and R 3b Each of the following is independently derived from the reactants of a fat-soluble vitamin (e.g., tocopherol) with a hydroxyl group and succinic anhydride: an aliphatic hydrocarbon group with 13 to 19 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl); a group with 50 or fewer carbon atoms represented by the formula (4) (R in formula (4)). 10 It is an alkyl group or an alkynyl group with 1 to 10 carbon atoms, wherein the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentane). Alternatively, it may be a group with 50 or fewer carbon atoms represented by formula (5) (R in formula (5)). 11 R is an alkylene group with 2 or 3 carbon atoms. 12 and R 13 Each is independently an alkyl group having 1 to 10 carbon atoms, and X 4 (It is an oxygen atom.)

[0312] [Ionic lipids (1-2b)]

[0313] An ionic lipid (1), wherein,

[0314] R 1a and R 1b Each is an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene);

[0315] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 3 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 tertiary amino group.

[0316] R 2aand R 2b Each is independently an alkylene group having 6 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0317] Y a and Y b Each is an ester bond or an amide bond, independently;

[0318] Z a and Z b Each of these can be independently derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and possibly heteroatoms, and is a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-).

[0319] n a and n b Each is independently 0 or 1; and

[0320] R 3a and R 3b Each of the following is independently derived from the reactants of a fat-soluble vitamin (e.g., tocopherol) with a hydroxyl group and succinic anhydride: an aliphatic hydrocarbon group with 13 to 19 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl); a group with 50 or fewer carbon atoms represented by the formula (4) (R in formula (4)). 10 It is an alkyl group with 1 to 10 carbon atoms, or a group with 50 or fewer carbon atoms represented by the formula (5) (R in formula (5)). 11 R is an alkylene group with 2 or 3 carbon atoms. 12 and R 13 Each is independently an alkyl group having 1 to 10 carbon atoms, and X 4 It's an oxygen atom.

[0321] [Ionic lipids (1-2c)]

[0322] An ionic lipid (1), wherein,

[0323] R 1a and R 1b Each is an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene);

[0324] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 3 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 tertiary amino group.

[0325] R 2a and R 2bEach is independently an alkylene group having 6 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0326] Y a and Y b Each is an ester bond or an amide bond, independently;

[0327] Z a and Z b Each of these can be independently derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and possibly heteroatoms, and is a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-).

[0328] n a and n b Each is independently 0 or 1; and

[0329] R 3a and R 3b Each of these residues is independently derived from the reaction product of a fat-soluble vitamin (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group with 13 to 19 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl).

[0330] [Ionic lipids (1-2 days)]

[0331] An ionic lipid (1), wherein,

[0332] R 1a and R 1b Each is an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene);

[0333] X a and X b Each is independently an acyclic alkyl tertiary amino group (e.g., -N(CH3)-) having 1 to 3 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group (e.g., piperidinium) having 2 to 5 carbon atoms and 1 tertiary amino group.

[0334] R 2a and R 2b Each is independently an alkylene group having 6 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0335] Y a and Y b Each is an ester bond or an amide bond, independently;

[0336] Z a and Z bEach of these can be independently derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and possibly heteroatoms, and is a divalent group (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-).

[0337] n a and n b Each is independently 0 or 1; and

[0338] R 3a and R 3b Each is independently an aliphatic hydrocarbon group with 13 to 19 carbon atoms (e.g., heptadecanyl, heptadecanidyl, 1-hexylnonyl), or a group with 50 or fewer carbon atoms represented by formula (4) (R in formula (4)). 10 It is an alkyl group or an alkynyl group having 1 to 10 carbon atoms, wherein the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentane).

[0339] [Ionic lipids (1-3a)]

[0340] An ionic lipid (1), wherein,

[0341] R 1a and R 1b Each is independently an alkylene group (i.e., methylene or ethylene) having 1 to 2 carbon atoms.

[0342] X a and X b X, independently 1 :

[0343]

Chemical Formula 12

[0344]

[0345] (where R) 5 It is an alkyl group with 1 to 3 carbon atoms (e.g., methyl).

[0346] Or X 2 :

[0347]

Chemical Formula 13

[0348]

[0349] (In the formula, p is 1 or 2.)

[0350] R 2a and R 2b Each is independently an alkylene group having 4 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0351] Y aand Y b Each is an ester bond or an amide bond, independently;

[0352] Z a and Z b Z independently 1 :

[0353]

Chemical Formula 14

[0354]

[0355] (In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (preferably 0), and u is an integer of R.) 4 Substituents are represented independently.

[0356] n a and n b Each is independently 0 or 1; and

[0357] R 3a and R 3b Each of the following is a residue derived independently from the reaction product of a fat-soluble vitamin (e.g., tocopherol) with hydroxyl group and succinic anhydride, an aliphatic hydrocarbon group with 13 to 17 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl), or a group with 50 or fewer carbon atoms represented by the formula (4) (R in formula (4)). 10 It is an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 2 to 10 carbon atoms, wherein the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentane). Alternatively, it may be a group having 50 or fewer carbon atoms represented by formula (5) (R in formula (5)). 11 R is an alkylene group with 2 or 3 carbon atoms. 12 and R 13 Each is independently an alkyl group having 1 to 10 carbon atoms, and X 4 It's an oxygen atom.

[0358] [Ionic lipids (1-3b)]

[0359] An ionic lipid (1), wherein,

[0360] R 1a and R 1b Each is independently an alkylene group (i.e., methylene or ethylene) having 1 to 2 carbon atoms.

[0361] X a and X b X, independently 1 :

[0362]

Chemical Formula 15

[0363]

[0364] (where R) 5 It is an alkyl group with 1 to 3 carbon atoms (e.g., methyl).

[0365] Or X 2 :

[0366] [Chemical Formula 16]

[0367]

[0368] (In the formula, p is 1 or 2.)

[0369] R 2a and R 2b Each is independently an alkylene group having 4 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0370] Y a and Y b Each is an ester bond or an amide bond, independently;

[0371] Z a and Z b Z independently 1 :

[0372]

Chemical Formula 17

[0373]

[0374] (In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (preferably 0), and u is an integer of R.) 4 Substituents are represented independently.

[0375] n a and n b Each is independently 0 or 1; and

[0376] R 3a and R 3b Each of the following is a residue derived independently from the reaction product of a fat-soluble vitamin (e.g., tocopherol) with hydroxyl group and succinic anhydride, an aliphatic hydrocarbon group with 13 to 17 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl), or a group with 50 or fewer carbon atoms represented by the formula (4) (R in formula (4)). 10 It is an alkyl group with 1 to 10 carbon atoms, or a group with 50 or fewer carbon atoms represented by the formula (5) (R in formula (5)). 11 R is an alkylene group with 2 or 3 carbon atoms. 12 and R 13 Each is independently an alkyl group having 1 to 10 carbon atoms, and X 4It's an oxygen atom.

[0377] [Ionic lipids (1-3c)]

[0378] An ionic lipid (1), wherein,

[0379] R 1a and R 1b Each is independently an alkylene group (i.e., methylene or ethylene) having 1 to 2 carbon atoms.

[0380] X a and X b X, independently 1 :

[0381] [Chemical Formula 18]

[0382]

[0383] (where R) 5 It is an alkyl group with 1 to 3 carbon atoms (e.g., methyl).

[0384] Or X 2 :

[0385] [Chemical Formula 19]

[0386]

[0387] (In the formula, p is 1 or 2.)

[0388] R 2a and R 2b Each is independently an alkylene group having 4 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0389] Y a and Y b Each is an ester bond or an amide bond, independently;

[0390] Z a and Z b Z independently 1 :

[0391]

Chemical Formula 20

[0392]

[0393] (In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (preferably 0), and u is an integer of R.) 4 Substituents are represented independently.

[0394] n a and n b Each is independently 0 or 1; and

[0395] R 3a and R 3b Each of these residues is independently derived from the reaction product of a fat-soluble vitamin (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group with 13 to 17 carbon atoms (e.g., heptadecanyl, heptadecanedienyl, 1-hexylnonyl).

[0396] [Ionic lipids (1-3d)]

[0397] An ionic lipid (1), wherein,

[0398] R 1a and R 1b Each is independently an alkylene group (i.e., methylene or ethylene) having 1 to 2 carbon atoms.

[0399] X a and X b X, independently 1 :

[0400]

Chemical Formula 21

[0401]

[0402] (where R) 5 It is an alkyl group with 1 to 3 carbon atoms (e.g., methyl).

[0403] Or X 2 :

[0404]

Chemical Formula 22

[0405]

[0406] (In the formula, p is 1 or 2.)

[0407] R 2a and R 2b Each is independently an alkylene group having 4 or fewer carbon atoms (e.g., methylene, ethylene, trimethylene);

[0408] Y a and Y b Each is an ester bond or an amide bond, independently;

[0409] Z a and Z b Z independently 1 :

[0410]

Chemical Formula 23

[0411]

[0412] (In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (preferably 0), and u is an integer of R.) 4 Substituents are represented independently.

[0413] n a and n b Each is independently 0 or 1; and

[0414] R 3a and R 3b Each is independently an aliphatic hydrocarbon group with 13 to 17 carbon atoms (e.g., heptadecanyl, heptadecanidyl, 1-hexylnonyl), or a group with 50 or fewer carbon atoms represented by formula (4) (R in formula (4)). 10 It is an alkyl group having 1 to 10 carbon atoms or an alkynyl group having 2 to 10 carbon atoms, wherein the alkyl group may be substituted with a 3 to 14-membered non-aromatic heterocyclic group (e.g., dithiopentane).

[0415] Specific examples of ionic lipids (1) include the compounds listed in Tables 1-1 to 1-5 below.

[0416] Table 1-1

[0417]

[0418] Table 1-2

[0419]

[0420] Table 1-3

[0421]

[0422] Table 1-4

[0423]

[0424] Table 1-5

[0425]

[0426] In one embodiment of the present invention, the ionic lipid (1) (i.e., the compound represented by formula (1))

[0427] Preferably, at least one compound is selected from the group consisting of compounds listed in Tables 1-1 to 1-5.

[0428] More preferably, it is selected from at least one of the group consisting of SS-OP, SS-EC, compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6.

[0429] In another embodiment of the present invention, ionic lipid (1) (i.e., compound represented by formula (1))

[0430] Preferably, at least one compound is selected from the group consisting of compounds listed in Tables 1-1 to 1-4.

[0431] More preferably, it is selected from at least one compound chosen from the group consisting of SS-OP, SS-EC, compound 1, and compound 2.

[0432] SS-OP is a further preferred option.

[0433] In one embodiment of the present invention, ionic lipids

[0434] Preferably, it is selected from at least one of the following: SS-OP, compound 3, compound 4, compound 5, compound 6, [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate.

[0435] More preferably, SS-OP, compound 3, compound 4, compound 5, compound 6, [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, or (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate,

[0436] Further preferred compounds are SS-OP, compound 3, compound 4, compound 5, or compound 6.

[0437] SS-OP is particularly preferred.

[0438] Ionic lipids can be commercially available (e.g., “ALC-0315” manufactured by Jenkem Technology USA; “SM-102” and “DLin-MC3-DMA” manufactured by Cayman Chemical). Furthermore, ionic lipid (1) (i.e., the compound represented by formula (1)) can be manufactured by known methods (e.g., methods described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, WO2021 / 195529A2). Additionally, compounds 1 and 2 can be manufactured by the following synthetic methods. Ionic lipids (1) other than compound 1 or compound 2 can also be manufactured by the same synthetic methods as compound 1 or compound 2.

[0439] (I) Synthesis method of compound 1

[0440] <Synthesis of Intermediate 1>

[0441] Intermediate 1 is synthesized via the following synthetic path.

[0442] [Chemical Formula 24]

[0443]

[0444] <Synthesis of Intermediate 1-A>

[0445] 30.0 g (197 mmol) of 4-hydroxyphenylacetic acid and 5.00 g (19.9 mmol) of p-toluenesulfonic acid pyridinium were dissolved in 120 mL of dichloromethane at room temperature. A mixture of 83.0 g (987 mmol) of 3,4-dihydro-2H-pyran and 31.1 mL of dichloromethane was added dropwise to the resulting mixture at room temperature for 2 hours. Then, 12.0 g (98.2 mmol) of DMAP was added to the reaction mixture for neutralization. 301 mL of 2-propanol and 160 g of 100 g / L NaOH aqueous solution were added to the resulting mixture, and the mixture was allowed to react at room temperature for 1 hour. The mixture was concentrated using an evaporator, the concentrate was washed with chloroform, and neutralized with 6 M hydrochloric acid. The resulting mixture was extracted with chloroform, and sodium sulfate was added to the organic layer for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to give 47.0 g of intermediate 1-A.

[0446] <Synthesis of Intermediate 1-B>

[0447] 36.0 g (95.6 mmol) of intermediate 57, 49.7 g (210 mmol) of intermediate 1-A, and 4.68 mg (38.3 mmol) of DMAP synthesized according to the method described in International Publication No. 2016 / 121942 were dissolved in 240 mL of chloroform at room temperature. 55.1 g (287 mmol) of EDC hydrochloride was added to the resulting mixture, and the mixture was allowed to react at room temperature for 2 hours. The reaction solution was then washed with 5 wt% sodium dihydrogen phosphate aqueous solution, 9 wt% sodium bicarbonate solution, and 20 wt% brine, followed by dehydration with sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator to give 77.9 g of intermediate 1-B.

[0448] <Synthesis of Intermediate 1>

[0449] 77.7 g (95.6 mmol) of intermediate 1-B was dissolved in 325 mL of THF at room temperature. 319 mL of 2-propanol and 38.2 g (201 mmol) of p-toluenesulfonic acid monohydrate were added to the resulting mixture, and the reaction was allowed to proceed at below 25 °C for 1 hour. Then, 25.0 g (205 mmol) of DMAP was added to the reaction mixture for neutralization. After removing DMAP by filtration, the filtrate was concentrated using an evaporator. The resulting residue was dissolved in 627 mL of chloroform, washed with 0.5 M phosphate buffer (pH=6.5) and 0.5 M glycine buffer (pH=9.5), and then dehydrated with sodium sulfate. After removing sodium sulfate by filtration, 1.56 L of toluene was added for crystallization. The crude product was washed with hexane and dried under vacuum to give 40.3 g of intermediate 1.

[0450] <Synthesis of Intermediate 1-1>

[0451] 5.11 g (18.1 mmol) of oleic acid, 16.6 g (25.8 mmol) of intermediate 1, and 630 mg (5.16 mmol) of DMAP were dissolved in 166 mL of chloroform at room temperature. 5.94 g (31.0 mmol) of EDC hydrochloride was added to the resulting mixture, and the mixture was allowed to react at room temperature for 2 hours. The reaction solution was then washed with 5% sodium hydrogen phosphate aqueous solution, 0.5 M phosphate buffer (pH=2.0), and 20% saline solution, followed by dehydration with sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator. The residue was purified by silica gel column chromatography (chloroform / ethanol = 88 / 12) to give 8.21 g of intermediate 1-1.

[0452] [Chemical Formula 25]

[0453]

[0454] <Synthesis of Intermediate 5-1>

[0455] 5.00 g (16.0 mmol) of methyl ricinoleate, 2.54 g (17.6 mmol) of caprylic acid, and 391 mg (3.20 mmol) of DMAP were dissolved in 50.0 g of chloroform at room temperature. 4.60 g (24.0 mmol) of EDC hydrochloride was added to the resulting mixture, and the mixture was allowed to react at room temperature for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt% sodium bicarbonate aqueous solution, and 20 wt% saline solution, followed by dehydration with sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to give 6.78 g of intermediate 5-1.

[0456]

Chemical Formula 26

[0457]

[0458] <Synthesis of Intermediate 5>

[0459] 6.40 g (14.6 mmol) of intermediate 5-1 was dissolved in 0.3 M tert-butanol at room temperature. 8.17 g of 2 M NaOH aqueous solution was added to the resulting mixture, and the reaction was allowed to proceed for 8 hours at room temperature. Then, 1 M hydrochloric acid was added, and the resulting mixture was extracted with hexane. The organic layer was washed with 20% brine, and then dehydrated with sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator. The residue was purified by silica gel column chromatography (chloroform / ethanol = 94 / 6 (v / v)) to give 5.93 g of intermediate 5.

[0460] [Chemical Formula 27]

[0461]

[0462] <Synthesis of Compound 1>

[0463] 492 mg (0.541 mmol) of intermediate 1-1 was dissolved in 2.91 g of chloroform, followed by the addition of 230 mg (0.541 mmol) of intermediate 5, 13.2 mg (0.108 mmol) of DMAP, and 207 mg (1.08 mmol) of EDC hydrochloride. The reaction mixture was reacted at room temperature for 2 hours. The resulting solution was washed with 5% saline solution, concentrated using an evaporator, and then purified by silica gel column chromatography using ethanol / chloroform to obtain 532 mg of compound 1.

[0464] (II) Synthesis method of compound 2

[0465] <Synthesis of Intermediate 11-1>

[0466] 25.0 g (189 mmol) of (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol and 19.3 g (284 mmol) of imidazole were dissolved in 251 g of dimethylformamide at room temperature. 57.3 g (208 mmol) of TBDPS-Cl was added to the resulting mixture, and the mixture was reacted at room temperature for 1 hour. Then, chloroform was added to the reaction solution, and the mixture was washed with 5% sodium hydrogen phosphate aqueous solution and deion-exchanged water, respectively. Sodium sulfate was then added for dehydration. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator to give 56.0 g of intermediate 11-1.

[0467] [Chemical Formula 28]

[0468]

[0469] <Synthesis of Intermediate 11-2>

[0470] 55.9 g (151 mmol) of intermediate 11-1 and 503 g of 0.5 M phosphate buffer (pH 1.0) were dissolved in 530 g of THF at room temperature and reacted at 50 °C for 12 hours. A 1.0 M NaOH aqueous solution was added to adjust the pH to 7.0. Chloroform was added to the resulting mixture, and after washing with 20% (w / w) saline solution, sodium sulfate was added for dehydration. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 44.9 g of intermediate 11-2.

[0471] [Chemical Formula 29]

[0472]

[0473] <Synthesis of Intermediate 14-1>

[0474] 6.90 g (20.9 mmol) of intermediate 11-2, 6.84 g (46.0 mmol) of heptanoic acid, and 0.511 g (4.18 mmol) of DMAP were dissolved in 69.5 g of chloroform at room temperature. 12.0 g (62.7 mmol) of EDC hydrochloride was added to the resulting mixture, and the mixture was allowed to react at room temperature for 1 hour. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt% sodium bicarbonate aqueous solution, and 20 wt% saline solution, followed by dehydration with sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to give 9.03 g of intermediate 14-1.

[0475]

Chemical Formula 30

[0476]

[0477] <Synthesis of Intermediate 14-2>

[0478] 9.41 g (17.9 mmol) of intermediate 14-1 and 3.92 g (65.3 mmol) of acetic acid were dissolved in 27.2 g of THF at room temperature. 59.9 g (65.2 mmol) of 1 M TBAF in THF was added to the resulting mixture, and the mixture was allowed to react at room temperature for 18 hours. Then, 94.3 g of ethyl acetate was added to the reaction solution. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0) and 20% saline solution, followed by dehydration with sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to give 4.90 g of intermediate 14-2.

[0479]

Chemical Formula 31

[0480]

[0481] <Intermediate 14>

[0482] 4.90 g (15.4 mmol) of intermediate 14-2, 3.73 g (32.7 mmol) of glutaric anhydride, 4.95 g (48.9 mmol) of triethylamine, and 0.398 g (3.26 mmol) of DMAP were dissolved in 51.6 g of chloroform at room temperature and allowed to react for 1 hour at room temperature. The reaction solution was then washed with 0.5 M phosphate buffer (pH 4.0), 7 wt% sodium bicarbonate aqueous solution, and 20 wt% saline solution, followed by dehydration with sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 3.16 g of intermediate 14.

[0483]

Chemical Formula 32

[0484]

[0485] <Synthesis of Compound 2>

[0486] Using intermediates 14 and 1-1, 0.215 g of compound 2 was synthesized in the same manner as the synthesis of compound 1 described above.

[0487] From the viewpoint of lipid solubility, the concentration of ionic lipid (1) in the alcohol solution (i.e., the lipid solution) of step a) in one embodiment of the present invention is preferably 0.05 to 20 mM, more preferably 0.1 to 15 mM, and even more preferably 1 to 10 mM.

[0488] From the viewpoint of lipid solubility, in another embodiment of the present invention, the concentration of ionic lipid (1) in the alcohol solution (i.e., the lipid solution) of step a) is preferably 0.05 to 30 mM, more preferably 0.1 to 25 mM, and even more preferably 0.3 to 20 mM.

[0489] Furthermore, when using two or more ionic lipids (1), the “concentration of ionic lipids (1)” in this specification refers to the sum of the concentrations of the two or more ionic lipids (1). The meaning of “concentration of other components” is the same as that of “concentration of ionic lipids (1)”.

[0490] From the perspective of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency and lipid nanoparticle stability, the content of ionic lipids (1) in the target lipid nanoparticles is preferably 20-75 mol% relative to the total lipids, more preferably 30-70 mol%, and even more preferably 35-60 mol%.

[0491] Furthermore, when two or more ionic lipids (1) are used, the “content of ionic lipids (1)” in this specification refers to the sum of the contents of the two or more ionic lipids (1). The meaning of “content of other components” is the same as that of “content of ionic lipids (1)”. In addition, the “content of B relative to A (mol%)” or “amount of B relative to A (mol%)” in this specification refers to “100 × amount of B (mol) / amount of A (mol)”.

[0492] The alcohol solution in step a) preferably further contains phospholipids. Only one type of phospholipid may be used, or two or more may be used in combination.

[0493] Examples of phospholipids include 1,2-diacyl-sn-glycerol-3-phosphocholine (PC), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycerol-3-phosphoserine (PS), 1,2-diacyl-sn-glycerol-3-phosphoglycerol (PG), 1,2-diacyl-sn-glycerol-3-phosphatidic acid (PA), and their lysophospholipids.

[0494] As a specific example of 1,2-diacyl-sn-glycerol-3-phosphocholine (PC), examples can be cited.

[0495] 1,2-Didecanoyl-sn-glycerol-3-phosphocholine (DDPC)

[0496] 1,2-Dilauroyl-sn-glycerol-3-phosphocholine (DLPC)

[0497] 1,2-Dimyristoyl-sn-glycerol-3-phosphocholine (DMPC)

[0498] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC)

[0499] 1,2-Distearyl-sn-glycerol-3-phosphocholine (DSPC)

[0500] 1,2-Dioleoyl-sn-glycerol-3-phosphocholine (DOPC)

[0501] 1,2-Dilinoleoyl-sn-glycerol-3-phosphocholine (DLoPC)

[0502] 1,2-Disorhoyl-sn-glycerol-3-phosphocholine (DEPC)

[0503] 1-Myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine (MPPC)

[0504] 1-Myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine (MSPC)

[0505] 1-Palmitoyl-2-myristoyl-sn-glycerol-3-phosphocholine (PMPC)

[0506] 1-Palmitoyl-2-stearoyl-sn-glycerol-3-phosphocholine (PSPC)

[0507] 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine (SOPC).

[0508] Alternatively, 1,2-diacyl-sn-glycerol-3-phosphocholine (PC) can be obtained by replacing the phosphocholine portion with phosphoethanolamine, phosphoserine, phosphoglycerol, or phosphatidic acid, respectively, to obtain 1,2-diacyl-sn-glycerol-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycerol-3-phosphoserine (PS), 1,2-diacyl-sn-glycerol-3-phosphoglycerol (PG), or 1,2-diacyl-sn-glycerol-3-phosphatidic acid (PA).

[0509] As phospholipids, PC and PE are preferred, DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) and POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine) are more preferred, DOPC, DSPC and DEPC are even more preferred, and DOPC is particularly preferred.

[0510] When using phospholipids, from the viewpoint of lipid solubility, the concentration of phospholipids in the alcohol solution (i.e., the lipid solution) of step a) in one embodiment of the present invention is preferably 0.01 to 5 mM, more preferably 0.05 to 3 mM, and even more preferably 0.1 to 2 mM.

[0511] When using phospholipids, from the viewpoint of lipid solubility, the concentration of phospholipids in the alcohol solution (i.e., the lipid solution) of step a) in another embodiment of the present invention is preferably 0.01 to 20 mM, more preferably 0.01 to 15 mM, and even more preferably 0.03 to 10 mM.

[0512] From the perspective of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and stability of lipid nanoparticles, the phospholipid content in the target lipid nanoparticles is preferably 0-35 mol% relative to the total lipids, more preferably 2.5-30 mol%, and even more preferably 5-30 mol%.

[0513] The alcohol solution (i.e., lipid solution) in step a) contains sterols. Only one type of sterol may be used, or two or more may be used in combination.

[0514] Examples of sterols include cholesterol, lanosterol, phytosterol, yeast sterol, zymostenol, chain sterol, stigmasterol, dihydrolanosterol, and 7-dehydrocholesterol. Cholesterol, lanosterol, and phytosterol are preferred, with cholesterol being more preferred.

[0515] From the viewpoint of sterol solubility, the concentration of sterol in the alcohol solution (i.e., lipid solution) of step a) in one embodiment of the present invention is preferably 0.05 to 15 mM, more preferably 0.1 to 10 mM, and even more preferably 0.5 to 10 mM.

[0516] From the viewpoint of sterol solubility, in step a) of another embodiment of the present invention, the concentration of sterol in the alcohol solution (i.e., lipid solution) is preferably 0.001 to 25 mM, more preferably 0.01 to 20 mM, and even more preferably 0.05 to 15 mM.

[0517] From the perspective of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency and lipid nanoparticle stability, the sterol content in the target lipid nanoparticles is preferably 5-60 mol% relative to the total lipids, more preferably 7-55 mol%, and even more preferably 10-50 mol%.

[0518] The alcohol solution (i.e., lipid solution) in step a) contains PEG lipids. In this specification, "PEG lipid" refers to lipids having polyethylene glycol (PEG) chains. Only one type of PEG lipid may be used, or two or more may be used in combination.

[0519] PEG lipids are used to inhibit the aggregation of lipid nanoparticles by coating the surface of lipid nanoparticles with hydrophilic polyethylene glycol (PEG), and to inhibit the interaction between biological components and particles when administered to organisms.

[0520] The PEG moiety in the PEG lipid can be a substance of any molecular weight. In some embodiments, the number average molecular weight of the PEG moiety is preferably 200 to 10,000, more preferably 1,000 to 10,000. The PEG moiety can be linear or branched.

[0521] Examples of PEG lipids include PEG-phospholipids (i.e., phospholipids bound to PEG), PEG-ceramides (i.e., ceramides bound to PEG), PEG-diacylglycerols (i.e., diacylglycerols bound to PEG), and PEG-cholesterols (i.e., cholesterol bound to PEG). Among these, PEG-diacylglycerols are preferred.

[0522] As a PEG lipid,

[0523] Preferably, the PEG-diacylglycerol (i.e., diacylglycerol bound to PEG) has a number average molecular weight of 1,000 to 10,000 for the PEG portion.

[0524] More preferably, PEG-dimyristoylglycerol (i.e., dimyristoylglycerol bound to PEG) with a number average molecular weight of 1,000 to 10,000 for the PEG portion and PEG-distearylglycerol (i.e., distearylglycerol bound to PEG) with a number average molecular weight of 1,000 to 10,000 for the PEG portion.

[0525] Further preferred are PEG-dimyristoylglycerols with a number average molecular weight of 1,000 to 10,000 for the PEG portion (e.g., DMG-PEG2000 used in the examples described later).

[0526] From the viewpoint of lipid solubility, the concentration of PEG lipid in the alcohol solution (i.e., lipid solution) of step a) is preferably 0.001~1mM, more preferably 0.005~0.8mM, and even more preferably 0.01~0.5mM.

[0527] From the viewpoints of nucleic acid encapsulation efficiency, intracellular nucleic acid release efficiency, and lipid nanoparticle stability, the content of PEG lipids in the target lipid nanoparticles relative to the total molar percentage of other lipids is preferably 0.1 to 6.0 mol%, more preferably 0.5 to 5 mol%, and even more preferably 0.7 to 3.0 mol%. Here, "PEG lipids" refers to the PEG lipids used in step a), and does not include ligand-bound PEG lipids (one type of ligand-bound lipids) that can be used in step c), etc. Furthermore, "other lipids" refers to lipids other than the PEG lipids used in step a). For example, when ionic lipids (1), sterols, and PEG lipids are used as lipids in step a), "the total of other lipids" refers to the total of ionic lipids (1) and sterols. Furthermore, when ionic lipids (1), phospholipids, sterols, and PEG lipids are used as lipids in step a), "the total of other lipids" refers to the total of ionic lipids (1), phospholipids, and sterols.

[0528] From the viewpoint of solubility of total lipids and encapsulation of nucleic acids, the concentration of total lipids is preferably 0.1 to 30 mM, more preferably 0.5 to 25 mM, and even more preferably 1 to 20 mM.

[0529] From the viewpoint of solubility of total lipids and encapsulation of nucleic acids, the concentration of total lipids in the alcohol solution (i.e., lipid solution) of step a) is preferably 0.1 to 40 mM, more preferably 0.5 to 30 mM, and even more preferably 1 to 25 mM.

[0530] In step a), an acidic buffer solution with a pH of 1 to 6.5 is used. Examples of such acidic buffer solutions include, for instance, HCl / KCl buffer, p-toluenesulfonic acid / sodium p-toluenesulfonate buffer, tartaric acid / NaOH buffer, citrate buffer, potassium hydrogen phthalate / HCl buffer, glycine / HCl buffer, trans-aconitine / NaOH buffer, formic acid / sodium formate buffer, 3,3-dimethylglutaric acid / NaOH buffer, and 3,3-dimethylglutaric acid / NaOH / 0.1M buffer. NaCl buffer, phenylacetic acid / sodium phenylacetate buffer, acetate / sodium acetate buffer, succinic acid / NaOH buffer, potassium hydrogen phthalate / NaOH buffer, sodium dimethylarsinate / HCl buffer, sodium hydrogen maleate / NaOH buffer, maleic acid / tris(hydroxymethyl)aminomethane (Tris) / NaOH buffer, phosphate buffer, KH₂PO₄ / NaOH buffer, imidazole / HCl buffer, s-collidine (2,4,6-trimethylpyridine) / HCl buffer, triethanolamine HCl / NaOH buffer, 5,5-diethyl... Sodium barbiturate / HCl buffer, N-methylmorpholine / HCl buffer, sodium pyrophosphate / HCl buffer, 2-morpholine ethanesulfonic acid (MES) buffer, malate buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, 1,4-piperazine diethanesulfonic acid (PIPES) buffer, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, Bis-Tris buffer, Bis-Tris propane buffer, anhydrous sodium carbonate buffer, glycylglycine buffer, 3-(N-morpholine)propanesulfonic acid (MOPS) buffer, 2-hydroxy-3-morpholine propanesulfonic acid (MOPSO) buffer, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer.

[0531] The acidic buffer solution is preferably malate buffer, citrate buffer, acetate / sodium acetate buffer, or 2-morpholine ethanesulfonic acid (MES) buffer, more preferably malate buffer or MES buffer, and even more preferably malate buffer. The pH of the acidic buffer solution is 1 to 6.5, preferably 3 to 6.5.

[0532] From the viewpoint of lipid nanoparticle formation, the total concentration of acid and its salt in the acidic buffer (hereinafter sometimes simply referred to as "buffer concentration") is preferably 10 to 100 mM, more preferably 10 to 80 mM.

[0533] <Exchange of Dispersion Media>

[0534] The dispersion medium of suspensions of lipid nanoparticles without nucleic acids, suspensions of lipid nanoparticles encapsulated with nucleic acids, suspensions of ligand-modified lipid nanoparticles without nucleic acids, or suspensions of ligand-modified lipid nanoparticles encapsulated with nucleic acids (hereinafter sometimes collectively referred to as "lipid nanoparticle suspensions") can be replaced with a different buffer solution than the acidic buffer solution used in step a). This new buffer solution contains water as a solvent, and the suspension after the dispersion medium exchange contains water as the dispersion medium.

[0535] Other examples of buffer solutions include, for instance, tartaric acid / NaOH buffer, potassium hydrogen phthalate / HCl buffer, glycine / HCl buffer, trans-aconitine / NaOH buffer, formic acid / sodium formate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer, phenylacetic acid / sodium phenylacetic acid buffer, acetate / sodium acetate buffer, succinic acid / NaOH buffer, potassium hydrogen phthalate / NaOH buffer, sodium dimethylarsinate / HCl buffer, sodium hydrogen maleate / NaOH buffer, maleic acid / tris(hydroxymethyl)aminomethane (Tris) / NaOH buffer, phosphate buffer, KH₂PO₄ / NaOH buffer, 2-morpholine ethanesulfonic acid (MES) buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartaric acid buffer, citrate buffer, Bis-Tris buffer, glycylglycine buffer, etc.

[0536] Another buffer solution is preferably malate buffer, citrate buffer, acetate / sodium acetate buffer, 2-morpholine ethanesulfonic acid (MES) buffer, or maleic acid / tris(hydroxymethyl)aminomethane (Tris) / NaOH buffer, more preferably malate buffer or MES buffer, and even more preferably MES buffer. The pH of the other buffer solution is preferably 4.5 to 6.9, more preferably 5.0 to 6.9, and even more preferably 5.5 to 6.9. From the viewpoint of encapsulating nucleic acid and lipid nanoparticles, the total concentration of the acid and its salt in the other buffer solution (hereinafter sometimes simply referred to as the "buffer concentration") is preferably 10 to 100 mM, more preferably 20 to 50 mM.

[0537] Preferably, the concentrations of each component in the suspension after the exchange of the dispersion medium are the same as the concentrations of each component in the suspension before the exchange of the dispersion medium (i.e., the suspension obtained in step a).

[0538] <Mixture of other ingredients>

[0539] The suspension of lipid nanoparticles can also be mixed with other components. Examples of these other components include sugars such as monosaccharides, sugar alcohols, disaccharides, oligosaccharides, and polysaccharides. Disaccharides are preferred, and sucrose is more preferred. The concentration of sugars in the suspension of lipid nanoparticles is preferably 0-320 mg / mL, and more preferably 0-160 mg / mL.

[0540] <Preservation of suspensions of nucleic acid-free lipid nanoparticles>

[0541] The suspension of nucleic acid-free lipid nanoparticles obtained in step a) can be preserved by freezing or freeze-drying before use in steps b), c'), or d), or directly as a suspension. Furthermore, suspensions of lipid nanoparticles obtained in other steps can be preserved by freezing or freeze-drying before use in the next step), or directly as a suspension. Preservation can be carried out in an atmospheric atmosphere or under an inert atmosphere (e.g., argon).

[0542] The preferred freezing temperature for the suspension of lipid nanoparticles without nucleic acid is -200℃ to -5℃, more preferably -85℃ to -10℃, and the preferred freezing time is 1 to 72 hours, more preferably 3 to 24 hours.

[0543] The preferred temperature for freeze-drying a suspension of lipid nanoparticles without nucleic acids is -80°C to 50°C, more preferably -60°C to 40°C. The preferred pressure for freeze-drying is 0 to 300 mTorr, more preferably 50 to 250 mTorr. The preferred freeze-drying time is 12 to 120 hours, more preferably 24 to 100 hours. Preferably, freeze-drying is initially carried out at a low temperature, and then the temperature is gradually increased in stages.

[0544] From the viewpoint of the stability of the constituent components of lipid nanoparticles, the storage temperature of frozen or freeze-dried suspensions of lipid nanoparticles that do not contain nucleic acids is preferably -80°C to 0°C, more preferably -80°C to -10°C, and even more preferably -80°C to -20°C.

[0545] From the viewpoint of the stability of the constituent components of lipid nanoparticles, the storage temperature for directly storing the suspension of lipid nanoparticles without nucleic acids is preferably 1℃~50℃, more preferably 1℃~30℃, further preferably 1℃~10℃, and most preferably 1℃~5℃.

[0546] When storing frozen or lyophilized suspensions of lipid nanoparticles that do not contain nucleic acids, the suspension is prepared by thawing the obtained frozen material at 0–100°C before use in step b) or c'), or by adding water to the obtained lyophilized material and maintaining the resulting mixture at 0–100°C to prepare the suspension. Preferably, water is added to the lyophilized material so that the concentrations of each component in the resulting suspension are the same as the concentrations of each component in the suspension before lyophilization.

[0547] From the viewpoint of the stability of the constituent components of lipid nanoparticles, the melting temperature of the frozen product or the maintenance temperature of the mixture of the freeze-dried product and water is preferably 0~95°C, more preferably 0~50°C, further preferably 0~30°C, and most preferably 0~10°C. The melting of the frozen product, the addition of water to the freeze-dried product, and the maintenance of the resulting mixture are preferably carried out at atmospheric pressure.

[0548] <Process b)>

[0549] In one embodiment of the manufacturing method of the present invention, step b) is performed after step a) and other steps performed as needed. In step b), a suspension of lipid nanoparticles encapsulated with nucleic acids is obtained by mixing lipid nanoparticles without nucleic acids with a nucleic acid solution. In step b), the substance mixed with the nucleic acid solution may include, for example, (i) a suspension of lipid nanoparticles without nucleic acids, and (ii) a freeze-dried product of lipid nanoparticles without nucleic acids obtained by freeze-drying said suspension. Preferably, in step b), the suspension of lipid nanoparticles without nucleic acids is mixed with a nucleic acid solution. The nucleic acid solution used in step b) contains water as a solvent, and the suspension of lipid nanoparticles encapsulated with nucleic acids obtained in step b) contains water as a dispersion medium. Furthermore, in a preferred embodiment of the present invention, the suspension of lipid nanoparticles without nucleic acids used in step b) contains water as a dispersion medium.

[0550] The mixing in step b) can be performed, for example, by pipetting or using a device or mixer that includes a microfluidic path. Examples of mixers include, but are not limited to, vortex mixers, T-joint mixers, and jet mixers.

[0551] Nucleic acids can be, for example, substances described below. The preferred nucleic acid is mRNA.

[0552] From the viewpoint of encapsulation efficiency and toxicity of nucleic acids, the molar ratio of all amino groups of ionic lipids in the nucleic acid-free lipid nanoparticles (e.g., a suspension of nucleic acid-free lipid nanoparticles or a freeze-dried product of nucleic acid-free lipid nanoparticles) to all phosphate groups of nucleic acids in the nucleic acid solution (amount of all amino groups of ionic lipids (mol) / amount of all phosphate groups of nucleic acids (mol)) (sometimes referred to as "N / P ratio" in this specification) in step b) is preferably 5 to 280, more preferably 10 to 140, and even more preferably 15 to 70.

[0553] The concentration of nucleic acid in the nucleic acid solution (i.e., the amount of nucleic acid (μg) / the volume of the solution (mL)) is not particularly limited as long as it can enable the introduction of the target nucleic acid into the cell, but it is preferably 0.25~2700μg / mL, more preferably 0.25~1000μg / mL, and even more preferably 10~450μg / mL.

[0554] The nucleic acid solution is preferably an aqueous nucleic acid solution. To improve the nucleic acid encapsulation efficiency of the lipid nanoparticles, the aqueous nucleic acid solution may contain an alcohol. Examples of alcohols include methanol, ethanol, n-butanol, and tert-butanol. Only one alcohol may be used, or two or more may be used together. Ethanol is preferred. The concentration of alcohol in the aqueous nucleic acid solution is preferably 0-50 v / v%, more preferably 0-30 v / v%, and even more preferably 0-25 v / v%.

[0555] The nucleic acid aqueous solution is preferably an acidic buffer containing nucleic acid. The pH of the acidic buffer is preferably 1 to 6.5, more preferably 3 to 6.5. Examples of the acidic buffer are the same as those in step a). The acidic buffer is preferably an acetate / sodium acetate buffer, maleic acid / tris(hydroxymethyl)aminomethane (Tris) / NaOH buffer, 2-morpholine ethanesulfonic acid (MES) buffer, malate buffer, or citrate buffer, more preferably MES buffer. From the viewpoint of nucleic acid stability, the total concentration of the acid and its salts in the acidic buffer (i.e., the concentration of the buffer) is preferably 10 to 100 mM, more preferably 10 to 80 mM.

[0556] The volume ratio of the suspension of lipid nanoparticles without nucleic acid to the nucleic acid solution in step b) (volume of the suspension of lipid nanoparticles without nucleic acid: volume of the nucleic acid solution) is not particularly limited as long as it is a volume ratio that can encapsulate nucleic acid. However, from the viewpoint of efficient encapsulation of nucleic acid, it is preferred to be 1:20 to 20:1, and more preferably 1:12 to 12:1.

[0557] In step b), the concentration of nucleic acid in the suspension of nucleic acid-encapsulated lipid nanoparticles obtained is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 1 to 450 μg / mL. In step b) of a preferred embodiment of the present invention, to obtain the suspension of the aforementioned concentration, lipid nanoparticles without nucleic acid (e.g., a suspension of lipid nanoparticles without nucleic acid or a freeze-dried product of lipid nanoparticles without nucleic acid) are mixed with a nucleic acid solution.

[0558] To improve nucleic acid encapsulation efficiency, a mixture of nucleic acid-free lipid nanoparticles (preferably a suspension of nucleic acid-free lipid nanoparticles) and a nucleic acid solution can be maintained at a specified temperature and for a specified time. The temperature is preferably 0-95°C, more preferably 0-60°C, and even more preferably 0-40°C; the time is preferably 1 minute to 2 hours, more preferably 1-60 minutes, and even more preferably 1-30 minutes. Furthermore, the mixture in the above embodiments may also contain other components (e.g., unbound lipids).

[0559] <Process c)>

[0560] In one embodiment of the manufacturing method of the present invention, step c) is performed after step b) and other steps performed as needed. In step c), a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids is obtained by mixing a suspension of lipid nanoparticles encapsulated with nucleic acids with lipids bound to ligands. The suspension of lipid nanoparticles encapsulated with nucleic acids used in step c) contains water as a dispersion medium, and the suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids obtained in step c) contains water.

[0561] The mixing in step c) can be performed, for example, by pipetting or using a device or mixer that includes a microfluidic path. Examples of mixers include, but are not limited to, vortex mixers, T-joint mixers, and jet mixers.

[0562] Examples of ligands include, for example, peptides (including proteins), antibodies, nucleic acids (including oligonucleotides), nucleic acid aptamers, sugars, lipids, etc. The ligand is preferably a peptide, antibody, or sugar that has a specific affinity for molecules expressed on the surface of target cells, more preferably a peptide or antibody, and even more preferably an antibody. Examples of peptides, antibodies, or sugars that can be used as ligands include those described below.

[0563] Specific examples of ligands include transferrin, erythropoietin, EGF, VEGF, PDGF, IL-8, SDF1, thrombin, etanercept, romiplostim, abatacept, HER2, transferrin receptor, glucose transporter, MDR1, muromonab, ibritumomab, tositumomab, rituximab, and baribizumab. asiliximab, cetuximab, brentuximab, margetuximab, daclizumab, trastuzumab, pertuzumab, gemtuzumab, alemtuzumab, natalizumab, tocilizumab, moxetumomab, obinutuzumab, vedol izumab, pembrolizumab, atezolizumab, occrelizumab, Inotuzumab, benralizumab, ibazumab, pembrolizumab, crizanlizumab, sacituzumab, inalizumab, tafacitamab, satralizumab b) Loncastuximab, Dostarlimab, Panitumumab, Ofatumumab, Ipilimumab, Ramucirumab, Nivolumab, Necitumumab, Daratumab, Brodalumab, Olaratumab, Avelumab, DurvalumabDupilumab, Sarilumab, Pertuzumab, Amivantamab, Anifrolumab, Tisotumab, Pabinafusp, PD-1, PD-L1, CTLA4, Glutamine, Leucine, Isoleucine, Valine, Phenylalanine, Tyrosine, Tryptophan, Methionine, Histidine, Fc domain-binding peptide, RGD peptide, D NA aptamers, RNA aptamers, AS1411, A10, Sgc8, HeA21, HeA23, estrone, anisamide, methotrexate, adenosine, thiamine, riboflavin, biotin, glucose, mannose, lactose, maltose, N-acetylglucosamine, sucrose, galactose, sialic acid Lewis X, sialic acid, hyaluronic acid.

[0564] The ligand is preferably a cell-targeting ligand. Here, "cell-targeting ligand" refers to a ligand that has a higher affinity for one or more specific cell types (including tissues and organs containing such cells) than for other cells. Ligand-modified lipid nanoparticles encapsulated with cell-targeting nucleic acids, obtained by the manufacturing method of the present invention, can be selectively delivered to specific cells.

[0565] Examples of cell-targeting peptides include integrin α, which is highly expressed in the vascular system or in some cancer cells. v RGD peptides containing the RGD motif and having affinity for β3 (such as GRGDS pentapeptide), peptides derived from the laminin α2 chain that have affinity for dystrophic proteoglycans highly expressed in muscle cells, etc., are not limited to these. Alternatively, polypeptides that are natural ligands for cell surface receptors such as cytokines and hormones can also be listed.

[0566] As a cell-targeting antibody, for example, any antibody targeting a molecule expressed on the surface of a target cell can be listed. Specific examples of such antibodies include, but are not limited to, anti-transferrin receptor antibodies, anti-glucose transporter antibodies, anti-insulin receptor antibodies, anti-LAT1 antibodies, anti-MDR1 antibodies, anti-BCRP antibodies, anti-SMVT1 antibodies, anti-Cluaudin5 antibodies, anti-LDL receptor antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD19 antibodies, anti-CD28 antibodies, and anti-CD147 antibodies. Alternatively, antibodies used as existing antibody drugs or antibodies recognizing the same antigens as these can be used as ligands for targeting cells expressing the antigen.

[0567] The antibodies used in this invention can be complete antibody molecules, but for example, any other antibody fragments such as single-chain antibodies (scFv), Fab, F(ab')2, Fab', Fv, reductive antibodies (rIgG), dsFv, sFv, bispecific antibodies (diabody), and trispecific antibodies (triabody) can preferably be used. In this specification, unless otherwise specified, "antibody" is used to mean that it includes an antibody fragment.

[0568] In addition, as nucleic acids, examples include double-stranded RNA, CpG oligonucleotides, and other ligands targeting Toll-like receptors; as sugars, examples include N-acetylgalactosamine, which has affinity for desialyl glycoprotein receptors expressed in hepatocytes; and as lipids, examples include various lipids that are natural ligands for G protein-coupled receptors (e.g., lysophosphatidylserine).

[0569] The ligand is more preferably a brain-cell-targeting ligand or a T-cell-targeting ligand. Examples of brain-cell-targeting ligands include, for example, anti-transferrin receptor antibodies, anti-glucose transporter antibodies, anti-insulin receptor antibodies, anti-LAT1 antibodies, anti-CD147 antibodies, anti-MDR1 antibodies, anti-BCRP antibodies, anti-SMVT1 antibodies, and anti-Cluaudin5 antibodies. Examples of T-cell-targeting ligands include, for example, anti-CD3 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD28 antibodies, and anti-LDL receptor antibodies.

[0570] From the viewpoint of balancing selectivity for target cells and preferred particulate matter properties, the amount of ligand in the target lipid nanoparticles is preferably 0.001 to 30 mol% relative to the total amount of lipids in the lipid nanoparticles, more preferably 0.001 to 10 mol%, and even more preferably 0.01 to 5 mol%. To achieve such a ligand amount, it is preferable to mix a suspension of lipid nanoparticles encapsulated with nucleic acids with lipids bound to ligands.

[0571] In this specification, "ligand-bound lipids" refers to lipids covalently bonded to ligands. Ligand-bound lipids are preferably PEG lipids. In this specification, "ligand-bound PEG lipids" refers to PEG lipids covalently bonded to ligands.

[0572] There are no particular limitations on the manufacturing method of ligand-bound PEG lipids; they can be manufactured using known methods. For example,

[0573] (i) The activated PEG lipid (i.e., a PEG lipid having a reactive group capable of binding ligands) as described in International Publication No. 2023 / 054243 can be reacted with ligands to produce ligand-bound PEG lipids.

[0574] (ii) Alternatively, a compound having a reactive group (e.g., dibenzocyclooctyne-N-hydroxysuccinimide ester) can be reacted with a ligand to form a ligand having a reactive group, and then a PEG lipid having a group capable of forming a covalent bond with the reactive group (e.g., an azide group) can be reacted with the ligand having the reactive group to produce a PEG lipid bound with the ligand. The latter method of production is preferred (hereinafter referred to as "production method (ii)").

[0575] The reaction between the compound having a reactive group and the ligand in manufacturing method (ii) is preferably carried out in a solvent. Examples of such solvents include water and buffer solutions. The reaction temperature is preferably 0 to 95°C, more preferably 0 to 40°C, and even more preferably 0 to 25°C. The reaction time is preferably 10 minutes to 24 hours, more preferably 10 hours to 24 hours. The pH during the reaction is preferably 6 to 9, more preferably 7 to 9.

[0576] In manufacturing method (ii), the reaction of the PEG lipid having a group capable of forming a covalent bond with the reactive group and the ligand having the reactive group is preferably carried out in a solvent. Examples of solvents include water and buffer solutions. The reaction temperature is preferably 0–95°C, more preferably 0–40°C, and even more preferably 0–25°C. The reaction time is preferably 1–24 hours, more preferably 10–24 hours, and even more preferably 16–20 hours.

[0577] The PEG moiety in the ligand-bound PEG lipid can be of any molecular weight. In some embodiments, the number average molecular weight of the PEG moiety is preferably 200 to 10,000, more preferably 1,000 to 10,000. The PEG moiety can be linear or branched.

[0578] Examples of PEG lipids that are ligand-bound include PEG-phospholipids (i.e., phospholipids bound to PEG), PEG-ceramides (i.e., ceramides bound to PEG), PEG-diacylglycerols (i.e., diacylglycerols bound to PEG), and PEG-cholesterols (i.e., cholesterol bound to PEG). Among these, PEG-phospholipids and PEG-diacylglycerols are preferred.

[0579] The PEG lipids in the ligand-bound PEG lipids are preferably PEG-phospholipids (i.e., phospholipids bound to PEG) and PEG-diacylglycerols (i.e., diacylglycerols bound to PEG) with a number average molecular weight of 1,000 to 10,000 in the PEG moiety. More preferably, they are PEG-phospholipids with a number average molecular weight of 1,000 to 10,000 in the PEG moiety. Further preferably, they are PEG-1,2-diacyl-sn-glycerol-3-phosphate ethanolamine (i.e. 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine bound to PEG) with a number average molecular weight of 1,000 to 10,000 in the PEG moiety. Particularly preferred are PEG-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (i.e. -1,2-distearyl-sn-glycerol-3-phosphate ethanolamine bound to PEG, such as PEG-DSPE used in the examples described later) with a number average molecular weight of 1,000 to 10,000 in the PEG moiety.

[0580] The ligand-bound PEG lipids are preferably used in the form of an aqueous solution containing them. From the viewpoint of efficiency in inserting ligands into lipid nanoparticles, the concentration of the ligand-bound PEG lipids in the aqueous solution is preferably 0.0001 to 0.12 mM, more preferably 0.001 to 0.06 mM.

[0581] During the mixing process in step c), other components may also be mixed. Examples of other components include, for instance, lipids without ligands.

[0582] In step c), a suspension of nucleic acid-encapsulated lipid nanoparticles, lipids bound with ligands, and lipids without ligands can be mixed. The order of mixing is not particularly limited; for example, (i) the suspension of nucleic acid-encapsulated lipid nanoparticles, lipids bound with ligands, and lipids without ligands can be mixed simultaneously; (ii) the suspension of nucleic acid-encapsulated lipid nanoparticles can be mixed with ligand-bound lipids first, and then the resulting mixture can be mixed with lipids without ligands; (iii) ligand-bound lipids can be mixed with lipids without ligands, and then the resulting mixture can be mixed with the suspension of nucleic acid-encapsulated lipid nanoparticles; (iv) an aqueous solution or aqueous dispersion containing ligand-bound and lipids without ligands can be prepared first, and then the resulting aqueous solution or aqueous dispersion can be mixed with the suspension of nucleic acid-encapsulated lipid nanoparticles. As an aqueous solution or aqueous dispersion of (iv), for example, an aqueous solution or aqueous dispersion (reaction mixture) containing ligand-bound lipids (products) and unligand-bound lipids (unreacted reactants) obtained in the synthesis reaction of ligand-bound lipids can be cited.

[0583] In step c), when using unbound lipids, from the viewpoint of balancing the preferred physical properties of ligand-modified lipid nanoparticles and selectivity for target cells, the amount of unbound lipids used in step c) is preferably 0.01 to 5.0 mol% relative to the total lipids, more preferably 0.1 to 3.0 mol%. Furthermore, the "amount of unbound lipids used in step c)" does not include the amount of unbound lipids used outside of step c) (e.g., ionic lipids used in step a).

[0584] The "unbound ligand lipids" used in step c) are preferably unbound PEG lipids. The description of unbound PEG lipids is the same as that of PEG lipids in step a).

[0585] As unbound PEG lipids in process c),

[0586] Preferably, the PEG-phospholipids (i.e., phospholipids bound to PEG) and PEG-diacylglycerols (i.e., diacylglycerols bound to PEG) are PEG-phospholipids with a number average molecular weight of 1,000 to 10,000.

[0587] More preferably, PEG-phospholipids with a number average molecular weight of 1,000 to 10,000 are preferred.

[0588] Further preferred is PEG-1,2-diacyl-sn-glycerol-3-phosphate ethanolamine (i.e. 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine bound to PEG) with a number average molecular weight of 1,000 to 10,000.

[0589] Particularly preferred are PEG-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (i.e., PEG-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine bound to PEG, such as PEG-DSPE used in the examples described later).

[0590] In step c), when using unbound PEG lipids, from the viewpoint of balancing the preferred physical properties of ligand-modified lipid nanoparticles and selectivity for target cells, the amount of unbound PEG lipids used in step c) is preferably 0.01 to 5.0 mol% relative to the total lipids, more preferably 0.1 to 3.0 mol%. Furthermore, the phrase "amount of unbound PEG lipids used in step c)" does not include the amount of unbound PEG lipids used outside of step c) (e.g., the PEG lipids used in step a).

[0591] When using ligand-bound and unbound PEG lipids in step c), an aqueous solution containing both is preferred. From the viewpoint of insertion efficiency into lipid nanoparticles, the concentration of ligand-bound PEG lipids in the aqueous solution is preferably 0.0001 to 0.12 mM, more preferably 0.001 to 0.06 mM, and the concentration of unbound PEG lipids in the aqueous solution is preferably 0.0001 to 0.12 mM, more preferably 0.001 to 0.06 mM.

[0592] To efficiently encapsulate nucleic acid-encapsulated lipid nanoparticles by modifying them with ligand-bound lipids, a suspension of nucleic acid-encapsulated lipid nanoparticles and a mixture of ligand-bound lipids can be maintained at a specified temperature and for a specified time. The maintaining temperature is preferably 0–95°C, more preferably 0–40°C, and even more preferably 0–25°C. The maintaining time is preferably 1 minute to 5 hours, more preferably 1 minute to 2 hours, and even more preferably 1 to 60 minutes. Furthermore, the mixture in the above embodiments may also contain other components (e.g., unbound lipids).

[0593] <Process c')>

[0594] In one embodiment of the manufacturing method of the present invention, step c') is performed after step a) and other steps performed as needed. In step c'), a suspension of ligand-modified lipid nanoparticles without nucleic acids is obtained by mixing lipid nanoparticles without nucleic acids, water, and lipids bound with ligands. The suspension of ligand-modified lipid nanoparticles without nucleic acids obtained in step c') contains water as a dispersion medium.

[0595] In step c'), as an embodiment of mixing water with other components, examples include (i) mixing a suspension of lipid nanoparticles without nucleic acids (i.e., a suspension containing lipid nanoparticles without nucleic acids and water) with lipids bound to ligands; (ii) mixing lipid nanoparticles without nucleic acids (e.g., a freeze-dried product of lipid nanoparticles without nucleic acids) with an aqueous solution or aqueous dispersion containing lipids bound to ligands (preferably an aqueous solution containing lipids bound to ligands); and (iii) simultaneously mixing lipid nanoparticles without nucleic acids (e.g., a freeze-dried product of lipid nanoparticles without nucleic acids), water, and lipids bound to ligands. Among these, embodiments (i) and (ii) are preferred, and embodiment (i) is more preferred.

[0596] The mixing in step c') can be performed, for example, by pipetting or using a device or mixer that includes a microfluidic path. Examples of mixers include, but are not limited to, vortex mixers, T-joint mixers, and jet mixers.

[0597] During the mixing process in step c'), other components may also be mixed. Examples of other components include, for instance, lipids without ligands.

[0598] In step c'), lipid nanoparticles without nucleic acids, water, lipids bound with ligands and lipids without ligands can be mixed. There is no particular restriction on their mixing order. For example, (i) lipid nanoparticles without nucleic acids, water, lipids bound with ligands and lipids without ligands can be mixed simultaneously; (ii) a suspension of lipid nanoparticles without nucleic acids (i.e., a suspension containing lipid nanoparticles without nucleic acids and water), lipids bound with ligands and lipids without ligands can be mixed simultaneously; (iii) a suspension of lipid nanoparticles without nucleic acids (i.e., a suspension containing lipid nanoparticles without nucleic acids and water) can be mixed with lipids bound with ligands first, and then the resulting mixture can be mixed with lipids without ligands; (iii) water, lipids bound with ligands and lipids without ligands can be mixed first, and then the resulting aqueous solution or aqueous dispersion can be mixed with lipid nanoparticles without nucleic acids; (iv) an aqueous solution or aqueous dispersion containing lipids bound with ligands and lipids without ligands can be prepared first, and then the resulting aqueous solution or aqueous dispersion can be mixed with lipid nanoparticles without nucleic acids. As an aqueous solution or aqueous dispersion of (iv), for example, an aqueous solution or aqueous dispersion (reaction mixture) containing ligand-bound lipids (products) and unligand-bound lipids (unreacted reactants) obtained in the synthesis reaction of ligand-bound lipids can be cited.

[0599] In step c'), when using unbound lipids, from the viewpoint of balancing the preferred physical properties of ligand-modified lipid nanoparticles and selectivity for target cells, the amount of unbound lipids used in step c') is preferably 0.01 to 5.0 mol% relative to the total lipids, more preferably 0.1 to 3.0 mol%. Furthermore, the "amount of unbound lipids used in step c')" does not include the amount of unbound lipids used outside of step c') (e.g., ionic lipids used in step a).

[0600] In step c'), when using unbound PEG lipids, from the viewpoint of balancing the preferred physical properties of ligand-modified lipid nanoparticles and selectivity for target cells, the amount of unbound PEG lipids used in step c') is preferably 0.01 to 5.0 mol% relative to the total lipids, more preferably 0.1 to 3.0 mol%. Furthermore, the phrase "amount of unbound PEG lipids used in step c')" does not include the amount of unbound PEG lipids used outside of step c') (e.g., the PEG lipids used in step a).

[0601] The descriptions of ligands, ligand-bound lipids, and ligand-unbound lipids in step c') are the same as those in step c).

[0602] From the viewpoint of balancing selectivity for target cells and preferred particulate matter properties, the amount of ligand in the target lipid nanoparticles is preferably 0.001 to 30 mol% relative to the total amount of lipids in the lipid nanoparticles described above, more preferably 0.001 to 10 mol%, and even more preferably 0.01 to 5 mol%. To achieve such a ligand amount, it is preferable to mix lipid nanoparticles without nucleic acids, water, and lipids bound with ligands.

[0603] To efficiently modify nucleic acid-free lipid nanoparticles with ligand-bound lipids, a mixture of nucleic acid-free lipid nanoparticles, water, and ligand-bound lipids (preferably a suspension of nucleic acid-free lipid nanoparticles and a mixture of ligand-bound lipids) can be maintained at a specified temperature and for a specified time. The temperature is preferably 0–95°C, more preferably 0–40°C, and even more preferably 0–25°C. The time is preferably 1 minute to 5 hours, more preferably 1 minute to 2 hours, and even more preferably 1 to 60 minutes. Furthermore, the mixture in the above embodiments may also contain other components (e.g., unbound lipids).

[0604] <Process b')>

[0605] In one embodiment of the manufacturing method of the present invention, step b') is performed after step c') and other steps performed as needed. In step b'), a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid is obtained by mixing ligand-modified lipid nanoparticles that do not contain nucleic acid with a nucleic acid solution. The nucleic acid solution used in step b') contains water as a solvent, and the suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid obtained in step b') contains water as a dispersion medium.

[0606] As a substance mixed with the nucleic acid solution in step b'), examples include (i) a suspension of ligand-modified lipid nanoparticles without nucleic acid; and (ii) a freeze-dried product of ligand-modified lipid nanoparticles without nucleic acid obtained by freeze-drying said suspension. In step b'), it is preferable to mix the suspension of ligand-modified lipid nanoparticles without nucleic acid with the nucleic acid solution. In a preferred embodiment of the present invention, the suspension of ligand-modified lipid nanoparticles without nucleic acid used in step b') contains water as a dispersion medium.

[0607] The mixing in step b') can be performed, for example, by pipetting or using a device or mixer that includes a microfluidic path. Examples of mixers include, but are not limited to, vortex mixers, T-joint mixers, and jet mixers.

[0608] As a nucleic acid, for example, substances described later can be listed. The nucleic acid is preferably mRNA. The description of the nucleic acid solution in step b') (e.g., the description of its concentration, etc.) is the same as the description of the nucleic acid solution in step b).

[0609] From the viewpoint of encapsulation efficiency and toxicity of nucleic acids, the molar ratio of all amino groups of ionic lipids in the ligand-modified lipid nanoparticles (e.g., a suspension of ligand-modified lipid nanoparticles without nucleic acids or a freeze-dried product of ligand-modified lipid nanoparticles without nucleic acids) to all phosphate groups of nucleic acids in the nucleic acid solution in step b') is preferably 5 to 280, more preferably 10 to 140, and even more preferably 15 to 70.

[0610] The volume ratio of the suspension of ligand-modified lipid nanoparticles without nucleic acid to the nucleic acid solution in step b') is not particularly limited as long as it is a volume ratio that can encapsulate nucleic acid. However, from the viewpoint of efficient encapsulation of nucleic acid, it is preferred to be 1:20 to 20:1, and more preferably 1:12 to 12:1.

[0611] To improve nucleic acid encapsulation efficiency, a mixture of nucleic acid-free ligand-modified lipid nanoparticles (preferably a suspension of nucleic acid-free ligand-modified lipid nanoparticles) and a nucleic acid solution is maintained at a specified temperature and for a specified time. The temperature is preferably 0–95°C, more preferably 0–60°C, and even more preferably 0–40°C; the time is preferably 1 minute to 2 hours, more preferably 1–60 minutes, and even more preferably 1–30 minutes. Furthermore, the mixture in the above embodiments may also contain other components (e.g., unbound lipids).

[0612] In step b'), the concentration of nucleic acid (i.e., the amount of nucleic acid (μg) / the volume of the suspension (mL)) in the suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid obtained in step b') is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 1 to 450 μg / mL. In step b') of a preferred embodiment of the present invention, ligand-modified lipid nanoparticles without nucleic acid are mixed with a nucleic acid solution to achieve the aforementioned concentration.

[0613] <Process d)>

[0614] In one embodiment of the manufacturing method of the present invention, step d) is performed after step a) and other steps performed as needed. In step d), a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids is obtained by mixing lipid nanoparticles without nucleic acids, a nucleic acid solution, and lipids bound with ligands. Examples of substances mixed with the nucleic acid solution and the lipids bound with ligands in step d) include (i) a suspension of lipid nanoparticles without nucleic acids; and (ii) a freeze-dried product of lipid nanoparticles without nucleic acids obtained by freeze-drying the suspension. Preferably, in step d), the suspension of lipid nanoparticles without nucleic acids, the nucleic acid solution, and the lipids bound with ligands are mixed.

[0615] The mixing in step d) can be performed, for example, by pipetting or using a device or mixer that includes a microfluidic path. Examples of mixers include, but are not limited to, vortex mixers, T-joint mixers, and jet mixers.

[0616] The mixing order in step d) is not particularly limited. For example, (i) lipid nanoparticles without nucleic acids (preferably a suspension of lipid nanoparticles without nucleic acids), a nucleic acid solution, and lipids bound with ligands can be mixed simultaneously; (ii) lipid nanoparticles without nucleic acids (preferably a suspension of lipid nanoparticles without nucleic acids) can be mixed with a nucleic acid solution first, and then the resulting mixture can be mixed simultaneously with lipids bound with ligands; (iii) a nucleic acid solution can be mixed with lipids bound with ligands first, and then a solution containing both nucleic acids and lipids bound with ligands can be mixed with lipid nanoparticles without nucleic acids (preferably a suspension of lipid nanoparticles without nucleic acids). In the above embodiments, the embodiment described in (iii) is preferred.

[0617] During the mixing process in step d), other components may also be mixed. Examples of other components include, for instance, lipids without ligands.

[0618] In step d), lipid nanoparticles without nucleic acids (preferably a suspension of lipid nanoparticles without nucleic acids), a nucleic acid solution, lipids bound with ligands, and lipids without ligands can be mixed. The order of mixing is not particularly limited; for example, (i) lipid nanoparticles without nucleic acids (preferably a suspension of lipid nanoparticles without nucleic acids), a nucleic acid solution, lipids bound with ligands, and lipids without ligands can be mixed simultaneously; (ii) an aqueous solution or aqueous dispersion containing both ligand-bound and unbound lipids can be prepared first, and then the resulting aqueous solution or aqueous dispersion can be mixed with a nucleic acid solution, and then the resulting mixture can be mixed with lipid nanoparticles without nucleic acids (preferably a suspension of lipid nanoparticles without nucleic acids). As an aqueous solution or aqueous dispersion in step (ii), for example, an aqueous solution or aqueous dispersion (reaction mixture) containing ligand-bound lipids (products) and unbound lipids (unreacted reactants) obtained in the synthesis reaction of ligand-bound lipids can be cited as an example.

[0619] In step d), when using unbound lipids, from the viewpoint of balancing the preferred physical properties of ligand-modified lipid nanoparticles and selectivity for target cells, the amount of unbound lipids used in step d) is preferably 0.01 to 5.0 mol% relative to the total lipids, more preferably 0.1 to 3.0 mol%. Furthermore, the "amount of unbound lipids used in step d)" does not include the amount of unbound lipids used outside of step d) (e.g., ionic lipids used in step a).

[0620] In step d), when using unbound PEG lipids, from the viewpoint of balancing the preferred physical properties of ligand-modified lipid nanoparticles and selectivity for target cells, the amount of unbound PEG lipids used in step d) is preferably 0.01 to 5.0 mol% relative to the total lipids, more preferably 0.1 to 3.0 mol%. Furthermore, the phrase "amount of unbound PEG lipids used in step d)" does not include the amount of unbound PEG lipids used outside of step d) (e.g., the PEG lipids used in step a).

[0621] The descriptions of ligands, ligand-bound lipids, and ligand-unbound lipids in step d) are the same as those in step c).

[0622] From the viewpoint of balancing selectivity for target cells and preferred particulate matter properties, the amount of ligand in the target lipid nanoparticles is preferably 0.001 to 30 mol% relative to the total amount of lipids in the lipid nanoparticles, more preferably 0.001 to 10 mol%, and even more preferably 0.01 to 5 mol%. To achieve such a ligand amount, it is preferable to mix lipid nanoparticles without nucleic acids, a nucleic acid solution, and lipids bound with ligands.

[0623] Nucleic acid can be, for example, substances described later. The preferred nucleic acid is mRNA. The description of the nucleic acid solution in step d) (e.g., its concentration) is the same as that in step b).

[0624] From the viewpoint of encapsulation efficiency and toxicity of nucleic acids, the molar ratio of all amino groups of ionic lipids in the nucleic acid-free lipid nanoparticles (e.g., a suspension of nucleic acid-free lipid nanoparticles or a freeze-dried product of nucleic acid-free lipid nanoparticles) to all phosphate groups of nucleic acids in the nucleic acid solution (amount of all amino groups of ionic lipids (mol) / amount of all phosphate groups of nucleic acids (mol)) (sometimes referred to as "N / P ratio" in this specification) in step d) is preferably 5 to 280, more preferably 10 to 140, and even more preferably 15 to 70.

[0625] In step d), the volume ratio of the suspension of nucleic acid-free lipid nanoparticles to the nucleic acid solution (volume ratio of the suspension of nucleic acid-free lipid nanoparticles to the nucleic acid solution) is not particularly limited as long as it allows for the encapsulation of nucleic acids. However, for the purpose of efficient nucleic acid encapsulation, a ratio of 200:1 to 1:200 is preferred, and 20:1 to 1:20 is more preferred. Furthermore, the "nucleic acid solution" in the volume ratio may also contain components other than nucleic acids (e.g., lipids bound to ligands or lipids not bound to ligands).

[0626] In step d), the concentration of nucleic acid (i.e., the amount of nucleic acid (μg) / the volume of the suspension (mL)) in the suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid obtained in step d) is preferably 0.25 to 2700 μg / mL, more preferably 0.25 to 1000 μg / mL, and even more preferably 1 to 450 μg / mL. In step d) of the preferred embodiment of the present invention, lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands are mixed to achieve the above concentration.

[0627] To improve nucleic acid encapsulation efficiency, a mixture of nucleic acid-free lipid nanoparticles (preferably a suspension of nucleic acid-free lipid nanoparticles), a nucleic acid solution, and ligand-bound lipids can be maintained at a specified temperature and for a specified time. The temperature is preferably 0–95°C, more preferably 0–60°C, and even more preferably 0–40°C. The time is preferably 1 minute to 2 hours, more preferably 1–60 minutes, and even more preferably 1–30 minutes. Furthermore, the mixture in the above embodiments may also contain other components (e.g., unbound lipids).

[0628] Methods for introducing nucleic acids into cells

[0629] This invention also provides:

[0630] (i) A method for introducing nucleic acids into cells, comprising the step of contacting ligand-modified lipid nanoparticles encapsulated with nucleic acids, obtained by the manufacturing method of the present invention, with cells in vitro; and

[0631] (ii) A method for delivering nucleic acids into target cells within said organism, comprising the step of administering ligand-modified lipid nanoparticles encapsulated with nucleic acids, obtained by the manufacturing method of the present invention, to the organism.

[0632] (These methods are sometimes collectively referred to as "the methods of this invention" below).

[0633] The method of this invention allows any nucleic acid to be introduced into a cell. Examples of nucleic acids include DNA, RNA, chimeric RNA nucleic acids, and DNA / RNA hybrids, but are not limited to these. Furthermore, any single-stranded to triple-stranded nucleic acid can be used, but single-stranded or double-stranded is preferred. Nucleic acids can also be: other types of nucleotides that are N-glycosides of purine or pyrimidine bases; or other oligomers with a non-nucleotide backbone (e.g., commercially available peptide nucleic acids (PNAs)); or other oligomers with specific bonds (but these oligomers contain nucleotides that allow for base pairing or base attachment configurations common in DNA and RNA), etc. Furthermore, the nucleic acid may also be, for example, a nucleic acid with known addition modifications, a nucleic acid with a known label in the field, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides are replaced by analogs, a nucleic acid modified with intramolecular nucleotides, a nucleic acid with non-charge-bonded (e.g., methanesulfonates, triphosphates, phosphoramides, carbamates, etc.), a nucleic acid with charged or sulfur-containing bonds (e.g., thiophosphates, dithiophosphates, etc.), a nucleic acid with side chains such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (e.g., monosaccharides, etc.), a nucleic acid with chimeric compounds (e.g., acridine, psoralen, etc.), a nucleic acid containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), a nucleic acid containing alkylating agents, or a nucleic acid with modified bonds (e.g., α-anomeric nucleic acids, etc.).

[0634] The types of DNA that can be used in this invention are not particularly limited and can be appropriately selected according to the intended use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligomers, etc., with plasmid DNA, cDNA, and antisense DNA being preferred, and plasmid DNA being more preferred. Circular DNA such as plasmid DNA can also be used as linear DNA after appropriate digestion with restriction endonucleases.

[0635] There are no particular limitations on the types of RNA that can be used in this invention, and they can be appropriately selected according to the intended use. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. are listed, with siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon being preferred.

[0636] The nucleic acids used in this invention are preferably purified using methods commonly used by those skilled in the art.

[0637] The ligand-modified lipid nanoparticles encapsulated with nucleic acids obtained by the manufacturing method of the present invention can be administered in vivo, for example, for the purpose of preventing and / or treating diseases. Therefore, the nucleic acids used in the present invention are preferably nucleic acids that have preventive and / or therapeutic activity against a specific disease (nucleic acids for prevention / treatment). Examples of such nucleic acids include, for example, nucleic acids used in so-called gene therapy.

[0638] There is no particular limitation on the particle size of the ligand-modified lipid nanoparticles encapsulated with nucleic acids, but it is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. The particle size can be determined, for example, using a particle size distribution measuring device such as Zetasizer Nano (Malvern).

[0639] The surface potential (Zeta potential) of ligand-modified lipid nanoparticles encapsulated with nucleic acids is not particularly limited, but is preferably -15 to +15 mV, more preferably -10 to +10 mV. In conventional gene delivery, particles with positively charged surface potentials have been primarily used. Positively charged particles can promote electrostatic interactions with heparan sulfate on negatively charged cell surfaces, which is useful as a method to promote intracellular uptake. However, the positive surface charge may inhibit nucleic acid release from the carrier due to interaction with the delivered nucleic acid within the cell, or inhibit protein synthesis due to the interaction between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface charge to the aforementioned range. The surface charge can be measured, for example, using a Zeta potential measuring device such as the Zetasizer Nano. The surface charge of ligand-modified lipid nanoparticles encapsulated with nucleic acids can be adjusted by the composition of their constituent components.

[0640] The following details the process of contacting ligand-modified lipid nanoparticles encapsulated with nucleic acids with cells in vitro.

[0641] Several days before contact with ligand-modified lipid nanoparticles encapsulated with nucleic acids, cells are suspended in a suitable culture medium and cultured under appropriate conditions. At this contact, the cells may or may not be in the proliferative phase.

[0642] The culture medium used for this contact can be a serum-containing medium or a serum-free medium. The serum concentration in the medium is preferably 30% by weight or less, more preferably 20% by weight or less. Excessive serum or other proteins in the culture medium may hinder the contact between the nucleic acid-encapsulated ligand-modified lipid nanoparticles and the cells.

[0643] There is no specific limit to the cell density at this contact point; it can be set appropriately based on factors such as cell type, but it is usually 1×10⁻⁶. 4~1×10 7 The range of cells / mL.

[0644] To cells prepared in this manner, for example, add a suspension of the above-described nucleic acid-encapsulated ligand-modified lipid nanoparticles. The amount of this suspension added is not particularly limited and can be appropriately set considering factors such as the number of cells. The concentration of the nucleic acid-encapsulated ligand-modified lipid nanoparticles when in contact with cells is not particularly limited as long as it enables the introduction of the target nucleic acid into the cells; however, the total lipid concentration is typically 0.0075–14 mM, preferably 0.05–10 mM, more preferably 0.075–7 mM, and the nucleic acid concentration is preferably 0.1–1000 μg / mL, more preferably 0.1–375 μg / mL, and even more preferably 4–170 μg / mL.

[0645] After adding the above suspension to the cells, the cells are cultured. The temperature, humidity, and CO2 concentration during culture are appropriately set considering the cell type. When the cells are derived from mammals, the typical temperature is approximately 37°C, humidity is approximately 95%, and CO2 concentration is approximately 5%. Furthermore, the culture time can also be appropriately set considering the type of cells used, but it is typically in the range of 0.1 to 96 hours, preferably in the range of 0.2 to 72 hours, and more preferably in the range of 0.5 to 48 hours. If the culture time is too short, nucleic acids cannot be fully introduced into the cells; if the culture time is too long, cell viability may decrease.

[0646] The nucleic acids are introduced into the cells through the above-described culture process, but it is preferable to replace the culture medium with fresh medium, or to add fresh medium to the culture medium for further culturing. When the cells are derived from mammalian cells, the fresh culture medium preferably contains serum or nutritional factors.

[0647] Furthermore, as described above, by using ligand-modified lipid nanoparticles encapsulated with nucleic acids, nucleic acids can be introduced into cells both in vitro and in vivo. That is, by administering ligand-modified lipid nanoparticles encapsulated with nucleic acids to a target, the lipid nanoparticles reach and contact the target cells, thereby introducing the nucleic acids encapsulated in the lipid nanoparticles into the cells within the organism. There are no particular limitations on the target organisms to which these lipid nanoparticles can be administered; examples include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cattle), birds (e.g., chickens, ostriches), amphibians (e.g., frogs), and fish (e.g., zebrafish, medaka), invertebrates such as insects (e.g., silkworms, moths, fruit flies), and plants. Humans or other mammals are preferred as the target organisms for administering these ligand-modified lipid nanoparticles encapsulated with nucleic acids.

[0648] There are no particular limitations on the types of target cells. By using ligand-modified lipid nanoparticles encapsulated with nucleic acids, nucleic acids can be introduced into cells of 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.).

[0649] The method of administering ligand-modified lipid nanoparticles encapsulated with nucleic acids to a target (e.g., vertebrates, invertebrates, etc.) is not particularly limited as long as it enables the lipid nanoparticles to reach and contact the target cells and introduce the compound into the lipid nanoparticles into the cells. The type of compound introduced, the type and site of the target cells, etc., can be considered, and a well-known administration method in the art (e.g., oral administration, non-oral administration (e.g., intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected. The dosage of the lipid nanoparticle is not particularly limited as long as it is sufficient to introduce the compound into the cells. The dosage can be appropriately selected considering the type of target, the administration method, the type of compound introduced, the type and site of the target cells, etc.

[0650] When using ligand-modified lipid nanoparticles encapsulated with nucleic acids obtained by the manufacturing method of the present invention as nucleic acid delivery agents, formulation can be carried out according to conventional methods.

[0651] When this nucleic acid delivery agent is provided as a research reagent, it may be provided (i) directly as ligand-modified lipid nanoparticles encapsulated with nucleic acids, or (ii) in the form of, for example, water or a mixture of physiologically acceptable liquids other than water (e.g., water-soluble solvents (e.g., malate buffer, etc.), organic solvents (e.g., ethanol, methanol, DMSO, tert-butanol, etc.), or mixtures of water-soluble and organic solvents, etc.). The nucleic acid delivery agent of the present invention may suitably contain physiologically acceptable additives known in the art (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0652] Furthermore, when this nucleic acid delivery agent is provided as a drug, it can be provided in the form of ligand-modified lipid nanoparticles encapsulating nucleic acids, or as a mixture with pharmaceutically acceptable and well-known additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.). The nucleic acid delivery agent can be manufactured as an oral dosage form (e.g., tablets, capsules, etc.) or a non-oral dosage form (e.g., injections, sprays, etc.), or a non-oral dosage form (more preferably an injection). In addition to adult use, the nucleic acid delivery agent can also be formulated for pediatric use.

[0653] <Method for manufacturing pharmaceutical compositions>

[0654] The ligand-modified lipid nanoparticles encapsulated with nucleic acids obtained by the manufacturing method of the present invention can be used as drug delivery systems for selectively delivering nucleic acids and the like into specific cells. For example, they are useful for DNA vaccines based on introducing antigen genes into dendritic cells, gene therapy drugs for tumors, and nucleic acid drugs that inhibit target gene expression using RNA interference. Therefore, the present invention also provides a method for manufacturing a pharmaceutical composition incorporating the manufacturing method of the present invention.

[0655]

Example

[0656] The following examples illustrate the present invention in detail, but the present invention is not limited to the following examples.

[0657] In the following examples, the ionic lipids represented by formula (1) are shown by the names described in the table above. Furthermore, the meanings of the abbreviations used in the following examples are as follows.

[0658] ALC-0315: [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (manufactured by Jenkem Technology USA)

[0659] Azide-PEG-DSPE: 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (manufactured by Avanti Polar Lipids)

[0660] cRGD: Cyclo [Arg-Gly-Asp-D-Phe-Lys (Cys)] (Produced by Peptide Research Institute Co., Ltd.)

[0661] DBCO-NHS: Dibenzocyclooctyne-N-hydroxysuccinimide ester (Merck-Millipore)

[0662] DSPE-PEG-Mannose: DSPE-PEG-Mannose, MW2,000 (manufactured by Biopharma PEG Scientific)

[0663] DMG-PEG2000: 1,2-Dimyristoyl-rac-glycerol-3-methylpolyoxyethylene (number average molecular weight of PEG chain: 2000) (manufactured by Nippon Oil Co., Ltd.)

[0664] DSPE-034GS: N-[N'-(succiniminooxyglutarate)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-distearate-sn-glycerol-3-phosphate ethanolamine sodium salt (number average molecular weight of PEG chain: 3400) (manufactured by Nippon Oil Co., Ltd.)

[0665] DSPE-050GS: N-[N'-(succinimideoxyglutarate)aminopropylpolyoxyethyleneoxycarbonyl]-1,2-distearate-sn-glycerol-3-phosphate ethanolamine sodium salt (number average molecular weight of PEG chain: 5000) (manufactured by Nippon Oil Co., Ltd.)

[0666] EDTA: Ethylenediaminetetraacetic acid

[0667] GS-020TS: 1,2-Distearate-rac-glycerol-3-(succinimide-oxycarbonyl)polyoxyethylene (number average molecular weight of PEG chain: 2000) (manufactured by Nippon Oil Co., Ltd.)

[0668] LNP: Lipid nanoparticles

[0669] MC3: DLin-MC3-DMA (manufactured by Cayman Chemical)

[0670] Methoxy-PEG: Methyl-PEG2000-DSPE (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0671] Nuclease free water:UltraPure TM DNase / RNase-Free Distilled Water (manufactured by Thermo Fisher Scientific)

[0672] PBS: Phosphate-buffered saline

[0673] SM-102: Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (Cayman Chemical)

[0674] Tris·HCl: Tris(hydroxymethyl)aminomethane hydrochloride

[0675] Triton X-100: Nonionic surfactant (manufactured by Nacalai Tesque Co., Ltd.)

[0676] [Preparation Example 1] Preparation of an aqueous solution containing ligand-bound PEG lipids

[0677] Using an Amicon Ultra-4 (MWCO: 50 kDa), the solvent for the aqueous solution of anti-transferrin receptor antibody (BioXCell) was replaced with 200 mM NaHCO3 aqueous solution (pH: 9.0). The antibody aqueous solution with the replaced buffer solvent was mixed with 2 equivalents of DBCO-NHS, and the resulting mixture was incubated at room temperature for 2 hours. The mixture was then further incubated overnight at 4°C. Using an Amicon Ultra-4 (3 kDa), the solvent for this mixture was replaced with ultrapure water to prepare an aqueous solution of DBCO-anti-transferrin receptor antibody.

[0678] 10.1 μL of 74 μM DBCO-anti-transferrin receptor antibody aqueous solution, 7.47 μL of 0.1 mM Azide-PEG-DSPE aqueous solution, and 32.5 μL of 2-morpholine ethanesulfonic acid buffer (pH: 6.0) were mixed and incubated at 4 °C overnight to prepare an aqueous solution containing PEG lipids bound to a ligand (ligand: anti-transferrin receptor antibody).

[0679] [Comparative Example 1] Preparation of LNPs encapsulated with mRNA but without ligand modification

[0680] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 20 mM of cholesterol in ethanol, and 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol at a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with 1 mM of DMG-PEG2000 in ethanol to prepare the lipid solution (DMG-PEG2000 amount: 1.5 mol% relative to the total of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine).

[0681] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0682] SS-OP concentration: 4.2 mM

[0683] Cholesterol concentration: 3.2 mM

[0684] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 0.6 mM

[0685] DMG-PEG2000 concentration: 0.12mM

[0686] Total lipid concentration: 8.12 mM

[0687] Using the nanoparticle manufacturing apparatus "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malate buffer (buffer concentration: 20 mM, pH: 3.0) was mixed with lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of LNP suspension without nucleic acid (hereinafter referred to as "empty LNP suspension") (step a) . The same operation was performed to obtain 2 mL of empty LNP suspension (step a) .

[0688] Under vortex mixing, 2.5 mL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was rapidly added to each of the two resulting 4.5 mL aliquots of the mixture. The resulting 9.0 mL aliquot of the mixture was then combined with 4 mL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) and ultrafiltered using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrate was diluted with 14 mL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the diluted concentrate was ultrafiltered. 100 μL of 320 mg / mL sucrose solution was added to 100 μL of the concentrate to prepare an empty LNP suspension with an exchange dispersion medium. This LNP suspension was stored at 4 °C under an argon atmosphere until use.

[0689] Prepare an mRNA solution by mixing 2.5 μL of aqueous solution (3 μg mRNA) encoding NanoLuc (registered trademark) luciferase with 132.5 μL of 2-morpholinoethanesulfonic acid buffer (20 mM, pH 6.0). Using a vortex mixer, mix the total volume of the resulting mRNA solution with 15 μL of empty LNP suspension in exchange dispersion medium, and incubate the mixture at 37°C for 5 minutes. Add 150 μL of phosphate buffer (9.6 mM, pH 7.4) to the incubated mixture to prepare an LNP suspension encapsulated with mRNA (N / P ratio: 36, mRNA concentration: 10 μg / mL) (step b).

[0690] Then, 7.47 μL of 0.1 mM Azide-PEG-DSPE aqueous solution was mixed with 42.6 μL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the resulting solution was incubated at 4 °C overnight. The total volume of the incubated solution was then mixed with the total volume of the LNP suspension encapsulating the above mRNA, and the resulting mixture was incubated at 4 °C for 30 minutes to prepare the Azide-PEG-DSPE modified LNP suspension encapsulating mRNA.

[0691] [Comparative Example 2] Preparation of LNPs modified with ligands encapsulated with mRNA

[0692] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 10 mM of cholesterol in ethanol, and 5 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol at a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with 1 mM of DMG-PEG in ethanol and 1 mM of Azide-PEG-DSPE in aqueous solution to prepare a lipid solution (relative to the total amount of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine, DMG-PEG2000 amount: 1.5 mol%, Azide-PEG-DSPE amount: 0.8 mol%).

[0693] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0694] SS-OP concentration: 2.1 mM

[0695] Cholesterol concentration: 1.6 mM

[0696] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 0.3 mM

[0697] DMG-PEG2000 concentration: 0.06 mM

[0698] Azide-PEG-DSPE concentration: 0.0032 mM

[0699] Total lipid concentration: 4.092 mM

[0700] Using the nanoparticle manufacturing apparatus "iLiNP" (Lilac Pharma Co., Ltd.), a 1 mL suspension of LNP encapsulated with mRNA was prepared by mixing a malate buffer containing mRNA encoding luciferase (mRNA concentration: 0.167 mg / mL, buffer concentration: 20 mM, pH 3.0) with a lipid solution at a flow rate of 250 μL / min for a total of 1 minute. The same procedure was then performed to prepare another 1 mL suspension of LNP encapsulated with mRNA.

[0701] While stirring in a vortex mixer, 1.0 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was rapidly added to each of the two 1 mL LNP suspensions encapsulated with mRNA, and the resulting 2.0 mL mixtures were combined. 4 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 5.5) was added to the resulting 4.0 mL mixture, and the solution was concentrated to below 0.2 mL using an Amicon Ultra-4 (MWCO: 100 kDa). After ultrafiltration, the concentrate was diluted with phosphate buffer (4 mL, buffer concentration: 9.6 mM, pH: 7.4), and the diluted solution was then subjected to ultrafiltration. Malic acid (20 mM, 30 mM, pH 5.0) was added, and the volume of the mixture was adjusted to 4.0 mL. The resulting mixture was then concentrated again to 200 μL by ultrafiltration to prepare an LNP suspension encapsulated with mRNA in an exchange dispersion medium.

[0702] To ensure that DBCO-NHS is 2 equivalents relative to the F(ab')2 fragment, an aqueous solution of DBCO-NHS was added to the F(ab')2 fragment. After incubation at room temperature for 2 hours, the mixture was incubated overnight at 4°C. Ultrapure water was added to bring the volume of the mixture to 0.5 mL, and the mixture was ultrafiltered using an Amicon Ultra-0.5 to remove unreacted DBCO-NHS, thus preparing the DBCO-binding F(ab')2 fragment. The DBCO import number of the DBCO-binding F(ab')2 fragment was calculated based on the absorbance at 280 nm and 309 nm of the aqueous solution of the DBCO-binding F(ab')2 fragment. To ensure that the DBCO-binding F(ab')2 fragment is equimolar relative to the azide group in the LNP encapsulated with mRNA, the DBCO-binding F(ab')2 fragment was added to the LNP suspension encapsulated with mRNA, and the mixture was incubated overnight at 4°C, thus preparing a ligand-modified LNP suspension encapsulated with mRNA (ligand amount relative to total lipids: 0.8 mol%).

[0703] [Example 1] Preparation of ligand-modified LNPs encapsulated with mRNA

[0704] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 2 mM of cholesterol in ethanol, and 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol at a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with 1 mM of DMG-PEG2000 in ethanol to prepare a lipid solution (total amount of DMG-PEG2000 relative to the sum of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 1.5 mol%).

[0705] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0706] SS-OP concentration: 4.2 mM

[0707] Cholesterol concentration: 3.2 mM

[0708] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 0.6 mM

[0709] DMG-PEG2000 concentration: 0.12mM

[0710] Total lipid concentration: 8.12 mM

[0711] Using the nanoparticle manufacturing apparatus "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malate buffer (concentration: 20 mM, pH: 3.0) was mixed with lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a). The same operation was performed to obtain 2 mL of empty LNP suspension (step a).

[0712] Under vortex mixing, 2.5 mL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) was rapidly added to each of the two resulting 4.5 mL aliquots of the mixture. The resulting 9.0 mL aliquot of the mixture was then combined with 4 mL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the mixture was ultrafiltered using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrate was diluted with 14 mL of 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0), and the diluted solution was ultrafiltered. 100 μL of a 320 mg / mL sucrose solution was added to 100 μL of the concentrate to prepare an empty LNP suspension with an exchange dispersion medium. This LNP suspension was stored at 4 °C under an argon atmosphere until use.

[0713] Prepare an mRNA solution by mixing an aqueous solution (2.5 μL, mRNA amount: 3 μg) of luciferase-encoding mRNA with 132.5 μL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0). Using a vortex mixer, mix the total volume of the resulting mRNA solution (135 μL) with 15 μL of empty LNP suspension exchanged for dispersion medium, and incubate the mixture at 37°C for 5 minutes. Add 150 μL of phosphate buffer (buffer concentration: 9.6 mM, pH 7.4) to the incubated mixture to prepare an LNP suspension encapsulated with mRNA (N / P ratio: 36, mRNA concentration: 10 μg / mL) (step b)

[0714] The total amount of the LNP suspension containing mRNA (150 μL) was mixed with the aqueous solution containing ligand-bound PEG lipids obtained in Preparation Example 1 by pipetting, and incubated at 4 °C for 30 minutes to prepare the ligand-modified LNP suspension containing mRNA (ligand amount relative to total lipids: 0.8 mol%) (step c) .

[0715] In addition, except for changing the incubation temperature (hereinafter referred to as "incubation temperature for ligand modification") when preparing the ligand-modified LNP suspension encapsulated with mRNA to 37°C or 50°C, the ligand-modified LNP suspension encapsulated with mRNA was prepared in the same manner as described above. Thus, three suspensions with different incubation temperatures for ligand modification were prepared.

[0716] [Experimental Example 1] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency

[0717] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA from Example 1 or Comparative Example 2 were analyzed. Particle size and PdI were determined using dynamic light scattering assay with a Zetasizer (registered trademark). mRNA encapsulation efficiency was determined using the Ribogreen (registered trademark) assay. The results are shown in Table 2 below. Furthermore, the LNP used in Example 1 was an LNP incubated at 4°C during ligand modification.

[0718] Table 2

[0719]

[0720] As shown in Table 2, compared with the ligand-modified LNPs encapsulated with mRNA in Comparative Example 2, the ligand-modified LNPs encapsulated with mRNA in Example 1 had a higher mRNA encapsulation rate.

[0721] [Comparative Example 3] Preparation of LNPs encapsulated with mRNA but without ligand modification

[0722] Except for (i) preparing a lipid solution by mixing 10 mM of SS-OP in ethanol, 20 mM of cholesterol in ethanol, 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol, 1 mM of DMG-PEG2000 in ethanol, and 0.5 mM of 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindocarbonylcyanine perchlorate (fluorescent dye) in ethanol at a molar ratio of 52.5 / 40 / 7.5 / 1.5 / 0.5; and

[0723] (ii) Use mRNA encoding luciferase instead of mRNA encoding NanoLuc (registered trademark) luciferase.

[0724] In addition, an Azide-PEG-DSPE modified LNP suspension encapsulated with mRNA was prepared using the same steps as in Comparative Example 1.

[0725] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0726] SS-OP concentration: 4.2 mM

[0727] Cholesterol concentration: 3.2 mM

[0728] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 0.6 mM

[0729] DMG-PEG2000 concentration: 0.12mM

[0730] Total lipid concentration: 8.12 mM

[0731] In addition, the incubation temperature for preparing the Azide-PEG-DSPE-modified LNP suspension encapsulated with mRNA (hereinafter referred to as "incubation temperature for Azide-PEG-DSPE modification") was changed to either 37°C or 50°C, and the Azide-PEG-DSPE-modified LNP suspension encapsulated with mRNA was prepared in the same manner as described above. By performing this operation, three suspensions with different incubation temperatures for Azide-PEG-DSPE modification were prepared.

[0732] [Example 2] Preparation of LNPs modified with ligands encapsulated with mRNA

[0733] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 20 mM of cholesterol in ethanol, 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol, 1 mM of DMG-PEG2000 in ethanol, and 0.5 mM of 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindolecarbonylcyanine perchlorate (fluorescent dye) in ethanol at a molar ratio of 52.5 / 40 / 7.5 / 1.5 / 0.5. A ligand-modified LNP suspension encapsulated with mRNA was prepared by the same procedure as in Example 1, except that the lipid solution was used and incubated at 4°C, 37°C, or 50°C for ligand modification.

[0734] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0735] SS-OP concentration: 4.2 mM

[0736] Cholesterol concentration: 3.2 mM

[0737] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 0.6 mM

[0738] DMG-PEG2000 concentration: 0.12mM

[0739] Total lipid concentration: 8.12 mM

[0740] [Experimental Example 2] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency, etc.

[0741] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Example 2 were measured. The results are shown in Table 3 below. Furthermore, "temperature" in Table 3 refers to the incubation temperature during ligand modification.

[0742] Table 3

[0743]

[0744] [Experimental Example 3] Evaluation of LNP uptake capacity of 4T1 cells derived from mouse breast cancer in vitro (FACS analysis)

[0745] 4T1 cells derived from mouse breast cancer cells were used to make 7.5 × 10⁻⁶ cells. 4 Cells were seeded in 12-well plates. After 24 hours, either the ligand-modified LNP suspension encapsulated with mRNA obtained in Example 2 or the unmodified LNP suspension encapsulated with mRNA obtained in Comparative Example 3 was added to each well to achieve a lipid concentration of 10 μM. The mixture in the wells was incubated for 2 hours. After incubation, the mixture in the wells was washed twice with phosphate-buffered saline (pH: 7.4) to remove LNPs that were not taken up by the 4T1 cells. The 4T1 cells were then peeled off with trypsin. The 4T1 cells were centrifuged at 500 × g and 4 °C for 5 minutes. The resulting cell pellet was resuspended in 1000 μL of FACS buffer (0.05 wt% sodium azide aqueous solution and 0.5 wt% PBS containing bovine fetal serum albumin) to prepare a suspension. Then, the resulting suspension was centrifuged for 5 minutes at 500×g and 4°C. The resulting cell pellet was used as a sample, and the fluorescence intensity of LNP-derived cells in 4T1 cells was measured using NovoCyte. The results are shown in Table 4 below. In addition, the term "temperature" in Example 2 refers to the incubation temperature during ligand modification, and in Comparative Example 3 refers to the incubation temperature during Azide-PEG-DSPE modification.

[0746] Table 4

[0747]

[0748] As shown in Table 4, it was confirmed that regardless of the incubation temperature during ligand modification or Azide-PEG-DSPE modification, compared with the LNP encapsulated with mRNA but not modified with ligand in Comparative Example 3, the ligand-modified LNP encapsulated with mRNA in Example 2 showed increased fluorescence intensity and significantly increased uptake into 4T1 cells.

[0749] [Example 3]

[0750] Except that the mRNA encoding NanoLuc (registered trademark) luciferase was used instead of the mRNA encoding luciferase, and the incubation temperature for ligand modification was set to 4°C, the ligand-modified LNP suspension encapsulated with mRNA was prepared in the same manner as in Example 1.

[0751] [Experimental Example 4] Evaluation of LNP gene expression activity in vivo

[0752] C57BL / 6 mice were administered via the tail vein either the ligand-modified LNP suspension containing mRNA from Example 3, or the LNP suspension containing mRNA from Comparative Example 1 but without ligand modification, or PBS (mRNA amount: 1.0 μg).

[0753] Twenty-one hours after the above-mentioned drug administration, the mice were euthanized and the entire brain was removed. 800 μL of homogenization buffer (pH: 7.4, 100 mM Tris·HCl, 2 mM EDTA, 0.1 w / v% Triton X-100) was added to the obtained whole brain, and the brain was homogenized using zirconia beads. 500 μL of the supernatant of the resulting suspension was centrifuged at 13000 rpm and 4 °C for 10 minutes. 20 μL of the supernatant was mixed with 50 μL of an aqueous solution of luciferase matrix, and the luminescence of the NanoLuc (trademarked) luciferase was measured using GloMax. Furthermore, the protein concentration of the solution after diluting the supernatant 100-fold with ultrapure water was determined using the BCA method, and the luminescence intensity was normalized to protein mass. The results are shown in Table 5 below.

[0754] Table 5

[0755]

[0756] As shown in Table 5, it was confirmed that compared with the LNP encapsulated with mRNA but without ligand modification in Comparative Example 1, the luminescence intensity of the ligand-modified LNP encapsulated with mRNA in Example 3 was increased, and the gene expression activity in the mouse brain was significantly increased.

[0757] [Example 4] Preparation of ligand-modified LNPs encapsulated with mRNA

[0758] Except that a 10 mM ethanol solution of SM-102, 10 mM ethanol solution of MC3, 10 mM ethanol solution of ALC-0315, 10 mM ethanol solution of compound 3, 10 mM ethanol solution of compound 4, 10 mM ethanol solution of compound 5, or 10 mM ethanol solution of compound 6 was used instead of a 10 mM ethanol solution of SS-OP, the ligand-modified LNP suspension encapsulated with mRNA was prepared in the same manner as in Example 1.

[0759] [Experimental Example 5] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency

[0760] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Example 4 were measured. The results are shown in Table 6 below.

[0761] Table 6

[0762]

[0763] [Preparation Example 2] Preparation of an aqueous solution containing ligand-bound PEG lipids and unbound PEG lipids

[0764] Using an Amicon Ultra-4 (MWCO: 50 kDa), the solvent for the anti-CD3 antibody (Invitrogen) aqueous solution was replaced with 100 mM NaHCO3 aqueous solution (pH: 9.0). The antibody aqueous solution with the replaced buffer solvent was mixed with 2 equivalents of DBCO-NHS, and the resulting mixture was incubated at room temperature for 2 hours. The mixture was then further incubated overnight at 4°C. Using an Amicon Ultra-4 (3 kDa), the solvent for this mixture was replaced with ultrapure water to prepare the DBCO-anti-CD3 antibody aqueous solution.

[0765] 20 μL of 12 μM DBCO-antiCD3 antibody aqueous solution, 12.0 μL of 0.02 mM Azide-PEG-DSPE aqueous solution, 14.4 μL of 0.1 mM Methoxy-PEG aqueous solution, and 103.6 μL of 2-morpholine ethanesulfonic acid buffer (pH: 6.0) were mixed and incubated at 4 °C overnight to prepare an aqueous solution containing ligand-bound PEG lipids (ligand: antiCD3 antibody) and unbound PEG lipids (Methoxy-PEG) (concentration of ligand-bound PEG lipids: 0.0016 mM, concentration of unbound PEG lipids: 0.0096 mM).

[0766] [Example 5] Preparation of LNPs modified with ligands encapsulated with mRNA

[0767] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 10 mM of cholesterol in ethanol, and 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol at a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with 1 mM of DMG-PEG2000 in ethanol to prepare a lipid solution (total amount of DMG-PEG2000 relative to the sum of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 1.0 mol%).

[0768] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0769] SS-OP concentration: 10.5 mM

[0770] Cholesterol concentration: 1.5 mM

[0771] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 8 mM

[0772] DMG-PEG2000 concentration: 0.3mM

[0773] Total lipid concentration: 20.3 mM

[0774] Using the nanoparticle manufacturing apparatus "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malate buffer (concentration: 20 mM, pH: 3.0) was mixed with lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a). The same operation was performed to obtain 2 mL of empty LNP suspension (step a).

[0775] While stirring in a vortex mixer, 2.5 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was rapidly added to each of the two resulting 2 mL empty LNP suspensions, and the two resulting 4.5 mL mixtures were combined. 4 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was added to the resulting 9.0 mL mixture, and the mixture was ultrafiltered using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrate was diluted with 14 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0), and the diluted solution was ultrafiltered. An empty LNP suspension for exchange dispersion was prepared by adding 60 μL of sucrose solution dissolved in 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) at a concentration of 320 mg / mL to 15 μL of the obtained concentrate (lipid mass: 300 nmol). This LNP suspension was cryopreserved at -20 °C under an argon atmosphere and thawed at room temperature before use.

[0776] Prepare an mRNA solution by mixing an aqueous solution of mRNA encoding luciferase (mRNA amount: 2.5 μg) with 2-morpholinoethanesulfonic acid buffer (75 μL, buffer concentration: 40 mM, pH: 6.0). Using a vortex mixer, mix the total volume of the resulting mRNA solution (75 μL) with an empty LNP suspension (75 μL) exchanged for dispersion medium, and incubate the mixture at 75°C for 5 minutes. Add phosphate buffer (150 μL, buffer concentration: 9.6 mM, pH 7.4) to the incubated mixture to prepare an LNP suspension encapsulated with mRNA (N / P ratio: 36, mRNA concentration: 13.3 μg / mL) (step b)

[0777] Using a vortex mixer, a 150 μL suspension of LNP encapsulated with mRNA was mixed with a 150 μL aqueous solution containing ligand-bound and unbound PEG lipids obtained by the method described in Preparation Example 2. The mixture was incubated at 4 °C for 30 minutes, and 300 μL of phosphate buffer (buffer concentration: 9.6 mM, pH 7.4) was added to prepare a ligand-modified LNP suspension encapsulated with mRNA (ligand amount relative to total lipids: 0.08 mol%, unbound PEG lipids relative to total lipids: 0.48 mol%) (step c) .

[0778] [Comparative Example 4] Preparation of LNPs encapsulated with mRNA but without ligand modification

[0779] The same steps as in Example 5 were performed: a) exchange of dispersion medium, cryopreservation and thawing, and b) to prepare an LNP suspension encapsulated with mRNA. Using a vortex mixer, the LNP suspension encapsulated with mRNA (150 μL) was mixed with 2-morpholine ethanesulfonic acid buffer (150 μL, buffer concentration: 20 mM, pH: 6.0), and phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was added to prepare an LNP suspension encapsulated with mRNA (N / P ratio: 36).

[0780] [Experimental Example 6] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency

[0781] Similar to Experimental Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA from Example 5 and the unmodified LNPs encapsulated with mRNA from Comparative Example 4 were measured. The results are shown in Table 7 below.

[0782] Table 7

[0783]

[0784] As shown in Table 7, although the ligand-modified LNP encapsulated with mRNA in Example 5 was ligand modified, it still exhibited the same ideal mRNA encapsulation efficiency as the LNP suspension encapsulated with mRNA in Comparative Example 4.

[0785] [Preparation Example 3] Preparation of freeze-dried LNP

[0786] A lipid solution was prepared by mixing 10 mM SS-OP ethanol solution, 10 mM cholesterol ethanol solution, and 10 mM 1,2-dioleoyl-sn-glycerol-3-phosphocholine ethanol solution in a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with 1 mM DMG-PEG2000 ethanol solution in a molar ratio of 52.5 / 40 / 7.5 / 1.5.

[0787] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0788] SS-OP concentration: 4.2 mM

[0789] Cholesterol concentration: 3.2 mM

[0790] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 0.6 mM

[0791] DMG-PEG2000 concentration: 0.12mM

[0792] Total lipid concentration: 8.12 mM

[0793] Using the nanoparticle fabrication apparatus “NanoAssemblr Ignite” (manufactured by Precision NanoSystems), malate buffer (concentration: 20 mM, pH: 3.0) was mixed with lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a). The same operation was performed to obtain 2 mL of empty LNP suspension (step a).

[0794] While stirring in a vortex mixer, 2.5 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was rapidly added to each of the two resulting 2 mL empty LNP suspensions, and the two resulting 4.5 mL mixtures were combined. 4 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was added to the resulting 9.0 mL mixture, and the mixture was ultrafiltered using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrate was diluted with 14 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0), and the diluted solution was ultrafiltered. Add 100 μL of sucrose solution dissolved in 2-morpholine ethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) at a concentration of 320 mg / mL to 10 μL of the obtained concentrate (lipid mass: 400 nmol) to prepare an empty LNP suspension for exchange dispersion medium.

[0795] The freeze-drying process is performed as follows: First, the empty LNP suspension containing the exchange dispersion medium is cooled to -40°C under atmospheric pressure to freeze it. Then, the pressure is reduced to 200 mTorr, and the temperature is increased as described below. Specifically, the frozen material is allowed to stand at -40°C for 1 hour at 200 mTorr, then heated, then allowed to stand at -30°C for 6 hours, then heated, then allowed to stand at -20°C for 6 hours, then heated, then allowed to stand at -10°C for 6 hours, then heated, then allowed to stand at 0°C for 6 hours, then heated, then allowed to stand at 10°C for 3 hours, then heated, then allowed to stand at 20°C for 3 hours, then maintained at 30°C for 3 hours, and finally the pressure is restored to atmospheric pressure to recover the freeze-dried LNP.

[0796] The obtained freeze-dried LNP was stored in an argon atmosphere at -80°C before use.

[0797] [Example 6] Preparation of LNPs modified with ligands encapsulated with mRNA

[0798] An aqueous solution of mRNA encoding luciferase (mRNA amount: 2.0 μg) was mixed with 200 μL of nuclease-free water to prepare an mRNA solution. The total volume of the resulting mRNA solution (200 μL) was then pipetted together with the freeze-dried LNP obtained in Preparation Example 3. The resulting mixture was incubated at 37°C or 75°C for 5 minutes, respectively. Phosphate buffer (200 μL, buffer concentration: 9.6 mM, pH 7.4) was added to the incubated mixture to prepare an LNP suspension encapsulated with mRNA (N / P ratio: 69, mRNA concentration: 5 μg / mL) (Step b)

[0799] The total volume of the LNP suspension encapsulated with mRNA (100 μL) was mixed with the aqueous solution containing ligand-bound and unbound PEG lipids obtained by the method described in Preparation Example 2, and incubated at 4°C for 30 minutes. Phosphate buffer (200 μL, buffer concentration: 9.6 mM, pH 7.4) was added to the incubated mixture to prepare a ligand-modified LNP suspension encapsulated with mRNA (ligand amount relative to total lipids: 0.08 mol%) (Step c)

[0800] [Example 7] Preparation of ligand-modified LNPs encapsulated with mRNA

[0801] The total amount of the freeze-dried LNP obtained in Preparation Example 3 was mixed with the aqueous solution containing ligand-bound and unbound PEG lipids obtained by the method described in Preparation Example 2 by pipetting, and incubated at 4°C for 30 minutes to prepare a ligand-modified LNP suspension without mRNA (ligand amount relative to total lipids: 0.8 mol%) (step c').

[0802] Prepare an mRNA solution by mixing an aqueous solution of mRNA encoding NanoLuc (registered trademark) luciferase (mRNA amount: 2.0 μg) with 200 μL of nuclease-free water. Using a vortex mixer, mix the total volume of the resulting mRNA solution (200 μL) with 100 μL of ligand-modified LNP suspension (containing no mRNA), and incubate the mixture at 75°C for 5 minutes. Add 200 μL of phosphate buffer (buffer concentration: 9.6 mM, pH: 7.4) to the incubated mixture to prepare a ligand-modified LNP suspension encapsulated with mRNA (N / P ratio: 69, mRNA concentration: 4 μg / mL) (step b')

[0803] [Experimental Example 7] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency

[0804] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Examples 6 and 7 were measured. The results are shown in Table 8 below. Furthermore, "temperature" in Table 8 below refers to the "incubation temperature during nucleic acid encapsulation".

[0805] Table 8

[0806]

[0807] As shown in Table 8, the ligand-modified LNPs encapsulated with mRNA in Examples 6 and 7 both exhibited ideal mRNA encapsulation rates.

[0808] [Preparation Example 4] Preparation of an aqueous solution containing ligand-bound PEG lipids

[0809] Except for changing the ligand used to cRGD and the lipid used to GS-020TS, DSPE-034GS, or DSPE-050GS, an aqueous solution containing the ligand-bound PEG lipid was prepared in the same manner as in Preparation Example 1. Furthermore, the ligand-bound PEG lipids obtained using GS-020TS, DSPE-034GS, or DSPE-050GS will be referred to below as “DSPE-034-RGD,” “DSPE-050-RGD,” or “GS-020-RGD,” respectively.

[0810] [Preparation Example 5] Preparation of Empty LNP Suspension

[0811] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 2 mM of cholesterol in ethanol, and 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol at a molar ratio of 52.5 / 40 / 7.5. The resulting mixture was then mixed with 1 mM of DMG-PEG2000 in ethanol to prepare a lipid solution (the amount of DMG-PEG2000 relative to the total amount of SS-OP, cholesterol, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 1.5 mol%).

[0812] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0813] SS-OP concentration: 10.5 mM

[0814] Cholesterol concentration: 1.5 mM

[0815] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 8 mM

[0816] DMG-PEG2000 concentration: 0.3mM

[0817] Total lipid concentration: 20.3 mM

[0818] Using the nanoparticle manufacturing apparatus "iLiNP" (manufactured by Lilac Pharma Co., Ltd.), malate buffer (concentration: 20 mM, pH: 3.0) was mixed with lipid solution at a flow rate of 125 μL / min at 25°C for 2 minutes to obtain 2 mL of empty LNP suspension (step a). The same operation was performed to obtain 2 mL of empty LNP suspension (step a).

[0819] Under vortex mixing, 2.5 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was rapidly added to each of the two 2 mL aliquots of empty LNP suspensions, and the resulting 4.5 mL mixtures were combined. 4 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0) was added to the resulting 9.0 mL mixture, and the mixture was ultrafiltered using an Amicon Ultra-15 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrate was diluted with 14 mL of 2-morpholine ethanesulfonate buffer (buffer concentration: 20 mM, pH: 6.0), and the diluted solution was ultrafiltered. 100 μL of a 320 mg / mL sucrose solution was added to 100 μL of the concentrate to prepare an empty LNP suspension with an exchange dispersion medium. This LNP suspension was stored at 4 °C under an argon atmosphere until use.

[0820] [Example 8] Preparation of ligand-modified LNPs encapsulated with mRNA

[0821] Except for incubation at 37°C with the empty LNP suspension obtained in Preparation Example 5 using the exchange dispersion medium, an mRNA-encapsulated LNP suspension (N / P ratio: 36, mRNA concentration: 8.3 μg / mL) was prepared by the same procedure as in Example 5 (step b).

[0822] The LNP suspension encapsulated with mRNA (75 μL) was mixed with an aqueous solution containing ligand-bound PEG lipids (i.e., DSPE-034-RGD, DSPE-050-RGD, or GS-020-RGD) obtained in Preparation Example 4, or an aqueous solution containing 0.032 mg / mL DSPE-PEG-Mannose (75 μL) and incubated at 4 °C for 30 minutes to prepare a ligand-modified LNP suspension encapsulated with mRNA (ligand amount relative to total lipids: 0.8 mol%) (Step c).

[0823] [Example 9] Preparation of ligand-modified LNPs encapsulated with mRNA

[0824] The empty LNP suspension (15 μL) of the exchange dispersion medium obtained in Preparation Example 5 was mixed with the aqueous solution containing DSPE-034-RGD or the aqueous solution containing DSPE-PEG-Mannose (150 μL) obtained in Preparation Example 4 by pipetting, and incubated at 4°C for 30 minutes to prepare a ligand-modified LNP suspension without mRNA (ligand amount relative to total lipids: 0.8 mol%) (step c') .

[0825] An aqueous solution of mRNA encoding luciferase (mRNA amount: 3 μg) was mixed with 132 μL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 6.0) to prepare an mRNA solution. Using a vortex mixer, the total volume of the resulting mRNA solution (135 μL) was mixed with a ligand-modified LNP suspension (165 μL) containing no mRNA, and the resulting mixture was incubated at 37 °C or 75 °C for 5 minutes, respectively. Phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was added to the incubated mixture to prepare an LNP suspension encapsulated with mRNA (N / P ratio: 69, mRNA concentration: 5 μg / mL) (step b')

[0826] [Example 10] Preparation of ligand-modified LNPs encapsulated with mRNA

[0827] An aqueous solution containing ligand-bound PEG lipids (i.e., DSPE-034-RGD, DSPE-050-RGD, or GS-020-RGD) obtained in Preparation Example 4, or an aqueous solution containing DSPE-PEG-Mannose (150 μL), was mixed with a solution of mRNA encoding luciferase (mRNA amount: 3 μg, 2-morpholinoethanesulfonic acid buffer: 132 μL, buffer concentration: 20 mM, pH: 6.0) to obtain an aqueous solution containing ligand-bound PEG lipids and mRNA.

[0828] Using a vortex mixer, the total volume (285 μL) of the aqueous solution containing ligand PEG lipids and mRNA was mixed with the empty LNP suspension (15 μL) of the exchange dispersion medium obtained in Preparation Example 5. The resulting mixture was incubated at 37°C for 5 minutes and then at 4°C for 30 minutes. Phosphate buffer (300 μL, buffer concentration: 9.6 mM, pH 7.4) was added to the incubated mixture to prepare a ligand-modified LNP suspension encapsulated with mRNA (N / P ratio: 69) (Step d).

[0829] [Experimental Example 8] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency

[0830] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Examples 8-10 were measured. The results are shown in Table 9 below. Table 9 also describes the ligand-bound PEG lipids used in Examples 8-10.

[0831] Table 9

[0832]

[0833] As shown in Table 9, regardless of the type of lipids bound by the PEG ligand, the ligand-modified LNPs encapsulated with mRNA in Examples 8-10 all exhibited ideal mRNA encapsulation rates.

[0834] [Example 11] Preparation of ligand-modified LNPs encapsulated with mRNA

[0835] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 20 mM of cholesterol in ethanol, 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol, and 1 mM of DMG-PEG2000 in ethanol at a molar ratio of 52.5 / 40 / 7.5 / 1.5. A ligand-modified LNP suspension encapsulated with mRNA was prepared by following the same steps as in Example 1, except that the incubation temperature for encapsulating nucleic acids in step b) was set to 4°C, 37°C, 60°C, or 95°C, and the incubation time for encapsulating nucleic acids was set to 1 minute, 5 minutes, 30 minutes, 60 minutes, or 120 minutes.

[0836] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0837] SS-OP concentration: 10.5 mM

[0838] Cholesterol concentration: 1.5 mM

[0839] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 8 mM

[0840] DMG-PEG2000 concentration: 0.3mM

[0841] Total lipid concentration: 20.3 mM

[0842] [Experimental Example 9] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency, etc.

[0843] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Example 11 were measured. The results are shown in Table 10 below. Furthermore, in Table 10 below, "temperature" refers to the incubation temperature for encapsulating nucleic acids, and "time" refers to the incubation time for encapsulating nucleic acids.

[0844] Table 10

[0845]

[0846] [Example 12] Preparation of ligand-modified LNPs encapsulated with mRNA

[0847] A lipid solution was prepared by mixing 10 mM of SS-OP in ethanol, 20 mM of cholesterol in ethanol, 10 mM of 1,2-dioleoyl-sn-glycerol-3-phosphocholine in ethanol, and 1 mM of DMG-PEG2000 in ethanol at a molar ratio of 52.5 / 40 / 7.5 / 1.5. A ligand-modified LNP suspension encapsulated with mRNA was prepared by the same steps as in Example 1, except that the incubation for ligand modification in step c) was performed at the temperatures and times shown in Table 11 below.

[0848] The concentrations of each lipid and the total lipid concentration in the lipid solution obtained as described above are as follows.

[0849] SS-OP concentration: 10.5 mM

[0850] Cholesterol concentration: 1.5 mM

[0851] Concentration of 1,2-dioleoyl-sn-glycerol-3-phosphocholine: 8 mM

[0852] DMG-PEG2000 concentration: 0.3mM

[0853] Total lipid concentration: 20.3 mM

[0854] [Experimental Example 10] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency, etc.

[0855] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Example 12 were measured. The results are shown in Table 11 below. In addition, "temperature" in Table 11 below refers to "incubation temperature during ligand modification", and "time" refers to "incubation time during ligand modification".

[0856] Table 11

[0857]

[0858] [Preparation Example 6] Preparation of aqueous solutions containing ligand-bound PEG lipids and aqueous solutions containing both ligand-bound and unbound PEG lipids.

[0859] Except for changing the concentrations of ligand-bound PEG lipids (ligand: anti-CD3 antibody) and unbound PEG lipids (Methoxy-PEG) in the aqueous solution as described in Table 12, aqueous solutions containing ligand-bound PEG lipids and aqueous solutions containing ligand-bound PEG lipids and unbound PEG lipids were prepared in the same manner as in Preparation Example 2 (hereinafter sometimes referred to as "Aqueous Solution 6-1" to "Aqueous Solution 6-7").

[0860] Table 12

[0861]

[0862] [Example 13]

[0863] In addition to adding 0.5 mM of 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindole carbonyl cyanine perchlorate (fluorescent dye) ethanol solution to the lipid solution of Example 1, a lipid solution containing fluorescent dye was prepared with the molar ratio of SS-OP / cholesterol / 1,2-dioleoyl-sn-glycerol-3-phosphocholine / DMG-PEG2000 / 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindole carbonyl cyanine perchlorate being 52.5 / 40 / 7.5 / 1.5 / 0.5.

[0864] A lipid solution containing the fluorescent dye was used instead of the lipid solution in Example 1; and

[0865] In step c), any one of the aqueous solutions 6-1 to 6-7 obtained in Preparation Example 6 can be used instead of the aqueous solution containing the ligand-bound PEG lipid obtained in Preparation Example 1.

[0866] In addition, ligand-modified LNP suspensions encapsulated with mRNA were prepared in the same manner as in Example 1 (hereinafter sometimes referred to as "Suspension 13-1" to "Suspension 13-7").

[0867] [Experimental Example 11] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency, etc.

[0868] Similar to Experimental Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA in Example 13 were measured. The results are shown in Table 13 below. Furthermore, Table 13 below records the ligand-modified LNP suspensions encapsulated with mRNA in Example 13 (suspensions 13-1 to 13-7) and the aqueous solutions used in Preparation Example 6 (aqueous solutions 6-1 to 6-7). Additionally, the columns for "Ligand Amount" and "Mass of Unbound PEG Lipids" in Table 13 below also record the amount of ligand (anti-CD3 antibody) relative to total lipids and the amount of unbound PEG lipids (Methoxy-PEG) used in step c) relative to total lipids, respectively.

[0869] Table 13

[0870]

[0871] [Experimental Example 12] Evaluation of LNP gene expression activity in vivo

[0872] The ligand-modified LNP suspensions No. 13-1 to No. 13-3 encapsulated with mRNA obtained in Example 13 or the LNP suspensions encapsulated with mRNA but not ligand-modified in Comparative Example 3 (mRNA amount: 1.0 μg) were administered to C57BL / 6 mice via the tail vein.

[0873] Twenty-one hours after drug administration, mice were euthanized and their spleens were removed. The resulting spleens were recovered and placed in a culture dish containing 3 mL of RPMI-1640 with L-Gln liquid (manufactured by Nacalai Tesque Co., Ltd.) (1% bovine fetal serum (manufactured by SIGMA)). Splenic cells were filtered out using forceps, and using a 5 mL syringe, the cells were resuspended in spleen cell culture medium. The resulting cell suspension was passed through a 70 μL cell filter, and the cells were recovered into a 15 mL test tube. The recovered cells were centrifuged at 4°C and 500 g for 4 minutes, and the supernatant was removed to obtain a cell pellet. 1 mL of Red Blood Cell Lysing Buffer was added to the cell pellet and the pellet was resuspended. The red blood cells were removed by incubating the cell suspension at room temperature for 5 minutes. The cell suspension, after removing red blood cells, was diluted 5-fold with FACS buffer (0.05 wt% sodium azide aqueous solution and 0.5 wt% PBS containing bovine fetal serum albumin). The diluted solution was centrifuged, and 5 mL of FACS buffer was added to the cell pellet after removing the supernatant. The mixture was centrifuged again, and the supernatant was removed. The resulting cell pellet was resuspended in 3 mL of FACS buffer. The resulting cell suspension was diluted 10-fold with FACS buffer. 18 μL of the diluted cell suspension was mixed with 2 μL of acridine orange to obtain a final mixture. Cell counting was performed using a LUNA-FL (Logos Biosystems) analyzer. FACS buffer was added to the cell suspension to achieve a cell concentration of 1 × 10⁻⁶ cells / mL. 6The cell suspension was then centrifuged at 4°C and 500g for 4 minutes, and the supernatant was removed to obtain a cell pellet. To prevent nonspecific staining, 2 μL of CD16 / 32 was mixed with 98 μL of FACS buffer to prepare a blocking solution. 50 μL of blocking solution was added to the cell pellet and mixed by tapping to obtain a cell suspension. The cell suspension was incubated at 4°C in the dark for 10 minutes. BV605-labeled anti-CD45 antibody, BV421-labeled anti-CD3ε antibody, BV510-labeled anti-CD19 antibody, PE-labeled anti-CD4 antibody, and AF488-labeled anti-CD8a antibody were diluted with blocking solution to the manufacturer's recommended concentration to obtain a staining antibody mixture. 54 μL of the staining antibody mixture was added to 50 μL of the cell suspension, and the resulting mixture was incubated at 4°C in the dark for 30 minutes. During the first 5 minutes of observation, 5 μL of 7-AAD Viability Staining Solution (BioLegend) was added sequentially to the mixture for dead cell staining. 1 mL of FACS buffer was added to the cell suspension containing the antibody mixture and 7-AAD, and the centrifugation and supernatant removal were repeated twice. The resulting cell pellet was resuspended in 500 μL of FACS buffer. The resulting suspension (sample) was analyzed using Novocyte assays, and the percentage of DiD-positive T cells (%) was calculated (=100 × number of DiD-fluorescent T cells / total number of T cells). Here, T cells exhibiting DiD fluorescence are those that have taken up LNP. Specifically, the cell population stained with BV421-labeled anti-CD3ε antibody in the cell suspension was considered T cells, and the proportion of DiD-fluorescent T cells (i.e., T cells that have taken up LNP) relative to the total number of T cells was calculated. The results are shown in Table 14 below.

[0874] Table 14

[0875]

[0876] As shown in Table 14, it was confirmed that the proportion (%) of DiD positive cells in T cells was increased in the ligand-modified LNPs containing mRNA encapsulated in Example 13 compared with the LNPs containing mRNA encapsulated but not modified with ligands in Comparative Example 3.

[0877] [Example 14] Preparation of ligand-modified LNPs encapsulated with mRNA

[0878] Except for adding 1 mM of 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole carbonylcyanine perchlorate (fluorescent dye) to the lipid solution of Preparation Example 5, a preparative lipid solution containing fluorescent dye was prepared with a molar ratio of 52.5 / 40 / 7.5 / 1.5 / 0.5 for SS-OP / cholesterol / 1,2-dioleoyl-sn-glycerol-3-phosphocholine / DMG-PEG2000 / 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole carbonylcyanine perchlorate; and

[0879] A lipid solution containing the fluorescent dye was used instead of the lipid solution used in Preparation Example 5.

[0880] In addition, the same steps a) and the exchange of dispersion medium were performed as in Preparation Example 5 to prepare an empty LNP suspension with exchanged dispersion medium.

[0881] Using the NanoAssemblr Ignite nanoparticle fabrication apparatus (manufactured by Precision NanoSystems), an empty LNP suspension with an exchange dispersion medium was mixed with a solution of mRNA encoding luciferase at a flow rate of 5 mL / min at 25°C for 55 seconds to obtain a 9.2 mL suspension (9200 μL, mRNA concentration: 184 μg). This solution was divided into 4.6 mL portions, and either unincubated or incubated at 37°C for 5 minutes to prepare LNP suspensions encapsulated with mRNA (N / P ratio: 36, mRNA concentration: 20 μg / mL) (step b)).

[0882] Using the NanoAssemblr Ignite nanoparticle fabrication apparatus (manufactured by Precision NanoSystems), the obtained LNP suspension encapsulated with mRNA was mixed with an aqueous solution of PEG lipids containing ligand-bound and unbound PEG lipids obtained in Preparation Example 2 at a flow rate of 0.5 mL / min at 25°C for 60 seconds. Alternatively, the obtained LNP suspension encapsulated with mRNA was mixed with an aqueous solution of PEG lipids containing ligand-bound and unbound PEG lipids obtained in Preparation Example 2 at a flow rate of 5 mL / min at 25°C for 6 seconds to obtain a 1 mL suspension. This suspension was divided into 0.5 mL portions and subjected to either no-incubation treatment or incubation at 4°C for 30 minutes. Add phosphate buffer (0.5 mL, buffer concentration: 9.6 mM, pH 7.4) to prepare a ligand-modified LNP suspension encapsulated with mRNA (ligand amount relative to total lipids: 0.1 mol%, 0.2 mol%, or 0.4 mol%) (step c) to the suspension.

[0883] [Experimental Example 13] Evaluation of LNP particle size, PdI, and mRNA encapsulation efficiency, etc.

[0884] Similar to Example 1, the particle size, PdI, and mRNA encapsulation efficiency of the ligand-modified LNPs encapsulated with mRNA from Example 14 were measured. The results are shown in Table 15 below. Table 15 also records the incubation treatments in steps b) and c), the flow rate of the LNP suspension encapsulated with mRNA in step c), and the flow rate of the aqueous solution containing ligand-bound PEG lipids and unbound PEG lipids. Furthermore, the two flow rates are combined in the "Flow Rate of Step c)" column of Table 15 below. Additionally, the "Amount of Ligand (Anti-CD3 Antibody) relative to total lipids" column of Table 15 below also records the "Amount of Ligand (Anti-CD3 Antibody)."

[0885] Table 15

[0886]

[0887] [Industry Applicability]

[0888] The nucleic acid-encapsulated lipid nanoparticles obtained by the manufacturing method of the present invention are useful for nucleic acid drugs, gene therapy, biochemical experiments, etc.

[0889] This application is based on Japanese Patent Application No. 2023-159114, the contents of which are fully contained in this specification.

Claims

1. A method for manufacturing ligand-modified lipid nanoparticles encapsulated with nucleic acids, comprising: Step a) involves mixing an alcoholic solution containing ionic lipids, sterols, and PEG lipids with an acidic buffer solution with a pH of 1–6.5 to obtain a suspension of lipid nanoparticles that do not contain nucleic acids; and includes Step b) involves mixing lipid nanoparticles that do not contain nucleic acids with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulated with nucleic acids. And step c), the suspension of lipid nanoparticles encapsulated with nucleic acids is mixed with lipids bound with ligands to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids. Or include Step c') involves mixing lipid nanoparticles without nucleic acids, water, and lipids bound with ligands to obtain a suspension of ligand-modified lipid nanoparticles without nucleic acids; and step b') involves mixing ligand-modified lipid nanoparticles without nucleic acids with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acids. Or include Step d) involves mixing lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

2. The method according to claim 1, wherein, In step b), a suspension of lipid nanoparticles without nucleic acid is mixed with a nucleic acid solution to obtain a suspension of lipid nanoparticles encapsulated with nucleic acid; or In step c'), a suspension of lipid nanoparticles without nucleic acid is mixed with lipids bound to ligands to obtain a suspension of ligand-modified lipid nanoparticles without nucleic acid; and in step b'), a suspension of ligand-modified lipid nanoparticles without nucleic acid is mixed with a nucleic acid solution to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid. or In step d), a suspension of lipid nanoparticles without nucleic acid, a nucleic acid solution, and lipids bound with ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

3. The method according to claim 2, wherein, Includes process a), process b), and process c), or, includes process a), process c'), and process b').

4. The method according to any one of claims 1 to 3, wherein, The process includes the following steps: mixing the nucleic acid-free lipid nanoparticles of step b) with a nucleic acid solution, or the nucleic acid-free ligand-modified lipid nanoparticles of step b') with a nucleic acid solution, and maintaining the mixture at 0~95℃ for 1 minute to 2 hours.

5. The method according to any one of claims 1 to 3, wherein, The process includes the following steps: 1) maintaining the suspension of nucleic acid-encapsulated lipid nanoparticles from step c) with a mixture of lipids bound to ligands, or the mixture of nucleic acid-free lipid nanoparticles, water, and lipids bound to ligands from step c') at 0-95°C for 1 minute to 5 hours.

6. The method according to claim 1 or 2, wherein, The process includes the following steps: maintaining a mixture of lipid nanoparticles without nucleic acid, nucleic acid solution, and lipids bound with ligands from step d) at 0~95℃ for 1 minute to 2 hours.

7. The method according to claim 1 or 2, wherein, In step c), a suspension of nucleic acid-encapsulated lipid nanoparticles, lipids bound to ligands, and lipids without bound ligands are mixed to obtain a suspension of nucleic acid-encapsulated ligand-modified lipid nanoparticles; or In step c'), lipid nanoparticles without nucleic acids, water, lipids bound to ligands, and lipids without bound ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles without nucleic acids; or In step d), lipid nanoparticles without nucleic acid, nucleic acid solution, lipids bound with ligands and lipids without ligands are mixed to obtain a suspension of ligand-modified lipid nanoparticles encapsulated with nucleic acid.

8. The method according to any one of claims 1 to 3, wherein, The alcohol solution in step a) further contains phospholipids.

9. The method according to any one of claims 1 to 3, wherein, Ionic lipids include at least one selected from the group consisting of compounds represented by formula (1), [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; and (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate. Compounds represented by formula (1): In equation (1), R 1a and R 1b Each of the alkylene groups having 1 to 6 carbon atoms can be independently represented. X a and X b Each of these can be used independently to represent a noncyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups. R 2a and R 2b Each can be used independently to represent an alkylene group or an oxadiene group having 8 or fewer carbon atoms. Y a and Y b Each can be used independently to represent an ester bond, amide bond, carbamate bond, ether bond, or urea bond. Z a and Z b Each of these groups independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and possibly heteroatoms. n a and n b Each can be independently 0 or 1. R 3a and R 3b Represented independently: Residues derived from the reaction products of fat-soluble vitamins with hydroxyl groups and succinic anhydride or glutaric anhydride; Residues derived from the reaction product of sterol derivatives with hydroxyl groups and succinic anhydride or glutaric anhydride; Aliphatic hydrocarbon groups with 1 to 40 carbon atoms; Alkyl groups having a cyclopropane ring with 3 to 40 carbon atoms; The group represented by formula (3); Groups with 50 or fewer carbon atoms represented by formula (4); or Groups with 50 or fewer carbon atoms represented by formula (5), Equation (3): R 9 -O-CO-(CH2) a -* (3) In equation (3), * indicates the bonding location. R 9 This refers to aliphatic hydrocarbon groups with 2 to 20 carbon atoms. a represents an integer from 2 to 10; Groups with 50 or fewer carbon atoms represented by formula (4): In equation (4), * indicates the bonding location, and R 10 It represents an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, or a hydrocarbon cyclic group having 3 to 12 carbon atoms, and R 10 The substituents selected from the group consisting of heterocyclic groups of 3 to 14 members and hydrocarbon cyclic groups of 3 to 12 carbon atoms are either substituted or not substituted; Groups with 50 or fewer carbon atoms represented by formula (5): In equation (5), * indicates the bonding location. R 11 It represents an alkylene group with 2 to 9 carbon atoms, an alkenyl group with 2 to 9 carbon atoms, or an alkynyl group with 2 to 9 carbon atoms, and R 11 The substituents selected from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups with 3 to 12 carbon atoms may be substituted or not substituted. R 12 and R 13 R represents an alkyl group having 1 to 17 carbon atoms, an alkenyl group having 2 to 17 carbon atoms, or an alkynyl group having 2 to 17 carbon atoms, respectively. 12 At least one ethylene or at least one trimethylene in R is replaced or not replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds. 13 At least one ethylene or at least one trimethylene in the form is replaced or not replaced by at least one bond selected from the group consisting of ester bonds, amide bonds, carbamate bonds, and carbonate bonds, and R 12 and R 13 The substituents selected independently from the group consisting of halogen atoms, hydroxyl groups, and hydrocarbon ring groups with 3 to 12 carbon atoms may be substituted or not substituted. X 4 It represents an oxygen atom, NH, or a sulfur atom.

10. The method according to claim 9, wherein, Ionic lipids include compounds represented by formula (1).

11. The method according to any one of claims 1 to 3, wherein, The ligands in lipids that are bound to ligands are either ligands that target brain cells or ligands that target T cells.

12. A method for introducing nucleic acids into cells, comprising the step of: contacting ligand-modified lipid nanoparticles encapsulated with nucleic acids, obtained by any one of claims 1 to 3, with cells in vitro.

13. A method for introducing nucleic acid into target cells within an organism, comprising the steps of: administering ligand-modified lipid nanoparticles encapsulated with nucleic acid, obtained by any one of claims 1 to 3, to the organism.

14. A method for manufacturing a pharmaceutical composition, comprising the method according to any one of claims 1 to 3.

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