Freeze-dried composition of lipid nanoparticles

Abstract

Provided is a lyophilized composition in which an arbitrary nucleic acid can be highly efficiently and easily encapsulated. This lipid nanoparticle lyophilized composition is free from nucleic acids and contains an ionic lipid, sterol, a PEG-lipid, an acidic buffer solution component having a buffering action at a pH of 1-6, and a cryoprotectant. The weight ratio of the cryoprotectant and all the lipids is 10:1 to 1000:1.

Description

[Technical field]

[0001] The present invention relates to a freeze-dried composition of lipid nanoparticles that does not contain nucleic acid, and a method for producing lipid nanoparticles that encapsulate nucleic acid using the same. [Background technology]

[0002] Effective and safe nucleic acid delivery carriers are required for practical application of nucleic acid therapy using oligonucleotides such as siRNA and gene therapy using mRNA, pDNA, etc. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but development of non-viral nucleic acid delivery carriers that are safer to use is underway.

[0003] Cationic liposomes using cationic lipids having quaternary amines are positively charged, and therefore can form a complex (lipoplex) with negatively charged nucleic acid through electrostatic interaction, and can deliver nucleic acid into cells. Furthermore, by utilizing the electrostatic interaction between quaternary amines and nucleic acid, a freeze-dried composition of cationic liposomes not containing nucleic acid can be prepared, and rehydrated with an aqueous solution of nucleic acid to form a lipoplex, so that it can also be used as a gene transfer reagent (Patent Documents 1 and 2).

[0004] However, it is difficult to control the particle size of lipoplexes prepared by such a method, and cytotoxicity derived from positively charged cationic lipids is a problem.

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

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

[0007] Although various non-viral carriers have been developed, nucleic acids are generally unstable compounds, and therefore there are still issues regarding their stability as formulations.

[0008] As one method for improving the stability of a formulation, attempts have been made to freeze-dry lipid nanoparticles encapsulating nucleic acids and then rehydrate them at the time of use to reconstitute the lipid nanoparticles (Patent Document 4 and Non-Patent Document 2).

[0009] These methods are useful for increasing the storage stability of lipid nanoparticles encapsulating a specific nucleic acid, but have issues as a method for more simply encapsulating any nucleic acid into lipid nanoparticles.

[0010] A method for easily encapsulating any nucleic acid in lipid nanoparticles includes the method described in Patent Documents 1 and 2, in which a freeze-dried composition not containing nucleic acid is prepared and then rehydrated with an aqueous solution of nucleic acid.

[0011] However, lipid nanoparticles obtained using ionic lipids having a tertiary amine in the molecule have a surface charge after preparation that ranges from a weak negative charge to neutral, and therefore do not interact electrostatically with nucleic acids. Therefore, it is not possible to prepare nucleic acid-encapsulated lipid nanoparticles using the method disclosed in Patent Documents 1 and 2 in which a freeze-dried composition not containing nucleic acid is produced and then rehydrated with an aqueous solution of nucleic acid.

[0012] As described above, there has been no means for easily encapsulating any nucleic acid in lipid nanoparticles with a high nucleic acid encapsulation rate. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Patent No. 4919397 [Patent Document 2] Patent No. 4598908 [Patent Document 3] Patent No. 6093710 [Patent Document 4] International Publication No. 2017 / 218704 [Non-patent literature]

[0014] [Non-Patent Document 1] Gene Therapy 6:271-281, 1999 [Non-Patent Document 2] Biol. Pharm. Bull. 41, 1291-1294(2018) Summary of the Invention [Problem to be solved by the invention]

[0015] The objective of the present invention is to provide a freeze-dried composition that can encapsulate any nucleic acid with high efficiency and ease, something that could not be achieved with conventional technology, and to provide a method for producing nucleic acid-encapsulated lipid nanoparticles using the same. [Means for solving the problem]

[0016] In view of the above problems, the present inventors have made extensive efforts and found that any nucleic acid can be encapsulated in lipid nanoparticles with high efficiency and ease by preparing lipid nanoparticles that do not contain nucleic acid in an acidic buffer, adding a cryoprotectant, lyophilizing the nanoparticles, and then rehydrating the nanoparticles with an aqueous solution containing nucleic acid. Furthermore, they carried out gene transfer experiments into cells using lipid nanoparticles prepared by this method and found that uniform gene transfer into cells is possible by increasing the concentration of the cryoprotectant before lyophilization, thus completing the present invention.

[0017] That is, the present invention encompasses the following: [1] A freeze-dried composition of lipid nanoparticles that does not contain nucleic acids and contains an ionic lipid, a sterol, a PEG lipid, an acidic buffer component having a buffering effect at pH 1 to 6, and a cryoprotectant, A freeze-dried composition, wherein the weight ratio of cryoprotectant to total lipid is 10:1 to 1000:1.

[0018] [2] The freeze-dried composition according to [1], further comprising a phospholipid.

[0019] [3] The freeze-dried composition according to [1] or [2], wherein the weight ratio of cryoprotectant to total lipid is 30:1 to 1000:1.

[0020] [4] The freeze-dried composition according to any one of [1] to [3], wherein the concentration of the cryoprotectant is 80 to 800 mg / mL in the composition before freeze-drying.

[0021] [5] The freeze-dried composition according to any one of [1] to [4], wherein the concentration of the cryoprotectant is 160 to 800 mg / mL in the composition before freeze-drying.

[0022] [6] The freeze-dried composition according to any one of [1] to [5], wherein the ionic lipid is a compound represented by formula (1):

[0023] [ka]

[0024] (In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups, R 2a and R 2b each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z a and Z beach independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; R 3a and R 3b each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0025] [7] The freeze-dried composition according to any one of [1] to [6], wherein the cryoprotectant is a disaccharide.

[0026] [8] The freeze-dried composition according to any one of [1] to [6], wherein the cryoprotectant is sucrose.

[0027] [9] A method for producing nucleic acid-encapsulated lipid nanoparticles, comprising the steps of: a) mixing an alcohol solution containing ionic lipids, sterols and PEG lipids with an acidic buffer solution having a buffering effect at pH 1 to 6 to prepare a suspension of lipid nanoparticles not containing nucleic acids; b) mixing a suspension of nucleic acid-free lipid nanoparticles with a cryoprotectant to obtain a mixture containing 80-800 mg / mL of cryoprotectant and having a pH of 1-6; c) freeze-drying the mixture obtained in step b to obtain a freeze-dried composition; d) mixing the lyophilized composition with an aqueous solution containing nucleic acid and optionally containing 0-25 v / v% alcohol, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles; and e) exchanging the external aqueous phase of the resulting nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration or dilution.

[0028]

[10] The method according to [9], further comprising, in step a, exchanging the external aqueous phase for another acidic buffer solution having a buffering effect at pH 1 to 6 by dialysis, ultrafiltration or dilution after preparation of the lipid nanoparticle suspension.

[0029]

[11] The method according to [9] or

[10] , wherein in step a, the alcohol solution further contains a phospholipid.

[0030]

[12] The method according to any one of [9] to

[11] , wherein in step b, the concentration of the cryoprotectant in the mixture is 160 to 800 mg / mL.

[0031]

[13] The method according to any one of [9] to

[12] , wherein the ionic lipid is a compound represented by the formula (1).

[0032]

[14] The method according to any one of [9] to

[13] , wherein the cryoprotectant is a disaccharide.

[0033]

[15] The method according to any one of [9] to

[13] , wherein the cryoprotectant is sucrose.

[0034]

[16] A method for producing a freeze-dried composition of lipid nanoparticles, comprising the step of freeze-drying a lipid nanoparticle composition that does not contain nucleic acids and contains an ionic lipid, a sterol, a PEG lipid, an acidic buffer solution having a buffering effect at pH 1 to 6, and 80 to 800 mg / mL of a cryoprotectant.

[0035]

[17] The method according to

[16] , wherein the lipid nanoparticle composition further comprises a phospholipid.

[0036]

[18] The method according to

[16] or

[17] , wherein the concentration of the cryoprotectant in the composition before lyophilization is 160 to 800 mg / mL.

[0037]

[19] The method according to any one of

[16] to

[18] , wherein the ionic lipid is a compound represented by the formula (1).

[0038]

[20] The method according to any one of

[16] to

[19] , wherein the cryoprotectant is a disaccharide.

[0039]

[21] The method according to any one of

[16] to

[19] , wherein the cryoprotectant is sucrose.

[0040]

[22] A nucleic acid transfer agent comprising the freeze-dried composition described in any one of [1] to [8].

[0041]

[23] A method for introducing a nucleic acid into a cell, comprising contacting the cell with the nucleic acid introduction agent according to

[22] containing the nucleic acid ex vivo.

[0042]

[24] A method for introducing a nucleic acid into a cell, comprising administering the nucleic acid introduction agent according to

[22] encapsulating a nucleic acid to a living body so as to deliver the nucleic acid to the target cell.

[0043]

[25] A method for introducing a nucleic acid into a cell, comprising the steps of: a) mixing an alcohol solution containing ionic lipids, sterols and PEG lipids with an acidic buffer solution having a buffering effect at pH 1 to 6 to prepare a suspension of lipid nanoparticles not containing nucleic acids; b) mixing a suspension of nucleic acid-free lipid nanoparticles with a cryoprotectant to obtain a mixture containing 80-800 mg / mL of cryoprotectant and having a pH of 1-6; c) freeze-drying the mixture obtained in step b to obtain a freeze-dried composition; d) mixing the freeze-dried composition with an aqueous solution containing the nucleic acid and optionally containing 0-25 v / v % alcohol, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles; e) exchanging the external aqueous phase of the resulting nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration or dilution; and f) contacting the resulting nucleic acid-encapsulated lipid nanoparticles with the cells in vitro.

[0044]

[26] The method according to

[25] , further comprising, in step a, exchanging the external aqueous phase for another acidic buffer solution having a buffering effect at pH 1 to 6 by dialysis, ultrafiltration or dilution after preparation of the lipid nanoparticle suspension.

[0045]

[27] A method for introducing a nucleic acid into a target cell, comprising the steps of: a) mixing an alcohol solution containing ionic lipids, sterols and PEG lipids with an acidic buffer solution having a buffering effect at pH 1 to 6 to prepare a suspension of lipid nanoparticles not containing nucleic acids; b) mixing a suspension of nucleic acid-free lipid nanoparticles with a cryoprotectant to obtain a mixture containing 80-800 mg / mL of cryoprotectant and having a pH of 1-6; c) freeze-drying the mixture obtained in step b to obtain a freeze-dried composition; d) mixing the freeze-dried composition with an aqueous solution containing the nucleic acid and optionally containing 0-25 v / v % alcohol, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles; e) exchanging the external aqueous phase of the resulting nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration or dilution; and f) administering the resulting nucleic acid-encapsulated lipid nanoparticles to a living body so that they are delivered to the target cells.

[0046]

[28] The method according to

[27] , further comprising, in step a, exchanging the external aqueous phase for another acidic buffer solution having a buffering effect at pH 1 to 6 by dialysis, ultrafiltration or dilution after preparation of the lipid nanoparticle suspension. Effect of the Invention

[0047] The present invention relates to a freeze-dried composition of nucleic acid-free lipid nanoparticles and a method for preparing lipid nanoparticles using the same. By rehydrating the freeze-dried composition of the present invention with an aqueous solution containing nucleic acid, lipid nanoparticles encapsulating any nucleic acid can be prepared highly efficiently and simply. In the method for preparing lipid nanoparticles of the present invention, the encapsulation rate of nucleic acid can be further increased by adding any aqueous solution of nucleic acid and further adding alcohol to the freeze-dried composition of the present invention and incubating the mixture. When gene transfer is performed using lipid nanoparticles prepared by the method of the present invention, genes can be transferred uniformly into cells. Furthermore, the lipid nanoparticles prepared by the method of the present invention have a particle size of 50 to 200 nm and a narrow particle size distribution, making them advantageous for gene transfer in vivo. [Brief description of the drawings]

[0048] [Figure 1] FIG. 1 is a graph evaluating the effect of sucrose concentration on the uniformity of gene transfer into cells. [Diagram 2] FIG. 1 is a graph evaluating the effect of sucrose concentration on the expression intensity of gene introduced into cells. [Diagram 3] FIG. 1 is a graph evaluating the effect of lipid composition on efficiency of gene transfer into cells when DOPC is used as the phospholipid. [Figure 4] FIG. 1 is a graph evaluating the effect of lipid composition on efficiency of gene transfer into cells when POPC is used as the phospholipid. [Diagram 5] FIG. 1 is a graph evaluating the effect of lipid composition on the efficiency of gene transfer into cells when DOPE is used as the phospholipid. [Figure 6] FIG. 1 shows an evaluation of the effect of lipid composition on the efficiency of gene transfer into cells when POPE was used as the phospholipid. [Figure 7] FIG. 1 is a graph showing the evaluation of mRNA expression efficiency in vivo in mice. [Figure 8] FIG. 1 shows the in vivo CTL activity in mice. [Figure 9] FIG. 1 is a graph comparing the efficiency of gene transfer into cells between particles prepared by a conventional method and the particles of the present invention. [Figure 10] FIG. 1 is a graph comparing the efficiency of mRNA expression in vivo in mice between particles prepared by a conventional method and the particles of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these.

[0050] The present invention relates to a freeze-dried composition of lipid nanoparticles that does not contain nucleic acids and contains an ionic lipid, a sterol, a PEG lipid, an acidic buffer component having a buffering effect at pH 1 to 6, and a cryoprotectant, The freeze-dried composition has a weight ratio of cryoprotectant to total lipid of 10:1 to 1000:1.

[0051] Lipid nanoparticles refer to particles having a membrane structure in which the hydrophilic group of amphiphilic lipid is arranged toward the aqueous phase side of the interface. "Amphiphilic lipid" refers to lipid having both a hydrophilic group that shows hydrophilicity and a hydrophobic group that shows hydrophobicity. Examples of amphiphilic lipids include ionic lipids, phospholipids, PEG lipids, etc. The lipid nanoparticles of the present invention contain ionic lipids, sterols and PEG lipids as membrane constituents, and may further contain phospholipids. The particle size of the lipid nanoparticles is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. The particle size can be measured using a particle size distribution measuring device such as Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted depending on the preparation method of the lipid nanoparticles. In the present invention, the particle size means the average particle size (number average) measured by dynamic light scattering. In the present invention, "total lipid" refers to the total amount of lipid. The lipid includes ionic lipid, sterol, PEG lipid, and phospholipid. In the present invention, "free of nucleic acids" or "free of nucleic acids" means substantially free of nucleic acids, and means that the content of nucleic acids is below the detection limit. In the present invention, the term "nucleic acid-encapsulated lipid nanoparticles" refers to lipid nanoparticles in which nucleic acid is encapsulated inside the lipid nanoparticles.

[0052] The freeze-dried composition of the present invention is obtained by dissolving ionic lipids, sterols, PEG lipids or even phospholipids in a water-soluble organic solvent, mixing with an acidic buffer solution to induce organization, and then adding a cryoprotectant to the lipid nanoparticles, followed by freeze-drying. Examples of the water-soluble organic solvent include alcohols such as tert-butanol and ethanol.

[0053] Ionic Lipids The ionic lipid that can be used in the present invention may be any lipid that is composed of a tertiary amino group and a hydrophobic group and is capable of forming lipid nanoparticles. Specific examples of ionic lipids include 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (referred to as DLin-MC3-DMA or MC3), and a compound of the following formula (1) or formula (2), preferably the compounds of the following formula (1) or formula (2), DODMA, and MC3, and most preferably the compound of the following formula (1). Formula (1)

[0054] [ka]

[0055] (In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X beach independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups, R 2a and R 2b each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z a and Z b each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; R 3a and R 3b each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0056] R 1a and R 1b are each independently an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 1 to 4, and more preferably 1 to 2. Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, and a neopentylene group. R 1a and R 1b are preferably each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each is an ethylene group.

[0057] R 1a is R1b may be the same as or different from, but preferably, R 1a is R 1b It is the same group as

[0058] X a and X b each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups, and preferably each independently represents a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups.

[0059] The alkyl group having 1 to 6 carbon atoms in the non-cyclic alkyl tertiary amino group having 1 tertiary amino group may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group, and preferably a methyl group, an ethyl group, a propyl group, or an isopropyl group, and most preferably a methyl group.

[0060] A preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is: 1 As shown in the figure.

[0061] [ka]

[0062] X 1 R 5represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group, among which a methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is most preferred.

[0063] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is preferably 4 to 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include an aziridylene group, an azetidylene group, a pyrrolidine group, a piperidylene group, an imidazolidylene group, and a piperaziylene group, preferably a pyrrolidine group, a piperidylene group, and a piperaziylene group, and most preferably a piperidylene group.

[0064] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is X 2 As shown in the figure.

[0065] [ka]

[0066] X 2 p is 1 or 2. When p is 1, X 2 is a pyrrolidine group, and when p is 2, X 2 is a piperidylene group. Preferably, p is 2.

[0067] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X3 As shown in the figure.

[0068] [ka]

[0069] X 3 In the formula, w is 1 or 2. When w is 1, X 3 is an imidazolidylene group, and when w is 2, X 3 is a piperazylene group.

[0070] X a isX b may be the same as or different from, but preferably, X a isX b It is the same group as

[0071] R 2a and R 2b each independently represents an alkylene group or oxydialkylene group having 8 or less carbon atoms, and preferably each independently represents an alkylene group having 8 or less carbon atoms.

[0072] The alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear. The number of carbon atoms contained in the alkylene group is preferably 6 or less, and most preferably 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include a methylene group, an ethylene group, a propylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group, and are preferably a methylene group, an ethylene group, a propylene group, or a tetramethylene group, and most preferably an ethylene group.

[0073] An oxydialkylene group having 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) via an ether bond, and the total number of carbon atoms of the two alkylene groups present is 8 or less. Here, the two alkylene groups present may be the same or different, but are preferably the same. Specific examples of the oxydialkylene group having 8 or less carbon atoms include an oxydimethylene group, an oxydiethylene group, an oxydipropylene group, and an oxydibutylene group. An oxydimethylene group, an oxydiethylene group, and an oxydipropylene group are preferred, and an oxydiethylene group is most preferred.

[0074] R 2a is R 2b may be the same as or different from each other, but preferably, R 2a is R 2b It is the same group as

[0075] Y a and Y b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or a carbamate bond, more preferably each independently an ester bond or an amide bond, and most preferably each an ester bond. a and Y b The bond orientation of Y is not restricted. a and Y b When -Z is an ester bond, it is preferably a -CO-OR 2a - and -Z b -CO-OR 2b - structure.

[0076] Y a is Y b may be the same as or different from, but preferably, Y a is Y b It is the same group as

[0077] Z a and Z beach independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom. The number of carbon atoms contained in the aromatic compound is preferably 6 to 12, and most preferably 6 to 7. The aromatic compound preferably contains one aromatic ring.

[0078] Examples of the aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms include, for the aromatic hydrocarbon ring, a benzene ring, a naphthalene ring, and an anthracene ring, and for the aromatic heterocycle, an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furanthiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring, among which a benzene ring, a naphthalene ring, and an anthracene ring are preferred, and a benzene ring is most preferred. The aromatic ring may have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a carbon Examples of the alkyl group include an aryl group having 6 to 10 carbon atoms and an aryloxy group having 6 to 10 carbon atoms. Preferred examples of the alkyl group include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group.

[0079] Z a and Z b Preferred specific structures of Z 1 Examples include:

[0080] [ka]

[0081] In the formula, s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R 4 each independently represents a substituent.

[0082] Z 1 In the above formula, s is preferably an integer of 0 to 1, and more preferably 0.

[0083] Z 1The letter t is preferably an integer of 0 to 2, and more preferably 1.

[0084] Z 1 The character u is preferably an integer of 0 to 2, and more preferably an integer of 0 to 1.

[0085] Z 1 R 4 is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms, which does not inhibit the reaction in the synthesis process of the cationic lipid. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, a phenyl group ... R is an aryl group having 6 to 10 carbon atoms, and the like, and preferred examples thereof include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group. 4 If there are multiple R 4 may be the same or different.

[0086] Z a Z b may be the same as or different from, but preferably, Z a Z b It is the same group as

[0087] R3a and R 3b each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, preferably each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and most preferably each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0088] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0089] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and preferably cholesterol or cholestanol.

[0090] The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The unsaturated bond includes a carbon-carbon double bond and a carbon-carbon triple bond, and is preferably a carbon-carbon double bond. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, and the like, and is preferably an alkyl group or an alkenyl group. Specific examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group, docosyl group, dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosyl group, docosenyl group, dodecadienyl group, tridecadienyl group, tetradecen ... tridecadienyl group, icosenyl group, henicosyl group, docosenyl group, tridecadienyl group, icosenyl group, henicosyl group, docosenyl group, tridecadienyl group, icosenyl group, henicosyl group, docosenyl group, tridec Examples of such alkyl groups include dienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group, and 1-decylundecyl group. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0091] In one embodiment of the present invention, R 3a and R 3bThe aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by the formula (1) is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). Specific examples of the aliphatic hydrocarbon group include a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.

[0092] R 3a is R 3b may be the same as or different from, but preferably, R 3a is R 3b It is the same group as

[0093] In one embodiment of the present invention, R 1a is R 1b is the same as X a isX b is the same as R 2a is R 2b is the same as Y a is Y b is the same as Z a Z b is the same as R 3a is R 3b is equivalent to.

[0094] Suitable examples of the cationic lipid represented by formula (1) include the following cationic lipids: [Cationic lipid (1-1)] R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); X a and X b each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., -N(CH 3 )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., a piperidylene group); R 2a and R 2beach independently represents an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y b are each independently an ester bond or an amide bond; Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C 6 H 4 -CH 2 -, -CH 2 -C 6 H 4 -CH 2 -) and; R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group); Cationic lipids (1).

[0095] [Cationic lipids (1-2)] R 1a and R 1b each independently represents an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); X a and X b each independently represents a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., -N(CH 3 )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., a piperidylene group); R 2a and R 2b each independently represents an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y bare each independently an ester bond or an amide bond; Z a and Z b are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C 6 H 4 -CH 2 -, -CH 2 -C 6 H 4 -CH 2 -) and; R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group); Cationic lipids (1).

[0096] [Cationic lipids (1-3)] R 1a and R 1b each independently represents an alkylene group having 1 to 2 carbon atoms (e.g., a methylene group, an ethylene group); X a and X b are independent of each other, X 1 :

[0097] [ka]

[0098] (In the formula, R 5 is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X 2 :

[0099] [ka]

[0100] (In the formula, p is 1 or 2.) and; R 2a and R 2b each independently represents an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y b are each independently an ester bond or an amide bond; Z a and Z b are independent of each other, Z 1 :

[0101] [ka]

[0102] (In the formula, s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R 4 each independently represents a substituent. and; R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group); Cationic lipids (1).

[0103] Specific examples of cationic lipids represented by formula (1) include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, and O-Ph-C3M.

[0104] [Table 1-1]

[0105] [Table 1-2]

[0106] Formula (2)

[0107] [ka]

[0108] In formula (2), X a and X b are independently 1 Or X 2 and preferably X 1 It is.

[0109] [ka]

[0110] X 1 R in 4 represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 3. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, a isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. R 4 is preferably a methyl group, an ethyl group, a propyl group or an isopropyl group, and most preferably a methyl group.

[0111] X 2 In the formula, s is either 1 or 2. When s is 1, X 2 is preferably a pyrrolidine group, and when s is 2, X 2 is preferably a piperidylene group. Preferably, s is 2.

[0112] X a isX bmay be the same as or different from, but preferably, X a isX b It is the same group as

[0113] n a and n b is independently 0 or 1, preferably 1. a If is 1, R 3a is Y a and R 2a Via X a Combines with n a If is 0, then R 3a -X a -R 1a ―S-structure. Similarly, n b If is 1, R 3b is Y b and R 2b Via X b Combines with n b If is 0, then R 3b -X b -R 1b -It has an S-structure.

[0114] n a is b may be the same as or different from, but preferably, a is b is equivalent to.

[0115] R 1a and R 1b R independently represents an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, and a neopentylene group. 1a and R 1b is preferably a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and is most preferably an ethylene group.

[0116] R 1a is R1b may be the same as or different from, but preferably, R 1a is R 1b It is the same group as

[0117] R 2a and R 2b are each independently an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear. As the alkylene group having 1 to 6 carbon atoms, R 1a and R 1b Examples of the alkylene group having 1 to 6 carbon atoms include those listed above. 2a and R 2b is preferably a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably a trimethylene group.

[0118] R 2a is R 2b may be the same as or different from, but preferably, R 2a is R 2b It is the same group as

[0119] Y a and Y b is independently an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, preferably an ester bond, an amide bond, or a carbamate bond, and most preferably an ester bond. Y a and Y b The bond orientation of Y is not restricted. a is an ester bond, preferably, R 3a -CO-OR 2a - structure, Y b is an ester bond, preferably, R 3b -CO-OR 2b - structure.

[0120] Y a is Y b may be the same as or different from, but preferably, Y a is Y b It is the same group as

[0121] R 3a and R 3b each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, preferably each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and most preferably each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0122] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0123] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and preferably cholesterol or cholestanol.

[0124] The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The unsaturated bond includes a carbon-carbon double bond and a carbon-carbon triple bond, and is preferably a carbon-carbon double bond. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, and the like, and is preferably an alkyl group or an alkenyl group. Specific examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group, docosyl group, dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosyl group, docosenyl group, dodecadienyl group, tridecadienyl group, tetradecen ... tridecadienyl group, icosenyl group, henicosyl group, docosenyl group, tridecadienyl group, icosenyl group, henicosyl group, docosenyl group, tridecadienyl group, icosenyl group, henicosyl group, docosenyl group, tridec Examples of such alkyl groups include dienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group, and 1-decylundecyl group. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0125] In one embodiment of the present invention, R 3a and R 3bThe aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by the formula (1) is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). Specific examples of the aliphatic hydrocarbon group include a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.

[0126] R 3a is R 3b may be the same as or different from, but preferably, R 3a is R 3b It is the same group as

[0127] In one embodiment of the present invention, R 1a is R 1b is the same as X a isX b is the same as R 2a is R 2b is the same as Y a is Y b is the same as R 3a is R 3b is equivalent to.

[0128] Specific examples of the cationic lipid represented by formula (2) include the following B-2, B-2-5, TS-P4C2, L-P4C2, and O-P4C2.

[0129] [Table 2]

[0130] Next, a method for producing the cationic lipid represented by formula (1) (hereinafter also referred to as cationic lipid (1)) will be described.

[0131] Cationic lipid (1) has a -SS- (disulfide) bond. Therefore, the production method is 3a -CO-OZ a -Y a -R 2a -X a -R 1a-SH (thiol) compounds having R 3b -CO-OZ b -Y b -R 2b -X b -R 1b Examples of methods include a method in which a -SH (thiol) compound having - is produced and then oxidized (coupled) to obtain cationic lipid (1) containing an -SS- bond, and a method in which necessary parts are sequentially synthesized on a compound containing an -SS- bond to finally obtain cationic lipid (1). The latter method is preferred.

[0132] Specific examples of the latter method are given below, but the production method is not limited to these.

[0133] Examples of the starting compound include terminal carboxylic acids containing --SS- bonds, terminal amines, terminal isocyanates, terminal alcohols, terminal alcohols having leaving groups such as methanesulfonyl groups, and terminal carbonates having leaving groups such as p-nitrophenyl carbonate groups.

[0134] For example, R 1a and R 1b are the same and R 1 and X a and X b are identical and X, and R 2a and R 2b are the same and R 2 and Y a and Y b are identical and Y, and Z a and Z b are identical and Z, and R 3a and R 3b are the same and R 3 When producing the cationic lipid (1) represented by the formula (1'), the target cationic lipid product represented by the formula (1') can be obtained by the synthetic route shown below.

[0135] [ka]

[0136] The functional groups at both ends of compound (I) containing -SS- bonds (FG 1 ) with a secondary amine and one terminal functional group (FG 2 The compound (III) is synthesized by reacting the secondary amine in the compound (II) having R 3 Compound (IV) having a hydroxyl group and a reactive functional group (FG 3 ) to synthesize compound (VI), and finally, the reactive functional group (FG 3 ) and the reactive functional group (FG 2 ) to form a -SS- bond, R 1 , X, R 2 , Y, Z and R 3 It is possible to obtain a cationic lipid of formula (1') comprising:

[0137] Among the above-mentioned preparation methods, compound (III) can be prepared by the method described in US2014 / 0335157A1 or WO2016 / 121942.

[0138] The reaction of compound (IV) with compound (V) may be carried out using a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, or 4-dimethylaminopyridine (hereinafter referred to as "DMAP"), or in the presence of an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid, or without a catalyst.

[0139] Alternatively, compound (IV) may be reacted directly with compound (V) using a condensing agent such as dicyclohexylcarbodiimide (hereinafter referred to as "DCC"), diisopropylcarbodiimide (hereinafter referred to as "DIC"), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as "EDC"), or compound (IV) may be converted to an anhydride or the like using a condensing agent, and then reacted with compound (V).

[0140] The amount of compound (IV) charged is usually 1 to 50 mol equivalents, and preferably 1 to 10 mol equivalents, relative to compound (V).

[0141] The catalyst used in the reaction of compound (IV) with compound (V) may be appropriately selected depending on the type of the compounds to be reacted.

[0142] The amount of the catalyst is usually 0.05 to 100 mol equivalents, preferably 0.1 to 20 mol equivalents, more preferably 0.1 to 5 mol equivalents, relative to compound (V).

[0143] The solvent used in the reaction of compound (IV) and compound (V) may be any solvent that does not inhibit the reaction and may be used without any particular limitation. Examples include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, toluene, etc. Among these, chloroform and toluene are preferred.

[0144] The reaction temperature is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0145] The reaction product (VI) obtained by the above reaction can be appropriately purified by a general purification method such as extraction purification, recrystallization, adsorption purification, reprecipitation, column chromatography, or ion exchange chromatography.

[0146] When reacting compound (III) with compound (VI), like the reaction of compound (IV) with compound (V), a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, 4-dimethylaminopyridine, etc. may be used as a catalyst, or the reaction may be carried out in the presence of an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid, or without a catalyst.

[0147] In addition, compound (III) may be reacted directly with compound (VI) using a condensing agent such as DCC, DIC, EDC, etc., or compound (VI) may be converted into an anhydride or the like using a condensing agent, and then reacted with compound (III).

[0148] The amount of compound (VI) charged is usually 1 to 50 mol equivalents, and preferably 1 to 10 mol equivalents, relative to compound (III).

[0149] The catalyst used in the reaction of compound (III) with compound (VI) may be appropriately selected depending on the type of the compounds to be reacted.

[0150] The amount of the catalyst is usually 0.05 to 100 mol equivalents, preferably 0.1 to 20 mol equivalents, more preferably 0.1 to 5 mol equivalents, relative to compound (III).

[0151] The solvent used in the reaction of compound (III) and compound (VI) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, toluene, etc. Among these, chloroform and toluene are preferred.

[0152] The reaction temperature is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0153] The cationic lipid (1) obtained by the above reaction can be appropriately purified by a general purification method such as extraction purification, recrystallization, adsorption purification, reprecipitation, column chromatography, or ion exchange chromatography.

[0154] The cationic lipid represented by formula (2) (hereinafter also referred to as cationic lipid (2)) can be produced by the method described in US2014 / 0335157A1.

[0155] Phospholipids Phospholipids can be used as lipid membrane components of lipid nanoparticles. Examples of phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphatidylethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphatidylserine (PS), 1,2-diacyl-sn-glycero-3-phosphatidylglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), or lyso forms thereof. in particular, 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-diuracilloyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-Diethylhexyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) is one example, and these PCs can be appropriately converted to PE, PS, PG, or PA for use.

[0156] The phospholipid used in the present invention is preferably PC or PE, and more preferably DOPC, POPC, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), or POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

[0157] Sterols Sterol can be used as a component that adjusts the fluidity of lipid membrane of lipid nanoparticle.Examples of sterol include cholesterol, lanosterol, phytosterol, zymosterol, zymostenol, desmosterol, stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol, preferably cholesterol, lanosterol, phytosterol, more preferably cholesterol.

[0158] PEGylated lipids PEG lipids are used as stabilizers by coating the surface of lipid nanoparticles with hydrophilic polyethylene glycol (PEG) to prevent aggregation of particles, and to suppress interactions between biological components and particles when administered to the body. The PEG region can be of any molecular weight, hi some embodiments, the PEG region has a molecular weight between 200 and 10,000 Da and can be linear or branched.

[0159] Examples of PEG lipids include PEG-phospholipids, PEG-ceramide, PEG-diacylglycerol, and PEG-cholesterol. Diacylglycerol PEG having a molecular weight of 1,000 to 10,000 is preferred, and dimyristoylglycerol PEG or distearoylglycerol PEG having a molecular weight of 1,000 to 10,000 is more preferred.

[0160] Acidic Buffers and Acidic Buffer Components A buffer or buffer component that has a buffering effect in the acidic range can be used. By "acidic buffer component" is meant an acidic buffer substantially excluding water. The level of water contained in the acidic buffer component can be less than about 5% w / v, or less than 4% w / v, or less than 3% w / v, or less than 2% w / v, or less than 1% w / v. Specifically, these include HCl / KCl buffer, p-toluenesulfonic acid / p-toluenesulfonic acid Na salt buffer, tartaric acid / NaOH buffer, citric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / Na formate buffer, citric acid / Na citrate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer, phenylacetic acid / phenylacetic acid Na salt buffer, acetic acid / Na acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, cacodylic acid Na / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH 2 PO 4 Examples of such buffers include / NaOH buffer, imidazole / HCl buffer, s-collidine (2,4,6-trimethylpyridine) / HCl buffer, triethanolamine HCl / NaOH buffer, 5,5-diethylbarbituric acid Na / HCl buffer, N-methylmorpholine / HCl buffer, pyrophosphate Na / HCl buffer, MES buffer, malic acid buffer, ADA buffer, PIPES buffer, ACES buffer, HEPES, BES, Bis-Tris buffer, Bis-Tris propane buffer, anhydrous sodium carbonate buffer, glycylglycine buffer, MOPS, MOPSO, and TES.

[0161] The acidic buffer or acidic buffer component preferably has a buffering effect at pH 1 to 6, more preferably at pH 3 to 6, and examples of the acidic buffer include tartaric acid / NaOH buffer, citric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / Na formate buffer, citric acid / Na citrate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer, phenylacetic acid / Na salt buffer, acetic acid / Na acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, cacodylic acid Na / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH 2 PO 4 Examples of the buffer include NaOH buffer, MES buffer, malic acid buffer, BisTris buffer, and glycylglycine buffer, and more preferably malic acid buffer or MES buffer.

[0162] The freeze-dried composition of lipid nanoparticles of the present invention preferably has a pH of 1 to 6, and more preferably a pH of 3 to 6, when 100 to 500 mg of the freeze-dried composition is suspended in 1 to 5 mL of distilled water for injection at 0 to 30°C.

[0163] Method for preparing lipid nanoparticles Examples of "operations to induce organization" for preparing lipid nanoparticles include the alcohol dilution method using a microchannel or vortex, the simple hydration method, ultrasonic treatment, heating, vortexing, ether injection, the French press method, the cholic acid method, and Ca 2+ Examples of known methods include fusion, freeze-thaw, and reverse phase evaporation, and are preferably the alcohol dilution method using a microchannel or vortex, and more preferably the alcohol dilution method using a microchannel. The particle preparation by the alcohol dilution method using a microchannel can be performed using, for example, NanoAssemblr (Precision NanoSystems). The prepared lipid nanoparticles can be exchanged for the buffer solution of the external aqueous phase by ultrafiltration, dialysis, dilution, and other operations.

[0164] Cryoprotectants The cryoprotectant that can be used in the present invention may be a monosaccharide, sugar alcohol, disaccharide, oligosaccharide, polysaccharide, or polymer. Specifically, glyceraldehyde, erythrose, threose, ribose, lyxose, xylose, arabinose, allose, talose, gulose, glucose, altrose, mannose, galactose, idose, erythrulose, ribulose, psicose, fructose, sorbose, tagatose, erythritol, glycerin, isomaltol, lactitol, maltitol, mannitol, sorbitol, xylitol, inositol, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, so Examples of suitable sugars include holose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiose, mannobiose, melibiose, melibiulose, neolactose, galactosucrose, scillabiose, neohesperidose, rutinose, rutinulose, vicianose, xylobiose, primeverose, trehalosamine, maltitol, lactosamine, lactitol, sucralose, raffinose, panose, maltotriose, melezitose, gentianose, stachyose, cyclodextrin, hydroxypropyl-β-cyclodextrin, dextran, polyvinylpyrrolidone, and polyethylene glycol. Disaccharides are preferred, non-reducing disaccharides are more preferred, and sucrose is the most preferred.

[0165] The concentration of the cryoprotectant is preferably 80 to 800 mg / mL, more preferably 160 to 800 mg / mL, before freeze-drying. The weight ratio of the cryoprotectant to the total lipids after freeze-drying is preferably 10 to 1000 times, more preferably 30 to 1000 times, the total lipids.

[0166] Freeze drying process The lyophilization process can be carried out in any suitable container known in the pharmaceutical art, such as a glass container (or for example a glass vial) or a dual-chamber container. The stabilized lipid nanoparticle composition of the present invention containing a cryoprotectant can be introduced into a glass container. The volume of the composition added to the container can be 0.1 to 20 mL, or 1 to 10 mL. Any lyophilization process may be used, including those known in the pharmaceutical art. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Penn. (1990). The lyophilization process can include freezing the cryoprotectant-stabilized lipid nanoparticle composition at a temperature of about −55° C. to about −30° C. The frozen composition can be a dried form of the lyophilized composition. In some embodiments, the freezing step may involve gradually increasing the temperature from room temperature to the final temperature over a period of several minutes. The temperature gradient may be about 1° C. / min. In some embodiments, the drying process may be carried out at a pressure of about 0 to 250 mTorr, or 50 to 150 mTorr, and at a temperature of about -55°C to about 40°C. The drying process may be continued at elevated temperatures up to room temperature for a period of time up to several days. The level of residual water in the solid lyophilized composition may be less than about 5% w / v, or less than 4% w / v, or less than 3% w / v, or less than 2% w / v, or less than 1% w / v.

[0167] Rehydration process The rehydration step is a step of preparing nucleic acid-encapsulated lipid nanoparticles by adding an aqueous solution containing nucleic acid to the freeze-dried composition of the present invention. The amount of nucleic acid is, for example, a ratio of total lipid to nucleic acid contained in the freeze-dried composition of total lipid / nucleic acid = 1 to 1000 nmol / μg, preferably total lipid / nucleic acid = 50 to 500 nmol / μg, and the nucleic acid-encapsulated lipid nanoparticles can be prepared by adding an aqueous solution containing nucleic acid and mixing by pipetting or vortexing. In addition, in order to increase the nucleic acid encapsulation rate, alcohol can be added during rehydration, and as the alcohol, methanol, ethanol, n-butanol, and t-butanol can be used, and ethanol is preferred. The concentration of alcohol in the aqueous solution containing nucleic acid is 0 to 50 v / v%, preferably 0 to 30 v / v%, and more preferably 0 to 25 v / v%. In addition, in the rehydration step, in order to further increase the nucleic acid encapsulation efficiency, the freeze-dried composition can be incubated after adding an aqueous solution containing nucleic acid and / or alcohol. The incubation conditions are, for example, 0 to 100° C. for 0 to 120 minutes, and preferably 0 to 95° C. for 0 to 60 minutes.

[0168] A step of exchanging the external aqueous phase with a neutral buffer solution By exchanging the external aqueous phase of the nucleic acid-encapsulating lipid nanoparticles obtained in the rehydration step with a neutral buffer solution, it is possible to prepare nucleic acid-encapsulating lipid nanoparticles that can be used as nucleic acid introduction agents. Methods for exchanging the external aqueous phase with a neutral buffer include dialysis, ultrafiltration, or dilution. Neutral buffer solutions include phosphate buffered saline (PBS), Tris-HCl buffer, ADA, PIPES, PIPES sesquisodium, ACES, MOPS, BES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, etc. The pH of neutral buffer solutions is 6 to 8.

[0169] By encapsulating a nucleic acid in the freeze-dried composition of lipid nanoparticles of the present invention and contacting the composition with a cell, the nucleic acid can be introduced into the cell in vivo and / or ex vivo. Therefore, the present invention provides a nucleic acid transfer agent comprising the freeze-dried composition of lipid nanoparticles of the present invention. The present invention also provides a nucleic acid transfer agent comprising a nucleic acid-encapsulating lipid nanoparticle prepared using the freeze-dried composition of lipid nanoparticles of the present invention.

[0170] The nucleic acid transfer agent of the present invention can transfer any nucleic acid into cells. The types of nucleic acids include, but are not limited to, DNA, RNA, chimeric nucleic acid of RNA, DNA / RNA hybrids, and the like. The nucleic acid can be any one of 1- to 3-stranded, but is preferably single-stranded or double-stranded. The nucleic acid may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers with non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs), etc.), or other oligomers with special bonds (however, the oligomers contain nucleotides with a configuration that allows base pairing or base attachment as found in DNA or RNA). Furthermore, the nucleic acid may be, for example, a nucleic acid having a known modification added thereto, a nucleic acid labeled as known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more naturally occurring nucleotides have been replaced with an analogue, a nucleic acid having an intramolecular nucleotide modification, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid having an intercurrent compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., an α-anomeric nucleic acid, etc.), etc.

[0171] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of use. For example, plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc. can be mentioned, preferably plasmid DNA, cDNA, antisense DNA, more preferably plasmid DNA. Circular DNA such as plasmid DNA can also be appropriately digested with restriction enzymes and used as linear DNA.

[0172] The type of RNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of 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. can be mentioned, and preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon.

[0173] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0174] The nucleic acid transfer agent of the present invention encapsulating a nucleic acid can be administered in vivo, for example, for the purpose of preventing and / or treating a disease. Therefore, the nucleic acid used in the present invention is preferably one that has preventive and / or therapeutic activity against a certain disease (prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0175] The nucleic acid transfer agent of the present invention encapsulating a nucleic acid can be used as a drug delivery system for selectively delivering nucleic acids, etc. into specific cells, and is useful, for example, in DNA vaccines and gene therapy drugs for tumors that involve the introduction of antigen genes into dendritic cells, and nucleic acid pharmaceuticals that suppress the expression of target genes by utilizing RNA interference.

[0176] The particle size of the lipid nanoparticles encapsulating the nucleic acid is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. The particle size can be measured using a particle size distribution measuring device such as Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted depending on the method for preparing the lipid nanoparticles.

[0177] The surface potential (zeta potential) of lipid nanoparticles encapsulating nucleic acid is not particularly limited, but is preferably -15 to +15 mV, more preferably -10 to +10 mV. In conventional gene transfer, particles with a positively charged surface potential have been mainly used. This is useful as a method for promoting electrostatic interaction with heparin sulfate on the negatively charged cell surface and promoting uptake into cells, but a positive surface charge may inhibit the release of nucleic acid from the carrier due to interaction with the delivered nucleic acid in cells, and may inhibit protein synthesis due to interaction between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the surface charge within the above range. The measurement of the surface charge can be performed using a zeta potential measuring device such as Zetasizer Nano. The surface charge of lipid nanoparticles can be adjusted by the composition of the constituent components of the lipid nanoparticles.

[0178] The step of contacting cells with lipid nanoparticles encapsulating nucleic acids in vitro is specifically described below.

[0179] The cells are suspended in a suitable medium and cultured under appropriate conditions several days before contact with the lipid nanoparticles, and the cells may or may not be in a proliferative phase at the time of contact with the lipid nanoparticles.

[0180] The culture medium during the contact may be a serum-containing or serum-free medium, but the serum concentration in the medium is preferably 30% by weight or less, more preferably 20% by weight or less. If the medium contains excess proteins such as serum, the contact between the lipid nanoparticles and the cells may be inhibited.

[0181] The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the type of cells, etc., but is usually 1×10 4 ~1×10 7 in the range of cells / mL.

[0182] To the cells thus prepared, for example, a suspension of lipid nanoparticles encapsulating the above-mentioned nucleic acid is added. The amount of the suspension added is not particularly limited, and can be appropriately set in consideration of the number of cells, etc. The concentration of lipid nanoparticles when contacting with cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved, but the lipid concentration is usually 1 to 100 nmol / mL, preferably 10 to 50 nmol / mL, and the nucleic acid concentration is usually 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL.

[0183] After the above-mentioned suspension is added to the cells, the cells are cultured. The temperature, humidity, and CO 2 The concentration and other factors are set appropriately taking into consideration the type of cells. When the cells are mammalian cells, the temperature is usually about 37°C, the humidity is about 95%, and the CO 2 The concentration is about 5%. The culture time can also be appropriately set in consideration of conditions such as the type of cells used, but is usually in the range of 0.1 to 76 hours, preferably in the range of 0.2 to 24 hours, and more preferably in the range of 0.5 to 12 hours. If the culture time is too short, the nucleic acid is not sufficiently introduced into the cells, and if the culture time is too long, the cells may become weak.

[0184] The nucleic acid is introduced into the cells by the above-mentioned culture, and preferably the culture medium is replaced with a fresh medium or a fresh medium is added to the medium and the culture is continued. When the cells are derived from a mammal, the fresh medium preferably contains serum or a nutritional factor.

[0185] As described above, the use of lipid nanoparticles encapsulating nucleic acids makes it possible to introduce nucleic acids into cells not only in vitro but also in vivo. That is, by administering lipid nanoparticles encapsulating nucleic acids to a subject, the lipid nanoparticles reach and contact target cells, and the nucleic acids encapsulated in the lipid nanoparticles are introduced into cells in vivo. The subjects to which the lipid nanoparticles can be administered are not particularly limited, and examples thereof include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), and fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), and plants. The subjects to which the lipid nanoparticles encapsulating nucleic acids are administered are preferably humans or other mammals.

[0186] The type of target cell is not particularly limited, and by using lipid nanoparticles encapsulating nucleic acid, it is possible to introduce nucleic acid into cells in various tissues (e.g., liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0187] The method of administration of lipid nanoparticles into which nucleic acids and / or compounds other than nucleic acids have been introduced to a subject (e.g., vertebrates, invertebrates, etc.) is not particularly limited as long as the lipid nanoparticles reach and contact target cells and the compound introduced into the lipid nanoparticles can be introduced into the cells, and a method of administration known per se (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected taking into consideration the type of compound introduced, the type and site of the target cells, etc. The dosage of the lipid nanoparticles is not particularly limited as long as it is within a range that allows introduction of the compound into the cells, and can be appropriately selected taking into consideration the type of subject to be administered, the administration method, the type of compound introduced, the type and site of the target cells, etc.

[0188] When lipid nanoparticles encapsulating nucleic acids are used as nucleic acid transfer agents, they can be formulated according to conventional methods.

[0189] When the nucleic acid transfer agent is provided as a research reagent, the nucleic acid transfer agent of the present invention may be provided as lipid nanoparticles encapsulating nucleic acids as is, or in the form of a sterile solution or suspension in, for example, water or other physiologically acceptable liquid (e.g., a water-soluble solvent (e.g., malic acid buffer, etc.), an organic solvent (e.g., ethanol, methanol, DMSO, tert-butanol, etc.), or a mixture of a water-soluble solvent and an organic solvent, etc.). The nucleic acid transfer agent of the present invention may contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.) as appropriate.

[0190] Furthermore, when the nucleic acid transfer agent is provided as a medicine, the nucleic acid transfer agent of the present invention can be manufactured as an oral agent (e.g., tablets, capsules, etc.) or a parenteral agent (e.g., injections, sprays, etc.), preferably as a parenteral agent (more preferably, injections), by using the lipid nanoparticles encapsulating the nucleic acid as is, or together with known pharma- ceutical acceptable additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.) and mixing them in a unit dose form required for generally accepted formulation practice.

[0191] The nucleic acid transfer agent of the present invention can be formulated for use in children as well as for use in adults. EXAMPLES

[0192] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0193] The meanings of the abbreviations used in this specification are as follows: Chol: Cholesterol DMG-PEG2k: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (PEG MW 2000) DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine PBS: Phosphate-buffered saline MES: 2-morpholinoethanesulfonic acid DPBS: Dulbecco's phosphate buffered saline

[0194] Preparation of freeze-dried compositions of Comparative Examples 1-3 As a comparative example, a freeze-dried composition was prepared by preparing neutral lipid nanoparticles as described below, adding a cryoprotectant, and freeze-drying the nanoparticles. The lipid composition shown in Table 4 was mixed in tert-butanol solution and malic acid buffer (pH 3, 20 mM) in NanoAssemblr (flow rate ratio: buffer / lipid = 6 / 1 (v / v), total flow rate: 1 mL / min). This solution was diluted with MES buffer (pH 5.5, 20 mM) and replaced with PBS at pH 7.4 while ultrafiltration using an Amicon. LNP was concentrated and collected so that the theoretical lipid concentration was 200 nmol / 100 μL, and 100 μL of sucrose solution was added and mixed. This solution was lyophilized using a VerTis AdVantage Plus EL-85 freeze dryer. For lyophilization, the solution was first frozen at normal pressure and -55°C for 14 to 21 hours, then the pressure was reduced to 200 mTorr and the temperature was increased by 10°C at a time. The temperature rise program was set to raise the temperature by 10°C over 3 hours until it reached -20°C, then raise the temperature by 10°C over 2 hours above -10°C and leave it at that temperature for 1 hour. Once the temperature had risen to 30°C, it was left at that temperature for 3 hours before returning the sample to normal pressure and collecting the sample.

[0195] Preparation of freeze-dried compositions of Examples 1-59 A lipid tert-butanol solution and malic acid buffer (pH 3.0, 20 mM) were mixed in NanoAssemblr (flow rate ratio: buffer / lipid = 6 / 1 (v / v), total flow rate: 1 mL / min). An equal amount of sucrose solution (final sucrose concentrations of 80 mg / mL, 160 mg / mL, 320 mg / mL, and 433 mg / mL) was added to this LNP solution and mixed, and the lipid amount was dispensed to 200 nmol and 600 nmol. This solution was lyophilized using a VerTis AdVantage Plus EL-85 freeze dryer. For lyophilization, the solution was first frozen at atmospheric pressure and -55°C for 14 to 21 hours, then the pressure was reduced to 200 mTorr and the temperature was increased by 10°C increments. The temperature rise program was set to raise the temperature by 10°C over 3 hours until it reached -20°C, then raise the temperature by 10°C over 2 hours above -10°C and leave it at that temperature for 1 hour. Once the temperature had risen to 30°C, it was left at that temperature for 3 hours before returning the sample to normal pressure and collecting the sample.

[0196] Preparation of freeze-dried compositions of Examples 60-70 A tert-butanol solution of lipids with the lipid composition shown in Table 5 and MES buffer (pH 5.0, 20 mM) were mixed in NanoAssemblr (flow rate ratio: buffer / lipid = 6 / 1 (v / v), total flow rate: 1 mL / min). The subsequent operations were performed in the same manner as in Examples 1-59.

[0197] Rehydration of the freeze-dried compositions of Comparative Examples 1-3 and Examples 1-59 Under the conditions shown in Tables 4 and 5, an aqueous solution of mRNA, siRNA, and pDNA (RNase free, containing 12.5% ​​or 25% EtOH) was added to the lyophilized composition at a ratio of total lipid / mRNA (or siRNA or pDNA) = 200 nmol / μg, and mixed by pipetting. The solution was diluted with MES buffer (pH 5.5, 20 mM) and replaced with PBS at pH 7.4 while performing ultrafiltration using an Amicon. The LNP was concentrated and recovered so that the theoretical value of the mRNA (or siRNA or pDNA) concentration was 2.5 g / mL, and the recovery rate and encapsulation rate of nucleic acid were measured using Ribogreen (registered trademark) reagent or Picogreen (registered trademark) reagent. Particle size and zeta potential were measured using a Zetasizer.

[0198] - Measurement of recovery rate and inclusion rate (1) Prepare 250 μL of the nucleic acid-encapsulated LNP solution by diluting it 2.5-fold with DPBS (theoretical value: 1 μg / mL). (2) For the standard curve, prepare 250 μL of each of mRNA solutions of known concentrations diluted with DPBS to obtain mRNA concentrations of 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, and 0 ng / mL. (3) Prepare RiboGreen or PicoGreen reagent according to the conditions in the table below (amount per well). For an analysis of n=2, prepare (number of calibration curves + number of samples + 1) x 2.

[0199] [Table 3]

[0200] (4) Add 50 μL of the solutions prepared in (1) and (2) to each well of a black 96-well plate, then add 50 μL of solution A or 50 μL of solution B. (5) Cover the plate with aluminum foil to protect it from light and incubate it on a shaker (100-500 rpm) for 5 minutes. (6) Measure fluorescence using a microplate reader (Excitation: 480 nm, Emission: 520 nm). (7) Calculate the recovery rate and inclusion rate using the following formula.

[0201]

number

[0202] Rehydration of the freeze-dried compositions of Examples 60-63 A solution of mRNA (RNase free) was added to the lyophilized composition at a ratio of total lipid / mRNA = 200 nmol / μg, mixed by pipetting, and then incubated at 95 ° C for 5 minutes. The solution was diluted with MES buffer (pH 5.5, 20 mM) and replaced with PBS at pH 7.4 while performing ultrafiltration using an Amicon. The LNP was concentrated and recovered so that the mRNA concentration was 2.5 μg / mL, and the recovery rate and encapsulation rate of nucleic acid were measured using Ribogreen reagent. Particle size and zeta potential were measured using a Zetasizer.

[0203] Rehydration of the freeze-dried compositions of Examples 64 and 65 An aqueous solution of mRNA (RNase free) was added to the lyophilized composition at a ratio of total lipid / mRNA = 200 nmol / μg, mixed by pipetting, and then incubated at 95 ° C for 5 minutes. The recovery rate and encapsulation rate of nucleic acid were measured using Ribogreen reagent. Particle size and zeta potential were measured using a Zetasizer.

[0204] Rehydration of the freeze-dried compositions of Examples 66-70 A solution of mRNA (RNase free) was added to the lyophilized composition at a ratio of total lipid / mRNA = 200 nmol / μg, mixed by pipetting, diluted with MES buffer (pH 5.5, 20 mM), and replaced with PBS at pH 7.4 while performing ultrafiltration using an Amicon. The LNP was concentrated and recovered so that the mRNA concentration was 2.5 μg / mL, and the recovery rate and encapsulation rate of nucleic acid were measured using Ribogreen reagent. Particle size and zeta potential were measured using a Zetasizer.

[0205] The sequences of the mRNA, siRNA, and pDNA used are shown below. mRNA: CleanCap® FLuc mRNA (TriLink) CleanCap® EGFP mRNA (TriLink) CleanCap® EPO mRNA (5moU) (TriLink) CleanCap® OVA mRNA (TriLink) pDNA: pcDNA3.1-luc (method described in Biomaterials 2011, 32, 6342.) siRNA: siFVII (2'-F) Sense 5'-GGAucAucucAAGucuuAcdT*dT-3' Antisense 5'-GuAAGAcuuGAGAuGAuccdT*dT-3' dT: deoxythymidine *: phosphorothioate linkage Capital letter: native (2'-OH) ribonucleotides Small letter: 2'-Fluoro-modified nucleotides

[0206] [Table 4]

[0207] [Table 5-1]

[0208] [Table 5-2]

[0209] [Table 5-3]

[0210] [Table 5-4]

[0211] [Table 5-5]

[0212] [Table 5-6]

[0213] [Table 5-7]

[0214] According to the procedure, the freeze-dried composition of neutral lipid nanoparticles of the comparative example was rehydrated, and the particle properties, nucleic acid recovery rate, and encapsulation rate were evaluated. The results are shown in Table 6. As shown in the evaluation results in Table 6, in the freeze-dried composition of neutral lipid nanoparticles, the nucleic acid and the ionic lipid did not undergo electrostatic interaction upon rehydration with an aqueous nucleic acid solution, and therefore it was not possible to encapsulate the nucleic acid in the lipid nanoparticles.

[0215] [Table 6]

[0216] According to the procedure, the freeze-dried composition of acidic lipid nanoparticles with varying concentrations of sucrose as a cryoprotectant was rehydrated, and the particle properties, nucleic acid recovery rate, and encapsulation rate were evaluated. The results are shown in Table 7. As shown in the evaluation results in Table 7, nucleic acids could be efficiently encapsulated in the acidic freeze-dried compositions of Examples 1 to 3. Furthermore, as shown in Table 7, the encapsulation efficiency of nucleic acids could be increased by increasing the concentration of sucrose, which is a cryoprotectant, and lipid nanoparticles with a narrow particle distribution in terms of particle properties could be prepared.

[0217] [Table 7]

[0218] According to the procedure, the ethanol concentration during rehydration was changed, and the particle properties, nucleic acid recovery rate, and encapsulation rate were evaluated. The results are shown in Table 8. As shown in the evaluation results in Table 8, the efficiency of encapsulating nucleic acid could be increased by adding ethanol.

[0219] [Table 8]

[0220] According to the procedure, the type of nucleic acid used during rehydration was changed, and the particle properties, nucleic acid recovery rate, and encapsulation rate were evaluated. The results are shown in Table 9. As shown by the evaluation results in Table 9, nucleic acids could be efficiently encapsulated regardless of the type of nucleic acid.

[0221] [Table 9]

[0222] According to the procedure, the type of phospholipid and the lipid composition were changed, and the particle properties, nucleic acid recovery rate, and encapsulation rate were evaluated. The results are shown in Table 10. As shown by the evaluation results in Table 10, nucleic acids could be efficiently encapsulated regardless of the type of phospholipid and the lipid composition.

[0223] [Table 10-1]

[0224] [Table 10-2]

[0225] According to the procedure, the type of ionic lipid was changed, and the particle properties, nucleic acid recovery rate, and encapsulation rate were evaluated. The results are shown in Table 11. As shown by the evaluation results in Table 11, nucleic acids could be efficiently encapsulated regardless of the type of ionic lipid.

[0226] [Table 11-1]

[0227] [Table 11-2] Test Example 1

[0228] Evaluation of expression intensity and expression efficiency per cell using EGFP-mRNA-encapsulated LNPs An LNP solution encapsulating mRNA expressing EGFP was prepared by the method described in the Examples. 24 hours before transfection, human leukemia T cell Jurkat cells were 4 The cells were seeded in a 1.2 cm well at 1 mL / well. After 24 hours, the prepared LNP solution was added to the 1.2 cm well at 0.1 μg of mRNA, and the cells were cultured in an incubator for 24 hours. The culture medium was diluted with FACS buffer (0.5% bovine serum albumin (BSA), 0.1% NaN 3 The transfected cells were analyzed by measurement using a flow cytometer (NovoCyte; ACEA Biosciences). The percentage of cells expressing EGFP is shown in Figure 1, and the intensity of EGFP expression is shown in Figure 2. As shown in Figures 1 and 2, the nucleic acid-encapsulated lipid nanoparticles prepared using the freeze-dried composition of the present invention can efficiently introduce genes into cells, and furthermore, by increasing the concentration of sucrose, a cryoprotectant, the uniformity of gene introduction into cells and the expression intensity can be increased.

[0229] ·Evaluation of gene transfer efficiency into cells using Luc-mRNA-encapsulated lipid nanoparticles An LNP solution encapsulating mRNA expressing luciferase was prepared by the method described in the Examples. 2.0 × 10 Jurkat cells, a human leukemia T cell, were cultured 24 hours before transfection. 5 The cells were seeded on a 3.5 cm dish at 1.9 mL / dish. After 24 hours, 100 μL of medium (RPMI1640) containing D-luciferin was added to each dish at a final concentration of 0.1 mM. The prepared LNP solution was added to the dish at 0.4 μg as mRNA, and the dish was set in an incubator-type luminometer KronosDio. The luminescence intensity of luciferase was measured for 2 minutes every 3 hours. The cumulative luminescence intensity over 24 or 48 hours was calculated from the obtained change in expression over time. The results are shown in Figures 3 to 6. As shown in Figures 3 to 6, genes could be efficiently transferred into cells using any of DOPC, POPC, DOPE, and POPE as the phospholipid, and the gene transfer efficiency was highest when POPE was used.

[0230] -Evaluation of mRNA expression efficiency in vivo in mice An LNP solution encapsulating erythropoietin-expressing mRNA was prepared by the method described in the Examples. The prepared LNP solution was diluted with PBS to a concentration of 5 μg / mL of mRNA. The diluted mRNA-encapsulated LNP was administered into the tail vein of 6-week-old female Balb / c mice at 10 μL per 1 g of body weight (mRNA dose: 0.05 mg / kg). 15 μL of blood was collected from the mouse tail vein 1, 3, 6, 9, and 24 hours after administration. The collected blood was immediately mixed with 0.3 μL of heparin solution (5000 U / 5 mL). Each blood sample was centrifuged under centrifugation conditions (25°C, 2000 g, 20 min), and the supernatant was collected. The erythropoietin concentration in the supernatant was measured using a Mouse Erythropoietin Quantikine ELISA Kit (R&D Systems) according to the method described in the kit's protocol. The results are shown in Figure 7. As shown in Figure 7, the activity of the particles prepared in Example 39 was the highest, but in all of Examples 39, 22, and 38, mRNA expression in mice in vivo was confirmed.

[0231] Evaluation of CTL activity using ovalbumin (OVA; model antigen) Antigen-specific cytotoxic T cell activity (CTL activity) was evaluated using OVA as a model antigen. LNPs containing mRNA expressing OVA were prepared by the method described in the Examples. The prepared LNP solution was diluted with PBS to a concentration of 1 μg / mL of mRNA. The diluted mRNA-containing LNPs were administered subcutaneously to the back of the neck of 6-week-old C57BL6 / J mice at 0.1 μg of mRNA per mouse. CTL assay was performed 7 days after administration. CTL assay: Spleens were collected from 6-week-old C57BL6 / J mice not sensitized to antigens, and were disaggregated in RPMI medium, first with a 5 mL syringe and then with tweezers, to prepare splenocytes. The cells were passed through a 40 μm cell strainer and then centrifuged (4°C, 500 g, 5 min). The supernatant was removed, and the cell mass was suspended in red blood cell lysis buffer and centrifuged again after 5 min (4°C, 500 g, 5 min). The supernatant was removed, RPMI medium was added, and the cell number was counted. The cells were diluted to 1.0 × 10 7The cells were suspended at 1.0×10 cells / mL and divided into two treatment groups: target cell group; OVA epitope (SIINFEKL peptide) was added at a 400-fold dilution, left to stand for 30 minutes, and then centrifuged (4°C, 500 g, 5 minutes). 7 The cells were suspended at 1.0 × 10 cells / mL and stained with 5 μM CFSE to prepare target cells. Control cells: 1.0 × 10 7 The target cell group and the control cell group were mixed in equal amounts to give a total of 1.0 × 10 7 The cell mixture containing the OVA-specific cytotoxic T cell activity was administered via the tail vein of mice immunized as described above. 20 hours after administration, the mouse spleens were collected and CFSE fluorescence was measured using a flow cytometer (NovoCyte; ACEA Biosciences). It was confirmed that the counts of the control cell group remained almost unchanged throughout the experiment. The survival rate of the target cell group relative to the control cell group was calculated for each group. OVA-specific cytotoxic T cell activity was evaluated by determining 0% of cytotoxic T cell activity from the survival rate of target cells in PBS-administered mice (non-immunized group) and calculating the cytotoxic T cell activity of each sample from the survival rate of target cells in immunized mice. As shown in Figure 8, the particles prepared in Examples 62 and 65 had the highest activity, but it was confirmed that all of Examples 60, 62, 64, and 65 were able to confer antigen-specific cytotoxic T cell activity.

[0232] Preparation of lipid nanoparticles of Comparative Example 4 Particles were prepared according to the following procedure using a conventional method that does not involve a freeze-drying step. The lipid composition was SS-OP / cholesterol / DOPC / DMG-PEG2k=52.5 / 40 / 7.5 / 1.5 (mol), and the ratio of total lipid / mRNA=200nmol / μg was a lipid ethanol solution and mRNA malic acid buffer solution (pH 3.0, 20mM, containing 30mM NaCl) mixed in NanoAssemblr (flow rate ratio: buffer / lipid=3 / 1 (v / v), total flow rate: 4mL / min). This solution was diluted with MES buffer (pH 5.5, 20mM) and replaced with PBS at pH 7.4 while performing ultrafiltration using an Amicon. The solution was diluted with PBS so that the theoretical value of mRNA was 10μg / mL, and lipid nanoparticles were obtained.

[0233] Preparation of the freeze-dried composition of Example 71 The lipid composition was SS-OP / cholesterol / DOPC / DMG-PEG2k=52.5 / 40 / 7.5 / 1.5 (mol), and the lipid ethanol solution and malic acid buffer (pH 3.0, 20 mM) were mixed in NanoAssemblr (flow rate ratio: buffer / lipid = 7 / 1 (v / v), total flow rate: 1 mL / min). This solution was diluted with MES buffer (pH 6, 20 mM) and replaced with MES buffer (pH 6, 20 mM) by ultrafiltration using an Amicon. The total lipid was diluted with MES buffer (pH 6, 20 mM) to 400 nmol / 100 μL and collected. An equal amount of sucrose solution (sucrose final concentration 160 mg / mL) was added to this solution and mixed with a vortex mixer. 200 μL was dispensed into vials and lyophilized using an EYELA freeze dryer. The freeze-drying was performed by first freezing the solution at normal pressure and -40°C for 3 hours, then reducing the pressure to 200 mTorr and leaving it at -40°C for 20 hours, at -30 to 0°C for 6 hours at 10°C intervals, and at 10 to 30°C for 3 hours at 10°C intervals. After the program was completed, the pressure was returned to normal and the samples were collected.

[0234] Rehydration of the freeze-dried composition of Example 71 The collected lyophilized sample was mixed with 200 μL of water containing 2 μg of Luc-mRNA so that the total lipid / mRNA ratio was 200 nmol / μg, and then incubated at 95° C. for 5 minutes and neutralized with 200 μL of PBS.

[0235] Preparation of the freeze-dried composition of Example 72 The lipid composition was SS-OP / cholesterol / DOPC / DMG-PEG2k=52.5 / 40 / 7.5 / 1.5 (mol), and the lipid ethanol solution and malic acid buffer (pH 3.0, 20 mM) were mixed in NanoAssemblr (flow rate ratio: buffer / lipid = 7 / 1 (v / v), total flow rate: 16 mL / min). This solution was diluted with MES buffer (pH 6, 20 mM) and replaced with MES buffer (pH 6, 20 mM) by ultrafiltration using an Amicon. The total lipid was diluted with MES buffer (pH 6, 20 mM) to 400 nmol / 100 μL and collected. An equal amount of sucrose solution (sucrose final concentration 160 mg / mL) was added to this solution and mixed with a vortex mixer. 200 μL was dispensed into vials and lyophilized using an EYELA freeze dryer. The freeze-drying was performed by first freezing the solution at normal pressure and -40°C for 3 hours, then reducing the pressure to 200 mTorr and leaving it at -40°C for 20 hours, at -30 to 0°C for 6 hours at 10°C intervals, and at 10 to 30°C for 3 hours at 10°C intervals. After the program was completed, the pressure was returned to normal and the samples were collected.

[0236] Rehydration of the freeze-dried composition of Example 72 The collected freeze-dried sample was mixed with 200 μL of water containing 2 μg of hEPO-mRNA so that the total lipid / mRNA ratio was 200 nmol / μg, and then incubated at 37° C. for 15 minutes and neutralized with 200 μL of PBS.

[0237] The particle properties and nucleic acid encapsulation rates of Comparative Example 4 and Examples 71 and 72 were measured, and the results are shown in Table 12.

[0238] [Table 12] Test Example 5

[0239] Evaluation of in vitro gene expression activity using HeLa cells Using the particles of Comparative Example 4 and Example 71 prepared according to the procedures, gene expression intensity in cells was evaluated according to the following procedures. 24 hours before transfection, HeLa cells were incubated at 5.0 × 10 4 The LNPs were seeded on a 3.5 cm dish at 1.9 mL / dish. After 24 hours, 100 μL of medium (RPMI1640) containing D-luciferin was added to each dish at a final concentration of 0.1 mM. The prepared LNP solution was added thereto at 0.4 μg as mRNA, and the dish was set in an incubator-type luminometer KronosDio. The luminescence intensity of luciferase was measured for 2 minutes every hour. The results are shown in FIG. 9. Compared to Comparative Example 4, the nanoparticles of Example 71 showed approximately 34 times higher gene expression activity in vitro. Test Example 6

[0240] Evaluation of gene expression activity in vivo in mice Using the particles of Comparative Example 4 and Example 72 prepared according to the procedure, gene expression intensity in mice was evaluated in the same manner as in Test Example 3. The results are shown in Figure 10. Compared with Comparative Example 4, the nanoparticles of Example 72 showed about 1.7 times higher gene expression activity in vivo.

[0241] [Production Example 1] Synthesis of O-Ph-P4C2 <Acid anhydride of oleic acid> 70.0 g (248 mmol) of oleic acid (manufactured by NOF Corporation) was dissolved in 560 g of chloroform at room temperature and cooled to 10 - 15°C. A suspension prepared by dissolving 25.1 g (121 mmol) of DCC (manufactured by Osaka Organic Chemical Industry Co., Ltd.) in 140 g of chloroform was added dropwise thereto, and the reaction was carried out at 10 - 25°C for 2 hours. After filtering the reaction solution, the filtrate was concentrated using an evaporator. The obtained concentrate was redissolved in 210 g of hexane, and the insoluble matter was removed by filtration. The obtained filtrate was concentrated using an evaporator to obtain 64.2 g of oleic anhydride.

[0242] <Synthesis of 4-Oleyloxyphenylacetic Acid> 43.1 g (78.9 mmol) of oleic anhydride and 6.00 g (39.4 mmol) of 4-hydroxyphenylacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) were dissolved in 647 g of chloroform. 1.93 g (15.8 mmol) of DMAP (manufactured by Koei Chemical Co., Ltd.) was added thereto, and the reaction was carried out at room temperature for 9 hours. The reaction solution was washed twice with 216 g of 10% aqueous acetic acid solution and twice with 216 g of ion-exchanged water, and then 12.9 g of magnesium sulfate (manufactured by Kanto Chemical Co., Ltd.) was added to the organic layer and stirred for 30 minutes. After filtering magnesium sulfate, the filtrate was concentrated using an evaporator. The concentrate was redissolved in 284 g of hexane, and after filtering the insoluble matter, extraction was carried out 6 times using 168 g of acetonitrile. The acetonitrile layer was collected and concentrated using an evaporator to obtain 18.1 g of a crude product. By subjecting 14.5 g of the obtained crude product to column purification, 3.66 g of 4-oleyloxyphenylacetic acid was obtained.

[0243] <Synthesis of O-Ph-P4C2> 0.350g (0.929mmol) of bis{2-[4-(2-hydroxyethyl)piperidyl]ethyl}disulfide (di-4PE body) synthesized by the method described in US2014 / 0335157A1, 0.813g (1.95mmol) of 4-oleoyloxyphenylacetic acid, and 0.0454g (0.372mmol) of DMAP were dissolved in 10.5g of chloroform at room temperature. 0.534g (2.79mmol) of EDC was added thereto and reacted at 30-35°C for 4 hours. The reaction solution was washed twice with 7.00g of 20% saline, and then dehydrated using 0.350g of magnesium sulfate. After filtering the magnesium sulfate, the filtrate was concentrated with an evaporator to obtain 1.10g of crude product. The obtained crude product was purified by column purification to obtain 0.722g of O-Ph-P4C2.

[0244] [Production Example 2] Synthesis of E-Ph-P4C2 <Acid anhydride of D-α-tocopherol succinate> 70.0g (132mmol) of D-α-tocopherol succinate (manufactured by SIGMA-ALDRICH) was dissolved in 560g of chloroform at room temperature and cooled to 10-15°C. A suspension of 13.7g (66mmol) of DCC (manufactured by Osaka Synthetic Organic Chemical Laboratory Co., Ltd.) dissolved in 140g of chloroform was added dropwise thereto and reacted at 10-25°C for 2 hours. After filtering the reaction solution, the filtrate was concentrated by an evaporator. The obtained concentrate was redissolved in 210g of hexane, and insoluble matter was removed by filtration. The obtained filtrate was concentrated by an evaporator to obtain 64.2g of D-α-tocopherol succinate anhydride.

[0245] <Synthesis of 4-(D-α-tocopherol hemisuccinyl)phenylacetic acid> 43.1 g (41.3 mmol) of anhydrous succinic acid D-α-tocopherol and 3.13 g (20.6 mmol) of 4-hydroxyphenylacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) were dissolved in 647 g of chloroform. 1.01 g (8.26 mmol) of DMAP (manufactured by Hiroei Chemical Co., Ltd.) was added thereto, and the reaction was carried out at room temperature for 9 hours. The reaction solution was washed twice with 216 g of 10% aqueous acetic acid solution and twice with 216 g of ion-exchanged water, and then 12.9 g of magnesium sulfate (manufactured by Kanto Chemical Co., Inc.) was added to the organic layer and stirred for 30 minutes. After filtering off the magnesium sulfate, the filtrate was concentrated using an evaporator. The concentrate was redissolved in 284 g of hexane, the insoluble matter was filtered off, and extraction was carried out 6 times using 168 g of acetonitrile. The acetonitrile layer was recovered and concentrated using an evaporator to obtain 17.0 g of a crude product. By subjecting 13.6 g of the obtained crude product to column purification, 3.44 g of 4-(D-α-tocopherol hemisuccinyl)phenylacetic acid was obtained.

[0246] <Synthesis of E-Ph-P4C2> 0.350 g (0.929 mmol) of the di-4PE compound, 1.04 g (1.95 mmol) of 4-(D-α-tocopherol hemisuccinyl)phenylacetic acid, and 0.0454 g (0.372 mmol) of DMAP were dissolved in 10.5 g of chloroform at room temperature. 0.534 g (2.79 mmol) of EDC was added thereto, and the reaction was carried out at 30 - 35 °C for 4 hours. After washing the reaction solution twice with 7.00 g of 20% brine, dehydration was carried out using 0.350 g of magnesium sulfate. After filtering off the magnesium sulfate, the filtrate was concentrated using an evaporator to obtain 1.31 g of a crude product. By subjecting the obtained crude product to column purification, 0.860 g of E-Ph-P4C2 was obtained.

Industrial Applicability

[0247] According to the present invention, since it is possible to introduce nucleic acids into cells with high efficiency, it is useful for nucleic acid pharmaceuticals, gene therapy, and biochemical experiments.

[0248] This application is based on Japanese Patent Application No. 2019-176253 filed in Japan, the content of which is incorporated herein in its entirety.

Claims

1. A lyophilized composition of lipid nanoparticles that does not contain nucleic acids and contains an ionic lipid, a sterol, a PEG lipid, an acidic buffer component having a buffering effect at pH 1 to 6, and a cryoprotectant, The weight ratio of cryoprotectant to total lipid is 10:1 to 1000:1; The ionic lipid is an ionic lipid containing a tertiary amino group; A freeze-dried composition used to prepare nucleic acid-encapsulated lipid nanoparticles by mixing with an aqueous solution containing nucleic acid.

2. The lyophilized composition of claim 1 further comprising a phospholipid.

3. The freeze-dried composition according to claim 1 or 2, wherein the weight ratio of cryoprotectant to total lipid is from 30:1 to 1000:

1.

4. The freeze-dried composition according to any one of claims 1 to 3, wherein the concentration of the cryoprotectant is 80 to 800 mg / mL in the composition before freeze-drying.

5. The freeze-dried composition according to any one of claims 1 to 4, wherein the concentration of the cryoprotectant is 160 to 800 mg / mL in the composition before freeze-drying.

6. The freeze-dried composition according to any one of claims 1 to 5, wherein the ionic lipid is a compound represented by formula (1): 【Chemistry 1】 (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms, X ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

7. The freeze-dried composition according to any one of claims 1 to 6, wherein the cryoprotectant is a disaccharide.

8. The freeze-dried composition according to any one of claims 1 to 6, wherein the cryoprotectant is sucrose.

9. A method for producing nucleic acid-encapsulated lipid nanoparticles, comprising the steps of: a) mixing an alcohol solution containing an ionic lipid, a sterol and a PEG lipid with an acidic buffer having a buffering effect at pH 1 to 6 to prepare a suspension of lipid nanoparticles that does not contain nucleic acid, wherein the ionic lipid is an ionic lipid that contains a tertiary amino group; b) mixing the suspension of nucleic acid-free lipid nanoparticles with a cryoprotectant to obtain a mixture containing 80-800 mg / mL of cryoprotectant and having a pH of 1-6; c) freeze-drying the mixture obtained in step b to obtain a freeze-dried composition; d) mixing the lyophilized composition with an aqueous solution containing nucleic acid and optionally containing 0-25% v / v alcohol, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles; and e) A step of exchanging the external aqueous phase of the obtained nucleic acid-encapsulated lipid nanoparticles with a neutral buffer solution by dialysis, ultrafiltration or dilution, wherein the zeta potential of the nucleic acid-encapsulated lipid nanoparticles after exchanging the external aqueous phase with a neutral buffer solution is −15 to +15 mV.

10. The method according to claim 9, further comprising exchanging the external aqueous phase for another acidic buffer solution having a buffering effect at pH 1 to 6 by dialysis, ultrafiltration or dilution after preparation of the lipid nanoparticle suspension in step a.

11. The method according to claim 9 or 10, wherein in step a, the alcoholic solution further comprises a phospholipid.

12. The method according to any one of claims 9 to 11, wherein in step b, the concentration of the cryoprotectant in the mixture is 160 to 800 mg / mL.

13. The method according to any one of claims 9 to 12, wherein the ionic lipid is a compound represented by formula (1): 【Chemistry 2】 (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms, X ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

14. The method according to any one of claims 9 to 13, wherein the cryoprotectant is a disaccharide.

15. The method according to any one of claims 9 to 13, wherein the cryoprotectant is sucrose.

16. A method for producing a freeze-dried composition of lipid nanoparticles, comprising the step of freeze-drying a composition of lipid nanoparticles that does not contain nucleic acids and that contains an ionic lipid, a sterol, a PEG lipid, an acidic buffer having a buffering effect at pH 1 to 6, and 80 to 800 mg / mL of a cryoprotectant, The method of production, wherein the ionic lipid is an ionic lipid that contains a tertiary amino group.

17. 17. The method of claim 16, wherein the lipid nanoparticle composition further comprises a phospholipid.

18. The method according to claim 16 or 17, wherein the concentration of the cryoprotectant in the composition before lyophilization is from 160 to 800 mg / mL.

19. The method according to any one of claims 16 to 18, wherein the ionic lipid is a compound represented by formula (1): 【Chemistry 3】 (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms, X ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

20. The method according to any one of claims 16 to 19, wherein the cryoprotectant is a disaccharide.

21. The method according to any one of claims 16 to 19, wherein the cryoprotectant is sucrose.

22. The freeze-dried composition according to any one of claims 1 to 8 is included. A nucleic acid transfer agent used to prepare nucleic acid-encapsulated lipid nanoparticles by mixing with an aqueous solution containing nucleic acid.

23. A method for introducing a nucleic acid into a cell, comprising contacting the cell with the nucleic acid introduction agent according to claim 22 encapsulating the nucleic acid in an ex vivo state.

24. A method for introducing a nucleic acid into a cell, comprising the steps of: a) mixing an alcohol solution containing an ionic lipid, a sterol and a PEG lipid with an acidic buffer having a buffering effect at pH 1 to 6 to prepare a suspension of lipid nanoparticles that does not contain nucleic acid, wherein the ionic lipid is an ionic lipid that contains a tertiary amino group; b) mixing the suspension of nucleic acid-free lipid nanoparticles with a cryoprotectant to obtain a mixture containing 80-800 mg / mL of cryoprotectant and having a pH of 1-6; c) freeze-drying the mixture obtained in step b to obtain a freeze-dried composition; d) mixing the lyophilized composition with an aqueous solution containing the nucleic acid, optionally containing 0-25% v / v alcohol, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles; e) exchanging the external aqueous phase of the resulting nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration or dilution, wherein the zeta potential of the nucleic acid-encapsulated lipid nanoparticles after exchanging the external aqueous phase with a neutral buffer is −15 to +15 mV; and f) contacting the resulting nucleic acid-encapsulated lipid nanoparticles with the cells in vitro.

25. The method according to claim 24, further comprising exchanging the external aqueous phase for another acidic buffer solution having a buffering effect at pH 1 to 6 by dialysis, ultrafiltration or dilution after preparation of the lipid nanoparticle suspension in step a.

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