Neutral lipids and lipid nanoparticles

Abstract

Provided are: a neutral lipid capable of suppressing phase change inhibition of an endosomal membrane caused by phosphatidylcholine; and lipid nanoparticles containing the neutral lipid. The present invention relates to an ionic neutral lipid comprising a compound represented by general formula (I): (In formula (I), R1 represents a hydrocarbon group having 1-22 carbon atoms; the three R1 groups in one molecule can be the same group or different groups; a1 and a2 are each independently an integer of 0-4; b1 and b2 are 0 or 1 satisfying b1 + b2 = 1; r2 and R3 each independently represent a hydrogen atom or an alkyl group having 1-3 carbon atoms; and R4 represents an anionic group. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a neutral lipid having a hydrophobic chain and a hydrophilic group, and a lipid nanoparticle containing the neutral lipid.

[0002] The present invention claims priority based on Japanese Patent Application No. 2022-115017 filed in Japan on July 19, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] Lipid nanoparticles (LNPs) are used as carriers for encapsulating lipophilic drugs, siRNA (short interfering RNA), mRNA and other nucleic acids and delivering them to target cells. For example, regarding lipid nanoparticles as carriers for efficiently delivering nucleic acids such as siRNA into target cells, a lipid nanoparticle has been reported, which contains pH-sensitive cationic lipids that are electrically neutral at physiological pH and change to cationic under weakly acidic pH environments such as endosomes as constituent lipids (Patent Document 1). Positively charged lipid nanoparticles in the blood interact nonspecifically with plasma proteins and are rapidly cleared from the blood by the reticuloendothelial system. In contrast, lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids are uncharged under neutral conditions in the blood and positively charged under weakly acidic conditions in the endosome, and can efficiently escape from the endosomal tract.

[0004] As pH-sensitive cationic lipids, Jayaraman et al. developed DLin-MC3-DMA, ED 50 The factor VII (F7) knockdown in mouse liver reached 0.005 mg siRNA / kg (Non-patent Document 1). The inventors of the present application have previously developed original pH-sensitive cationic lipids YSK05 and YSK13-C3 as ED inhibitors for F7 knockdown. 50 , reaching 0.06 and 0.015 mg siRNA / kg respectively (Non-patent Documents 2 to 4). The inventors of the present application also developed compounds having two hydrocarbon chains and one hydrophilic group such as 7-(4-(diisopropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6) as pH-sensitive cationic lipids (Patent Document 1).

[0005] Lipid nanoparticles are taken into cells by endocytosis. Therefore, for improving the efficiency of lipid nanoparticles to be taken into cells, it is important that the membrane fusion of lipid nanoparticles and endosomal membranes is easy to carry out this point. On the other hand, the phosphatidylcholine (PC) as the main constituent lipid of endosomal membranes is owing to taking a cylindrical type, and therefore the lamellar (L) phase of lipid bilayer membrane (planar membrane) is stabilized, and the transformation of the anti-hexagonal (HII) phase necessary for membrane fusion is strongly suppressed. For example, it has been known for a long time that, with regard to DOPE as a general constituent lipid in lipid nanoparticles, owing to taking a cone, the transformation from lamellar phase to the anti-hexagonal phase is promoted, and on the other hand, the effect is greatly suppressed by phosphatidylcholine (non-patent literature 5).

[0006] On the other hand, as the manufacture method of the lipid nanoparticles containing nucleic acid etc., there is the method for principle with the alcohol dilution method using stream.For example, report that by using the built-in micro-flow path of the three-dimensional micro-mixer that can realize 2 liquid instantaneous mixing, can reproducibly manufacture the lipid nanoparticles (non-patent literature 6) of diameter 30nm or so.In addition, also report that by using the stream path structure body of the simple two-dimensional structure that the baffle (baffle) that the relative stream path width is a certain width is staggeredly configured from both sides in the stream path of the micro-size that the stock solution flows, compared with the stream path structure body of the three-dimensional mixer that uses in the past, can form the lipid granule formation system (patent documentation 2) of the nanometer size that particle diameter controllability is high.The manufacture method of these nanoparticle preparations is mainly used to manufacture the lipid nanoparticles (LNP) that load has the nucleic acid such as fat-soluble drug, siRNA (short interfering RNA) or mRNA in recent years.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2018 / 230710

[0010] Patent Document 2: International Publication No. 2018 / 190423

[0011] Non-patent literature

[0012] Non-patent document 1: Jayaraman et al., Angewandte Chemie International Edition, 2012, vol. 51, p. 8529-8533.

[0013] Non-patent document 2: Watanabe et al., Scientific Reports, 2014, 4: 4750, DOI: 10.1038 / srep04750.

[0014] Non-patent document 3: Yamamoto et al., Journal of Hepatology, 2016, vol. 64, p. 547-555.

[0015] Non-patent document 4: Sato et al., Molecular Therapy, 2016, vol. 24, p. 788-795.

[0016] Non-patent document 5: Cullis et al., Biochimica et Biophysica Acta, 1979, vol. 559, p. 399-420.

[0017] Non-patent document 6: Leung et al., Journal of Physical Chemistry C Nanomater Interfaces, 2012, vol. 116 (34), p. 18440-18450. Summary of the invention

[0018] Problems to be solved by the invention

[0019] In the uptake of lipid nanoparticles containing pH-sensitive cationic lipids into cells, the phase transition driven by the interaction between the pH-sensitive cationic lipids in the lipid nanoparticles and the anionic lipids in the endosomal membrane is important, but this phase transition is inhibited by neutral phospholipids such as phosphatidylcholine. Therefore, it is necessary to develop a constituent lipid of lipid nanoparticles that can inhibit the phase transition caused by the neutral phospholipids.

[0020] An object of the present invention is to provide a neutral lipid capable of suppressing the phase transition of endosomal membranes caused by phosphatidylcholine, and a lipid nanoparticle containing the neutral lipid.

[0021] Means for solving problems

[0022] The inventors of the present application discovered that a compound obtained by bonding the three hydroxyl groups of trishydroxymethylaminomethane (CAS No.: 77-86-1) (hereinafter referred to as "Tris") to a hydrocarbon chain and bonding the remaining amino group to a hydrophilic group having a cationic portion at the front end and an anionic group at the end is an ionic neutral lipid suitable as a constituent lipid of lipid nanoparticles, thereby completing the present invention.

[0023] That is, the present invention provides the following ionic neutral lipids and the like.

[0024] [1] An ionic neutral lipid comprising a compound represented by the following formula (I):

[0025] [Chemical formula 1]

[0026]

[0027] In formula (I), R 1 is a hydrocarbon group having 1 to 22 carbon atoms; the three R 1 They may be the same group or different groups; a1 and a2 are each independently an integer of 0 to 4; b1 and b2 are 0 or 1 satisfying b1+b2=1; R 2 and R 3 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R 4 It is an anionic group.

[0028] [2] The ionic neutral lipid according to [1], wherein in the general formula (I), R 4 It is a carboxylic acid group, a sulfonic acid group, a sulfate group or a phosphoric acid group.

[0029] [3] The ionic neutral lipid according to [1] or [2], wherein the three R 1 All are the same group.

[0030] [4] The ionic neutral lipid according to any one of [1] to [3], wherein R 1 It is a hydrocarbon group having 15 to 22 carbon atoms.

[0031] [5] The ionic neutral lipid according to any one of [1] to [4] above, which is a compound represented by any one of the following general formulas (Z-9) to (Z-15):

[0032] [Chemical formula 2]

[0033]

[0034] [Chemical formula 3]

[0035]

[0036] [Where R 1 Same as general formula (I)].

[0037] [6] The ionic neutral lipid according to any one of [1] to [5], which is (S)-4-amino-5-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-5-oxopentanoic acid, 2-((4-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-4-oxobutyl)dimethylammonio)acetate, 2-((3-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-3-oxopropyl)dimethylammonio)acetate, 2-((3-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-3-oxopropyl)(methyl)ammonio)acetate, (S)-3-amino-4-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-4-oxobutanoic acid, N4-(1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)-L-asparagine, or N5-(1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)-L-glutamine.

[0038] [7] Ionic neutral lipids having a Tris backbone,

[0039] A fatty acid residue is bonded to each of the three oxygen atoms of the Tris skeleton. The number of carbon atoms in the hydrocarbon chain of the fatty acid residue is 1 to 22, and the fatty acid residue may have one or more unsaturated bonds. The three fatty acid residues in one molecule may be the same group or two or more different groups.

[0040] An anionic group is linked to one nitrogen atom of the Tris skeleton via a divalent linking group having an ammonium cationic group or a quaternary ammonium cationic nitrogen atom.

[0041] [8] A lipid nanoparticle comprising the ionic neutral lipid according to any one of [1] to [7] above.

[0042] [9] The lipid nanoparticles described in [8] above, further comprising sterol- and polyalkylene glycol-modified lipids.

[0043]

[10] The lipid nanoparticles according to [8] or [9] above, further comprising a pH-sensitive cationic lipid having a pKa of 6.6 or less.

[0044]

[11] The lipid nanoparticle according to any one of [8] to

[10] above, comprising a nucleic acid.

[0045]

[12] The lipid nanoparticle described in

[11] above, wherein the nucleic acid is siRNA, mRNA or plasmid DNA.

[0046]

[13] A pharmaceutical composition comprising lipid nanoparticles containing the ionic neutral lipid according to any one of [1] to [7] above as an active ingredient.

[0047]

[14] A pharmaceutical composition comprising lipid nanoparticles containing the ionic neutral lipid according to any one of [1] to [7] above and a nucleic acid as an active ingredient.

[0048]

[15] The pharmaceutical composition according to

[13] or

[14] , which is used for gene therapy.

[0049]

[16] A method for expressing exogenous genes, wherein the lipid nanoparticles described in

[11] above, which encapsulate a target exogenous gene that can be expressed in target cells, are administered to a subject animal (excluding humans), so that the exogenous gene is expressed in the target cells of the subject animal.

[0050] Effects of the Invention

[0051] The ionic neutral lipids of the present invention have an inhibitory effect on the stabilization of the lipid bilayer membrane caused by phosphatidylcholine. Therefore, the ionic neutral lipids are suitable as constituent lipids of lipid nanoparticles for intracellular uptake, and are particularly useful as constituent lipids of lipid nanoparticles used as active ingredients of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] [ Figure 1 ] is the DOPC / DOPE membrane in Example 8 ( Figure 1 (A)) and DOPC / TOT-10 membrane ( Figure 1 (B) 31 PNMR spectral data.

[0053] [ Figure 2 ] is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver and spleen of mice administered with lipid nanoparticles loaded with Fluc mRNA in Example 9.

[0054] [ Figure 3 ] is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver of mice administered with lipid nanoparticles loaded with Fluc mRNA in Example 10.

[0055] [ Figure 4] is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the quadriceps muscle of mice to which lipid nanoparticles loaded with Fluc mRNA were administered in Example 11.

[0056] [ Figure 5 ] is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the brain of mice administered with lipid nanoparticles loaded with Fluc mRNA in Example 12.

[0057] [ Figure 6 ] is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver and spleen of mice administered with lipid nanoparticles loaded with Fluc mRNA in Example 13.

[0058] [ Figure 7 ] is a graph showing the measurement results of the charge rate (%) of lipid nanoparticles prepared from CL4F6, TOT-9 to TOT-15 or DSPC, cholesterol, and PEG-DMG under various pH conditions in Example 14.

[0059] [ Figure 8 ] is a graph showing the results of measuring the hemolytic activity of lipid nanoparticles prepared from CL4F6, TOT-9 to TOT-15 or DSPC, cholesterol, and PEG-DMG in Example 14.

[0060] [ Fig. 9 ] is a graph showing the measurement results of the charge rate (%) of lipid nanoparticles prepared in Example 14 using CL4F6, ALC-0315, MC3, SM-102, CL15F6 or CL15H6 as cationic lipids and TOT-15 or DSPC as ionic neutral lipids under various pH conditions.

[0061] [ Fig.10 ] is a graph showing the results of measuring the hemolytic activity of lipid nanoparticles prepared using CL4F6, ALC-0315, MC3, SM-102, CL15F6 or CL15H6 as cationic lipids and TOT-15 or DSPC as ionic neutral lipids in Example 14.

[0062] [ Fig.11 ] is a graph showing the results of measuring the amount of Fluc mRNA in the liver of mice to which lipid nanoparticles loaded with Fluc mRNA were administered in Example 15.

[0063] [ Fig.12is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver of a mouse administered with lipid nanoparticles loaded with Fluc mRNA in Example 15.

[0064] Fig.13 is a graph showing the results of measuring the knockdown efficiency of TTR in a mouse administered with lipid nanoparticles for TTR target genome editing loaded with Cas9-mRNA and TTR-gRNA in Example 16.

[0065] Fig.14 is a graph showing the results of time-course measurement of the average particle size and encapsulation efficiency of lipid nanoparticles loaded with Fluc mRNA prepared from CL4F6, TOT-15 or DSPC, cholesterol, and PEG-DMG when stored at 4°C in Example 17.

[0066] Fig.15 is a graph showing the results of measuring the Fluc activity (RLU / mg protein) in the liver of a mouse administered with lipid nanoparticles loaded with Fluc mRNA immediately after manufacture or stored at 4°C for 4 weeks in Example 17.

[0067] Fig.16 is a microscopic image obtained by observing the expression of EGFP in the liver of lipid nanoparticles loaded with EGFP mRNA prepared from CL4F6, TOT-15 or DSPC, cholesterol, and PEG-DMG in Example 18. Detailed Description of the Invention

[0068] Hereinafter, embodiments of the present invention will be specifically described. In the present application specification, "X1 to X2 (X1 and X2 are real numbers satisfying X1 < X2)" means "X1 or more and X2 or less". In addition, "C X3-X4 (X3 and X4 are integers satisfying X3 < X4)" means "the number of carbon atoms is X3 or more and X4 or less".

[0069] [Ionic Neutral Lipid]

[0070] The ionic neutral lipid of the present invention is a compound obtained by bonding each of the three hydroxyl groups of Tris to a hydrocarbon chain and bonding the amino group of Tris to an anionic group containing a cationic moiety. It is a compound having three hydrocarbon chains and a hydrophilic moiety, and due to the similarity of its structure, it has a high affinity for lipids such as phospholipids and is suitable as a constituent lipid of lipid nanoparticles.

[0071] ​​​​As an example of the ionic neutral lipid involved in the present invention, there can be cited a compound in which a fatty acid residue is bonded to each of the three oxygen atoms in the Tris skeleton, and an anionic group is connected to one nitrogen atom in the Tris skeleton via a linking group having an ammonium cationic group or a quaternary ammonium cationic nitrogen atom. Here, the "Tris skeleton" refers to a structure in which one hydrogen atom is removed from each of the three hydroxyl groups and the amino group of trishydroxymethylaminomethane. Among the three fatty acid residues bonded to the Tris skeleton, the number of carbon atoms in the hydrocarbon chain part is 1 to 22, and it may also have one or more unsaturated bonds. In addition, the multiple fatty acid residues present in one molecule may be the same group or may be two or more different groups. As the ionic neutral lipid involved in the present invention, it is preferred that all the multiple fatty acid residues present in one molecule are the same group. As an anionic group connected to one nitrogen atom of the Tris skeleton via a divalent linking group, R described later can be used. 4 As a divalent connecting group connecting one nitrogen atom of the Tris skeleton to the anionic group, there is no particular limitation as long as it is a divalent group having an ammonium cationic group inserted in the side chain, or a group having a quaternary ammonium cationic nitrogen atom inserted between carbon atoms constituting the main chain. For example, it is preferably a group in which one hydrogen atom bonded to a carbon atom of an alkylene group having 1 to 6 carbon atoms is substituted by an ammonium cationic group, or a group in which a quaternary ammonium cationic nitrogen atom is inserted between carbon atoms of an alkylene group having 1 to 6 carbon atoms. As the ammonium cationic group and the quaternary ammonium cationic nitrogen atom, the groups listed below can be used.

[0072] An example of the ionic neutral lipid according to the present invention includes a compound represented by the following general formula (I).

[0073] [Chemical formula 4]

[0074]

[0075] In the general formula (I), R 1 is a hydrocarbon group having 1 to 22 carbon atoms (C 1-22 The C 1-22 The hydrocarbon group may be straight-chain or branched. The hydrocarbon group may be an alkyl group or an alkenyl group. The alkenyl group may have one unsaturated bond, two unsaturated bonds, or three unsaturated bonds. 1 , preferably a hydrocarbon group having 15 to 22 carbon atoms (C 15-22 Hydrocarbon).

[0076] In R 1 C 1-22 In the case of an alkyl group, examples of the alkyl group include:

[0077] Methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl;

[0078] n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1-ethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl;

[0079] n-Hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1-methyl-2,2-dimethylbutyl;

[0080] n-heptyl, 1-methylhexyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 1-ethylpentyl, 1,1-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1-methyl-3,3-dimethylbutyl, 2-methyl-3,3-dimethylbutyl;

[0081] n-octyl, 1-methylheptyl, 2-methylheptyl, 3-methylheptyl, 4-methylheptyl, 5-methylheptyl, 6-methylheptyl, 1-ethylhexyl, 1,1-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 5,5-dimethylhexyl, 1-methyl-4,4-dimethylpentyl, 2-methyl-4,4-dimethylpentyl, 3-methyl-4,4-dimethylpentyl;

[0082] n-nonyl, 1-methyloctyl, 2-methyloctyl, 3-methyloctyl, 4-methyloctyl, 5-methyloctyl, 6-methyloctyl, 7-methyloctyl, 1-ethylheptyl, 1,1-dimethylheptyl, 2,2-dimethylheptyl, 3,3-dimethylheptyl, 4,4-dimethylheptyl, 5,5-dimethylheptyl, 6,6-dimethylheptyl, 1-methyl-5,5-dimethylhexyl, 2-methyl-5,5-dimethylhexyl, 3-methyl-5,5-dimethylhexyl, 4-methyl-5,5-dimethylhexyl;

[0083] n-Decyl, 1-methylnonyl, 2-methylnonyl, 3-methylnonyl, 4-methylnonyl, 5-methylnonyl, 6-methylnonyl, 7-methylnonyl, 8-methylnonyl, 1-ethyloctyl, 1,1-dimethyloctyl, 2,2-dimethyloctyl, 3,3-dimethyloctyl, 4,4-dimethyloctyl, 5,5-dimethyloctyl, 6,6-dimethyloctyl, 7,7-dimethyloctyl, 1-methyl-6,6-dimethylheptyl, 2-methyl-6,6-dimethylheptyl, 3-methyl-6,6-dimethylheptyl, 4-methyl-6,6-dimethylheptyl, 5-methyl-6,6-dimethylheptyl;

[0084] n-Undecyl, 1-methyldecyl, 2-methyldecyl, 3-methyldecyl, 4-methyldecyl, 5-methyldecyl, 6-methyldecyl, 7-methyldecyl, 8-methyldecyl, 9-methyldecyl, 1-ethylnonyl, 1,1-dimethylnonyl, 2,2-dimethylnonyl, 3,3-dimethylnonyl, 4,4-dimethylnonyl, 5,5-dimethylnonyl, 6,6-dimethylnonyl, 7,7-dimethylnonyl, 8,8-dimethylnonyl, 1-methyl-7,7-dimethyloctyl, 2-methyl-7,7-dimethyloctyl, 3-methyl-7,7-dimethyloctyl, 4-methyl-7,7-dimethyloctyl, 5-methyl-7,7-dimethyloctyl, 6-methyl-7,7-dimethyloctyl;

[0085] n-dodecyl, 1-methylundecyl, 2-methylundecyl, 3-methylundecyl, 4-methylundecyl, 5-methylundecyl, 6-methylundecyl, 7-methylundecyl, 8-methylundecyl, 9-methylundecyl, 10-methylundecyl, 1-ethyldecyl, 1,1-dimethyldecyl, 2,2-dimethyldecyl, 3,3-dimethyldecyl, 4,4-dimethyldecyl, 5,5-dimethyldecyl, 6,6-dimethyldecyl, 7,7-dimethyldecyl, 8,8-dimethyldecyl, 9,9-dimethyldecyl, 1-methyl-8,8-dimethylnonyl, 2-methyl-8,8-dimethylnonyl, 3-methyl-8,8-dimethylnonyl, 4-methyl-8,8-dimethylnonyl, 5-methyl-8,8-dimethylnonyl, 6-methyl-8,8-dimethylnonyl, 7-methyl-8,8-dimethylnonyl;

[0086] n-Tridecyl, 1-methyldodecyl, 2-methyldodecyl, 3-methyldodecyl, 4-methyldodecyl, 5-methyldodecyl, 6-methyldodecyl, 7-methyldodecyl, 8-methyldodecyl, 9-methyldodecyl, 10-methyldodecyl, 11-methyldodecyl, 1-ethylundecyl, 1,1-dimethylundecyl, 2,2-dimethylundecyl, 3,3-dimethylundecyl, 4,4-dimethylundecyl, 5,5-dimethylundecyl, 6,6 -dimethylundecyl, 7,7-dimethylundecyl, 8,8-dimethylundecyl, 9,9-dimethylundecyl, 10,10-dimethylundecyl, 1-methyl-9,9-dimethyldecyl, 2-methyl-9,9-dimethyldecyl, 3-methyl-9,9-dimethyldecyl, 4-methyl-9,9-dimethyldecyl, 5-methyl-9,9-dimethyldecyl, 6-methyl-9,9-dimethyldecyl, 7-methyl-9,9-dimethyldecyl, 8-methyl-9,9-dimethyldecyl;

[0087] n-Tetradecyl, 1-methyltridecyl, 2-methyltridecyl, 3-methyltridecyl, 4-methyltridecyl, 5-methyltridecyl, 6-methyltridecyl, 7-methyltridecyl, 8-methyltridecyl, 9-methyltridecyl, 10-methyltridecyl, 11-methyltridecyl, 12-methyltridecyl, 1-ethyldodecyl, 1,1-dimethyldodecyl, 2,2-dimethyldodecyl, 3,3-dimethyldodecyl, 4,4-dimethyldodecyl, 5,5-dimethyldodecyl, 6,6-dimethyldodecyl, 7,7-dimethyldodecyl, 8,8-dimethyldodecyl 1-methyl-10,10-dimethylundecyl, 2-methyl-10,10-dimethylundecyl, 3-methyl-10,10-dimethylundecyl, 4-methyl-10,10-dimethylundecyl, 5-methyl-10,10-dimethylundecyl, 6-methyl-10,10-dimethylundecyl, 7-methyl-10,10-dimethylundecyl, 8-methyl-10,10-dimethylundecyl, 9-methyl-10,10-dimethylundecyl;

[0088] Pentadecyl, 1-methyltetradecyl, 2-methyltetradecyl, 3-methyltetradecyl, 4-methyltetradecyl, 5-methyltetradecyl, 6-methyltetradecyl, 7-methyltetradecyl, 8-methyltetradecyl, 9-methyltetradecyl, 10-methyltetradecyl, 11-methyltetradecyl, 12-methyltetradecyl, 13-methyltetradecyl, 1-ethyltridecyl, 1,1-dimethyltridecyl, 2,2-dimethyltridecyl, 3,3-dimethyltridecyl, 4,4-dimethyltridecyl, 5,5-dimethyltridecyl, 6,6-dimethyltridecyl, 7,7-dimethyltridecyl, 8,8-dimethyltridecyl, 9,9-dimethyl 1-methyl-11,11-dimethyldodecyl, 2-methyl-11,11-dimethyldodecyl, 3-methyl-11,11-dimethyldodecyl, 4-methyl-11,11-dimethyldodecyl, 5-methyl-11,11-dimethyldodecyl, 6-methyl-11,11-dimethyldodecyl, 7-methyl-11,11-dimethyldodecyl, 8-methyl-11,11-dimethyldodecyl, 9-methyl-11,11-dimethyldodecyl, 10-methyl-11,11-dimethyldodecyl;

[0089] n-Hexadecyl, 1-methylpentadecyl, 2-methylpentadecyl, 3-methylpentadecyl, 4-methylpentadecyl, 5-methylpentadecyl, 6-methylpentadecyl, 7-methylpentadecyl, 8-methylpentadecyl, 9-methylpentadecyl, 10-methylpentadecyl, 11-methylpentadecyl, 12-methylpentadecyl, 13-methylpentadecyl, 14-methylpentadecyl;

[0090] n-Heptadecanyl, 1-methylhexadecyl, 2-methylhexadecyl, 3-methylhexadecyl, 4-methylhexadecyl, 5-methylhexadecyl, 6-methylhexadecyl, 7-methylhexadecyl, 8-methylhexadecyl, 9-methylhexadecyl, 10-methylhexadecyl, 11-methylhexadecyl, 12-methylhexadecyl, 13-methylhexadecyl, 14-methylhexadecyl, 15-methylhexadecyl;

[0091] n-octadecyl, 1-methylheptadecyl, 2-methylheptadecyl, 3-methylheptadecyl, 4-methylheptadecyl, 5-methylheptadecyl, 6-methylheptadecyl, 7-methylheptadecyl, 8-methylheptadecyl, 9-methylheptadecyl, 10-methylheptadecyl, 11-methylheptadecyl, 12-methylheptadecyl, 13-methylheptadecyl, 14-methylheptadecyl, 15-methylheptadecyl, 16-methylheptadecyl;

[0092] n-nonadecyl, 1-methyloctadecyl, 2-methyloctadecyl, 3-methyloctadecyl, 4-methyloctadecyl, 5-methyloctadecyl, 6-methyloctadecyl, 7-methyloctadecyl, 8-methyloctadecyl, 9-methyloctadecyl, 10-methyloctadecyl, 11-methyloctadecyl, 12-methyloctadecyl, 13-methyloctadecyl, 14-methyloctadecyl, 15-methyloctadecyl, 16-methyloctadecyl, 17-methyloctadecyl;

[0093] n-eicosyl, 1-methylnonadecyl, 2-methylnonadecyl, 3-methylnonadecyl, 4-methylnonadecyl, 5-methylnonadecyl, 6-methylnonadecyl, 7-methylnonadecyl, 8-methylnonadecyl, 9-methylnonadecyl, 10-methylnonadecyl, 11-methylnonadecyl, 12-methylnonadecyl, 13-methylnonadecyl, 14-methylnonadecyl, 15-methylnonadecyl, 16-methylnonadecyl, 17-methylnonadecyl, 18-methylnonadecyl;

[0094] n-heicosyl, 1-methyleicosyl, 2-methyleicosyl, 3-methyleicosyl, 4-methyleicosyl, 5-methyleicosyl, 6-methyleicosyl, 7-methyleicosyl, 8-methyleicosyl, 9-methyleicosyl, 10-methyleicosyl, 11-methyleicosyl, 12-methyleicosyl, 13-methyleicosyl, 14-methyleicosyl, 15-methyleicosyl, 16-methyleicosyl, 17-methyleicosyl, 18-methyleicosyl, 19-methyleicosyl;

[0095] n-docosanyl, 1-methylheicosanyl, 2-methylheicosanyl, 3-methylheicosanyl, 4-methylheicosanyl, 5-methylheicosanyl, 6-methylheicosanyl, 7-methylheicosanyl, 8-methylheicosanyl, 9-methylheicosanyl, 10-methylheicosanyl, 11-methylheicosanyl, 12-methylheicosanyl, 13-methylheicosanyl, 14-methylheicosanyl, 15-methylheicosanyl, 16-methylheicosanyl, 17-methylheicosanyl, 18-methylheicosanyl, 19-methylheicosanyl, 20-methylheicosanyl; and the like.

[0096] In R 1 C 2-22 In the case of an alkenyl group, examples of the alkenyl group include a C 2-22 The alkyl group refers to a group in which 1 to 3 of the saturated bonds between carbon molecules in the group are unsaturated bonds.

[0097] In the general formula (I), the three R 1 The three R groups in one molecule are preferably 1 All are groups of the same kind.

[0098] As the ionic neutral lipid of the present invention, R 1 Preferably, the straight chain C 1-22 Alkyl or linear C 2-22 Alkenyl, more preferably a straight-chain C 15-22 Alkyl or linear C 15-22 As the ionic neutral lipid of the present invention, more preferably three R 1 It is the same group or different groups selected from the group consisting of n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-eicosyl, n-heneicosyl, n-docosyl, n-pentadecenyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecenyl, n-eicosenyl, n-heneicosenyl and n-docosenyl. It is more preferred that 3 R 1 All of them are the same group selected from the group consisting of n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-eicosyl, n-heneicosyl, n-docosyl, n-pentadecenyl, n-hexadecenyl, n-heptadecenyl, n-octadecenyl, n-nonadecenyl, n-eicosenyl, n-heneicosenyl and n-docosenyl.

[0099] In the general formula (I), a1 and a2 are each independently an integer of 0 to 4. When a1 is 0, -(CH2)a1- means a single bond. Similarly, when a2 is 0, -(CH2)a2- means a single bond. As the ionic neutral lipid involved in the present invention, a1 is preferably an integer of 0 to 3. As the ionic neutral lipid involved in the present invention, a2 is preferably an integer of 0 to 3, and more preferably an integer of 0 to 2. As the ionic neutral lipid involved in the present invention, a1+a2 is preferably an integer of 1 to 6, and more preferably an integer of 1 to 4.

[0100] In the general formula (I), b1 and b2 are 0 or 1 satisfying b1+b2=1. When b1 is 1, b2 is 0, and when b1 is 0, b2 is 1. That is, the ionic neutral lipid represented by the general formula (I) must have -NH3 in one molecule. + and-(N + (R 2 )(R 3 ))-. It should be noted that, below -NH3 +and-(N + (R 2 )(R 3 )) are collectively referred to as "ammonium cationic part".

[0101] In the general formula (I), R 2 and R 3 are each independently a hydrogen atom or a C 1-3 Alkyl. As the C 1-3 The alkyl group includes methyl, ethyl, n-propyl or isopropyl. As the ionic neutral lipid of the present invention, R 2 and R 3 Each independently represents a hydrogen atom, a methyl group or an ethyl group, and more preferably R 2 and R 3 Each independently represents a hydrogen atom or a methyl group, and more preferably R 2 is a hydrogen atom or a methyl group and R 3 It is methyl.

[0102] In the general formula (I), R 4 is an anionic group. Examples of the anionic group include a carboxylic acid group (-C(=O)O-), a sulfonic acid group (-S(=O)2O-), a phosphate group (-OP(=O)O2-), and a sulfate group (-OS(=O)O2-). As the ionic neutral lipid of the present invention, R 4 It is a carboxylic acid group.

[0103] Examples of the ionic neutral lipids of the present invention include compounds represented by the following general formulae (Z-9) to (Z-15). 1 Same as general formula (I).

[0104] [Chemical formula 5]

[0105]

[0106] [Chemical formula 6]

[0107]

[0108] The ionic neutral lipids of the present invention reduce the stability of the lipid membrane containing phosphatidylcholine and easily cause phase transition. Therefore, by using the ionic neutral lipids of the present invention as the constituent lipids of lipid nanoparticles, the uptake efficiency of lipid nanoparticles in cell membranes containing phosphatidylcholine can be improved. Although the reason why the ionic neutral lipids of the present invention have an inhibitory effect on the phase transition inhibition of such phosphatidylcholine is not clear, it is speculated as follows.

[0109] The ionic neutral lipid of the present invention extends from one sp3 carbon to three strands including R1 Hydrocarbon chain, on which the sp3 carbon is also connected a hydrophilic group having an anionic group at the end and an ammonium cationic part at the front end. On the other hand, phosphatidylcholine has two hydrocarbon chains and a phosphorylcholine group, which has a trimethylammonium group at the end and an anionic phosphate group at the front end. Therefore, the ammonium cationic part and the anionic group in the hydrophilic group of the ionic neutral lipids of the present invention can form ion pairs with the phosphate group and the trimethylammonium group in the phosphorylcholine group of phosphatidylcholine, respectively. By making the hydrophilic group of the ionic neutral lipids of the present invention form an ion pair with the phosphorylcholine group of phosphatidylcholine in the endosomal membrane, the apparent shape of phosphatidylcholine is changed to a cone, which promotes the transition to a non-lamellar phase with negative curvature such as an inverse hexagonal phase. By changing the phase into the non-lamellar phase, the membrane fusion of lipid nanoparticles and endosomal membranes is promoted, and the uptake of the lipid nanoparticles into cells is promoted.

[0110] The ionic neutral lipids according to the present invention can be produced, for example, by condensing a compound represented by the following general formula (A) (hereinafter sometimes referred to as "compound (A)"), a compound represented by the following general formula (B) (hereinafter sometimes referred to as "compound (B)"), and trishydroxymethylaminomethane.

[0111] [Chemical formula 7]

[0112]

[0113] By dehydrating and condensing the carboxylic acid group of compound (A) with the hydroxyl group of Tris to form an ester bond, further, dehydrating and condensing the carboxylic acid group of compound (B) with the amino group of Tris to form an amide bond, an ionic neutral lipid represented by general formula (I) is synthesized. The dehydration condensation reaction of the carboxylic acid group of compound (A) and the hydroxyl group of Tris can usually be carried out under the same reaction conditions as the dehydration condensation reaction when forming an ester of carboxylic acid and alcohol. The dehydration condensation reaction of the carboxylic acid group of compound (B) and the amino group of Tris can usually be carried out under the same reaction conditions as the dehydration condensation reaction when forming an amidate of carboxylic acid and amine. That is, the ionic neutral lipid represented by general formula (I) can be synthesized relatively simply by using Tris as a linker and utilizing a common dehydration condensation reaction.

[0114] The ionic neutral lipid represented by the general formula (I) can be synthesized by reacting compound (A) with Tris to carry out a dehydration condensation reaction with the hydroxyl group of Tris, and then reacting compound (B) with the obtained esterified product to carry out a dehydration condensation reaction with the amino group derived from Tris. Alternatively, the ionic neutral lipid represented by the general formula (I) can be synthesized by reacting compound (B) with Tris to carry out a dehydration condensation reaction with the amino group of Tris, and then reacting compound (A) with the obtained amide product to carry out a dehydration condensation reaction with the hydroxyl group derived from Tris.

[0115] The ionic neutral lipid represented by the general formula (I) can be easily produced by the method specifically shown in the examples of this specification, for example. Those skilled in the art can easily produce any lipid included in the scope of the general formula (I) by referring to the production method and appropriately selecting raw material compounds, reagents and reaction conditions, etc.

[0116] [Lipid Nanoparticles]

[0117] The lipid nanoparticles of the present invention use the ionic neutral lipids of the present invention as constituent lipids. The ionic neutral lipids of the present invention that constitute the lipid nanoparticles of the present invention may be only one kind or may be two or more kinds. When the ionic neutral lipids of the present invention that constitute the lipid nanoparticles of the present invention are two or more kinds, the amount of the ionic neutral lipids of the present invention refers to the total amount of lipid molecules equivalent to the ionic neutral lipids of the present invention among the lipid molecules constituting the lipid nanoparticles.

[0118] The proportion of the ionic neutral lipids involved in the present invention in the lipid molecules constituting the lipid nanoparticles is not particularly limited. For example, in the lipid nanoparticles involved in the present invention, the ratio of the amount of the ionic neutral lipids involved in the present invention relative to the amount of the total lipids constituting the lipid nanoparticles ([the amount of the ionic neutral lipids involved in the present invention (mol)] / ([the amount of the total lipids constituting the lipid nanoparticles (mol)])×100%) is preferably 1 mol% or more, more preferably 3 mol% or more, and further preferably 5 mol% or more. On the other hand, if the proportion of the ionic neutral lipids in the lipid molecules constituting the lipid nanoparticles is too large, sometimes the content of other lipids used to impart the desired properties to the lipid nanoparticles is too small. Therefore, the ratio of the amount of the ionic neutral lipids involved in the present invention in the lipid nanoparticles involved in the present invention relative to the amount of the total lipids constituting the lipid nanoparticles is preferably 90 mol% or less, more preferably 80 mol% or less, and further preferably 70 mol% or less. In the case of lipid nanoparticles used as carriers for delivery to target cells in a living body, in order to easily obtain higher delivery efficiency, the ratio of the amount of the ionic neutral lipids involved in the present invention in the lipid nanoparticles involved in the present invention to the amount of the total lipids constituting the lipid nanoparticles is preferably 50 mol% or less, more preferably 30 mol% or less, and further preferably 5 to 20 mol%.

[0119] As the constituent lipids of the lipid nanoparticles of the present invention, it is preferred that a pH-sensitive cationic lipid is contained in addition to the ionic neutral lipids of the present invention. As the pH-sensitive cationic lipid, there is no particular limitation, and known pH-sensitive cationic lipids and modified compounds thereof can be used appropriately. As known pH-sensitive cationic lipids, for example, pH-sensitive cationic lipids described in non-patent literature 1 such as DLin-MC3-DMA, pH-sensitive cationic lipids described in non-patent literature 2 to 4 such as YSK05 and YSK13-C3, pH-sensitive cationic lipids described in patent literature 1 such as CL4H6, etc. can be cited. As the pH-sensitive cationic lipid that becomes the constituent lipid of the lipid nanoparticles of the present invention together with the ionic neutral lipids of the present invention, it is preferred that the pKa is 6.6 or less, more preferably the pKa is 5.8 to 6.6, and further preferably the pKa is 6.0 to 6.6.

[0120] As the lipid constituting lipid of the lipid nanoparticles of the present invention, except the ionic neutral lipids and pH-sensitive cationic lipids of the present invention, the lipids used when forming liposomes can be generally used. As such lipids, for example, phospholipids, sterols, glycolipids, or saturated or unsaturated fatty acids can be cited. They can be used alone or in combination of two or more.

[0121] As phospholipids, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerolphosphate, phosphatidic acid and other glycerophospholipids; sphingomyelin, ceramide phosphorylglycerol, ceramide phosphorylethanolamine and other sphingomyelins; and the like. In addition, phospholipids derived from natural substances such as egg yolk lecithin and soybean lecithin can also be used. The fatty acid residues in glycerophospholipids and sphingomyelin are not particularly limited, and for example, saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms can be mentioned, and saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms are preferably mentioned. Specifically, acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid can be mentioned. When these glycerolipids or sphingolipids have two or more fatty acid residues, all the fatty acid residues may be the same group or different groups.

[0122] Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, 24-dehydrocholesterol (desmosterol), and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and rapeseed sterol; and microorganism-derived sterols such as yeast sterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, and glycosyl diglyceride; and sphingolipids such as galactosyl cerebroside, lactosyl cerebroside, and ganglioside; and the like. Examples of the saturated or unsaturated fatty acid include saturated or unsaturated fatty acids having 12 to 20 carbon atoms, such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0123] As the constituent lipids of the lipid nanoparticles according to the present invention, in addition to the ionic neutral lipids according to the present invention, other neutral lipids are preferably contained, more preferably phospholipids or sterols are contained, further preferably sterols are contained, and further preferably cholesterol is contained.

[0124] The lipid nanoparticles of the present invention preferably contain polyalkylene glycol modified lipids as lipid components. Polyalkylene glycol is a hydrophilic polymer. By using polyalkylene glycol modified lipids as lipid membrane constituent lipids to construct lipid nanoparticles, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. By surface modification with polyalkylene glycol, the stability of the lipid nanoparticles such as blood retention can sometimes be improved.

[0125] Examples of the polyalkylene glycol include polyethylene glycol, polypropylene glycol, polybutylene glycol, and polyhexylene glycol. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.

[0126] For example, polyethylene glycol-based modification of lipids can use stearoyl polyethylene glycol (e.g., stearic acid PEG45 (STR-PEG45), etc.). Others can also use N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxy polyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol- The polyalkylene glycol lipids may be selected from polyethylene glycol derivatives such as [[(1,2-distearoyl-sn-glycero-3-phosphoethanolamine]]-2,2-distearoyl-sn-glycero-3-phosphoethanolamine], N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine], and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG), but are not limited thereto.

[0127] The ratio of the polyalkylene glycol modified lipid to the total lipid amount constituting the lipid nanoparticles of the present invention is not particularly limited as long as it is an amount that does not impair the inhibitory effect of the ionic neutral lipid of the present invention on the phase transition inhibition of phosphatidylcholine. For example, the ratio of the polyalkylene glycol modified lipid to the total lipid amount constituting the lipid nanoparticles is preferably set to 0.5 to 3 mol%.

[0128] The lipid nanoparticles involved in the present invention can be subjected to appropriate surface modification etc. as required.

[0129] The lipid nanoparticles of the present invention can be improved in blood retention by modifying the surface with a hydrophilic polymer, etc. The surface modification may also be performed by using lipids modified with these modifying groups as constituent lipids of the lipid nanoparticles.

[0130] When manufacturing the lipid nanoparticles of the present invention, as lipid derivatives for improving blood retention, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerol phospholipid derivatives, etc. can also be used. In addition, as hydrophilic polymers for improving blood retention, in addition to polyalkylene glycol, dextran, pullulan, polysucrose (ficoll), polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc. can also be used for surface modification.

[0131] In addition, in order to promote the nuclear translocation of the lipid nanoparticles involved in the present invention, for example, the lipid nanoparticles can also be surface-modified with oligosaccharide compounds of more than 3 sugars. The types of oligosaccharide compounds of more than 3 sugars are not particularly limited. For example, oligosaccharide compounds formed by the combination of about 3 to 10 sugar units can be used, and preferably oligosaccharide compounds formed by the combination of about 3 to 6 sugar units can be used. Among them, oligosaccharide compounds of 3-mers or 6-mers of glucose can be preferably used, and oligosaccharide compounds of 3-mers or 4-mers of glucose can be further preferably used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose or maltohexaose can be preferably used, among which maltotriose, maltotetraose, maltopentaose or maltohexaose formed by glucose combined with α1-4 are further preferred. Maltotriose or maltotetraose is particularly preferred, and maltotriose is most preferred. The amount of surface modification of lipid nanoparticles with an oligosaccharide compound is not particularly limited, and is, for example, about 1 to 30 mol %, preferably about 2 to 20 mol %, and more preferably about 5 to 10 mol % relative to the amount of total lipids.

[0132] The method for surface modification of lipid nanoparticles with oligosaccharide compounds is not particularly limited, for example, it is known that liposomes (International Publication No. 2007 / 102481) with surface modification of lipid nanoparticles with monosaccharides such as galactose and mannose are used, so the surface modification method described in the publication can be adopted. The entire disclosure of the above publication is included in the disclosure of the specification of the present application by reference.

[0133] In addition, the lipid nanoparticles of the present invention can be endowed with any one or more functions such as temperature change sensitivity function, membrane permeation function, gene expression function, and pH sensitivity function. By appropriately adding these functions, the retention of lipid nanoparticles in the blood can be improved, and the lipid nanoparticles can be efficiently escaped from the endosome after endocytosis of the target cells, so that the encapsulated nucleic acid can be more efficiently expressed in the target cells.

[0134] The lipid nanoparticles of the present invention can also include one or more materials selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate or butylated hydroxytoluene, charged substances, and membrane polypeptides. As charged substances that give positive charge, for example, saturated or unsaturated aliphatic amines such as stearylamine and oleylamine can be enumerated, and as charged substances that give negative charge, for example, dicetyl phosphate, cholesterol succinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc. can be enumerated. As membrane polypeptides, for example, membrane peripheral polypeptides or membrane intrinsic polypeptides, etc. can be enumerated. The amount of these substances is not particularly limited and can be appropriately selected according to the purpose.

[0135] With regard to the size of the lipid nanoparticles involved in the present invention, from the perspective of easily obtaining a high delivery efficiency to target cells in vivo, the average particle size is preferably less than 400nm, the average particle size is more preferably less than 300nm, the average particle size is further preferably less than 200nm, and further preferably less than 150nm. It should be noted that the average particle size of lipid nanoparticles refers to the number average particle size measured by dynamic light scattering (DLS). The determination based on the dynamic light scattering method can be performed by conventional methods using a commercially available DLS device, etc.

[0136] The polydispersity index (PDI) of the lipid nanoparticles of the present invention is about 0.01 to 0.7, preferably about 0.01 to 0.6, and more preferably about 0.01 to 0.3. The zeta potential may be in the range of -50 mV to 5 mV, preferably in the range of -10 mV to 5 mV.

[0137] The form of the lipid nanoparticles of the present invention is not particularly limited, and for example, as the form dispersed in an aqueous solvent, there can be mentioned unilamellar vesicles, multilamellar vesicles, spherical micelles, or amorphous lamellar structures, etc. As the lipid nanoparticles of the present invention, unilamellar vesicles and multilamellar vesicles are preferred.

[0138] The lipid nanoparticles of the present invention preferably contain the target component delivered to the target cell in the particle interior covered by the lipid membrane. As the component contained in the particle interior of the lipid nanoparticles of the present invention, as long as it is a size that can be contained, there is no particular limitation. Any substance such as nucleic acid, sugar, peptide, low molecular compound, metal compound can be encapsulated in the lipid nanoparticles of the present invention.

[0139] As the component contained in the lipid nanoparticles of the present invention, preferably nucleic acid. As nucleic acid, it can be DNA, RNA, or their analogs or derivatives (e.g., peptide nucleic acid (PNA), thiophosphorylated DNA, etc.). The nucleic acid contained in the lipid nanoparticles of the present invention can be single-stranded nucleic acid, double-stranded nucleic acid, linear, or cyclic.

[0140] The nucleic acid contained in the lipid nanoparticles of the present invention preferably comprises the exogenous gene to be expressed in the target cell, more preferably the nucleic acid that plays the function for making the exogenous gene expressed in the cell by being taken into the cell. The exogenous gene can be the gene originally included in the genomic DNA of the target cell, or the gene not contained in the genomic DNA. As such nucleic acid, the gene expression vector comprising the nucleic acid consisting of the base sequence of the target gene to be expressed can be enumerated. The gene expression vector can exist as an extrachromosomal gene in the cell introduced, or it can be integrated into the genomic DNA by homologous recombination.

[0141] As the gene expression vector contained in the lipid nanoparticles of the present invention, there is no particular limitation, and the vector used in conventional gene therapy etc. can be used. As the gene expression vector contained in the lipid nanoparticles of the present invention, it is preferably a nucleic acid vector such as a plasmid vector. The plasmid vector can be kept in a circular shape or can be encapsulated in the lipid nanoparticles of the present invention in a state of being cut into a linear shape in advance. The gene expression vector can be designed by conventional methods using commonly used molecular biology tools based on the base sequence information of the target gene to be expressed, and can be manufactured using known various methods.

[0142] The nucleic acid contained in the lipid nanoparticle to which the present invention relates is also preferably a functional nucleic acid that the expression of the target gene present in the target cell is regulated. As this functional nucleic acid, antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, mRNA etc. can be enumerated. In addition, it can also be a plasmid DNA (pDNA) as a siRNA expression vector that makes siRNA expressed in the cell. As the siRNA expression vector, it can be prepared by commercially available siRNA expression vectors, and it can also be appropriately modified in addition. As the nucleic acid contained in the lipid nanoparticle to which the present invention relates, particularly from the aspect of the good selectivity of the tissue in the organism, it is preferably mRNA or pDNA.

[0143] The method for manufacturing the lipid nanoparticles to which the present invention relates is not particularly limited, and any method that can be used by those skilled in the art can be used. In one example, it can be manufactured in the following manner: after all lipid components are dissolved in an organic solvent such as chloroform and dried under reduced pressure based on an evaporator, and a lipid film is formed by spray drying based on a spray dryer, an aqueous solvent containing components (such as nucleic acids, etc.) to be encapsulated in the lipid nanoparticles is added to the dried mixture, and further emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier. In addition, as a method for manufacturing liposomes, it is also possible to manufacture using a well-known method, such as a reverse phase evaporation method, etc. When the size of the lipid nanoparticles is to be controlled, it is sufficient to extrude (extrusion filtration) under high pressure using a membrane filter with a consistent pore size.

[0144] The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include buffers such as phosphate buffer, citrate buffer, and phosphate buffered saline, physiological saline, and cell culture medium, etc. These aqueous solvents (dispersion mediums) can stably disperse the lipid nanoparticles, and can further include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melezitose, polysaccharides such as cyclodextrin, sugars (aqueous solutions) such as sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and polyols (aqueous solutions) such as 1,3-butanediol, etc. In order to stably store the lipid nanoparticles dispersed in the aqueous solvent for a long time, it is preferred to exclude electrolytes in the aqueous solvent as much as possible from the perspective of physical stability such as suppressing aggregation. In addition, from the perspective of the chemical stability of the lipid, it is preferred to set the pH of the aqueous solvent to a weakly acidic to neutral range (pH 3.0 to 8.0) and / or remove dissolved oxygen by nitrogen bubbling.

[0145] The lipid nanoparticles involved in the present invention can also be manufactured using an alcohol dilution method using a flow path. The method is to introduce a solution obtained by dissolving a lipid component in an alcohol solvent and a solution obtained by dissolving a water-soluble component to be contained in the lipid nanoparticles in an aqueous solvent from different flow paths, and merge them to manufacture lipid nanoparticles. By using a three-dimensional micromixer with a built-in microflow path that can achieve instantaneous mixing of two liquids, lipid nanoparticles with a diameter of about 30nm can be manufactured with good reproducibility (non-patent document 6). As the flow path used in the manufacture, from the aspect of being able to form a nano-sized lipid particle formation system with high particle size controllability, it is preferred to use a flow path structure with a simple two-dimensional structure as described in patent document 2, in which baffles (baffles) with a certain width relative to the flow path width are arranged alternately from both sides in a micro-sized flow path for the raw material solution to flow. As an aqueous solvent used in the alcohol dilution method, the aforementioned aqueous solvent can be used.

[0146] In the case of freeze-drying or spray-drying the aqueous dispersion of the obtained lipid nanoparticles, if sugars (aqueous solutions) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, maltose, trisaccharides such as raffinose, melezitose, polysaccharides such as cyclodextrin, erythritol, xylitol, sorbitol, mannitol, sugar alcohols such as maltitol are used, stability can sometimes be improved. In addition, in the case of freezing the above-mentioned aqueous dispersion, if polyols (aqueous solutions) such as the aforementioned sugars, glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol are used, stability can sometimes be improved.

[0147] The lipid nanoparticles of the present invention function as gene expression vectors for target tissues. After the target exogenous gene that can be expressed in target cells is encapsulated in the lipid nanoparticles of the present invention, it is administered to the test animal, so that the exogenous gene is expressed in the target tissue of the test animal. Therefore, the lipid nanoparticles of the present invention are useful as the active ingredient of the pharmaceutical composition used in gene therapy.

[0148] The animal to which the lipid nanoparticles of the present invention can be applied is not particularly limited, and can be a human or an animal other than a human. As non-human animals, mammals such as cattle, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, birds such as chickens, quails, ducks, etc. can be enumerated. In addition, the administration route when applying the lipid nanoparticles of the present invention to animals is not particularly limited, and preferably intravenous administration, enteral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, non-oral administration such as pulmonary administration are administered.

[0149] Example

[0150] Next, examples are shown to further illustrate the present invention in detail, but the present invention is not limited to the following examples.

[0151] <NMR (Nuclear Magnetic Resonance)>

[0152] In the following experiments, unless otherwise specified, 1 H and 13 C NMR spectra were obtained using an NMR apparatus (ECA500 apparatus, ECZ400 apparatus, or ECX400P apparatus) manufactured by JEOL Ltd., with tetramethylsilane as the internal standard (0 ppm). d is the abbreviation for doublet, t is the abbreviation for triplet, and m is the abbreviation for multiplet. 1 The chemical shift of the H NMR spectrum is reported in ppm on the σ scale at a lower magnetic field relative to tetramethylsilane.

[0153] <Constituent lipids of lipid nanoparticles>

[0154] 7-(4-(Diisopropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6) and 7-hydroxy-7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diyl dioleate (CL15H6) were synthesized using the method described in Patent Document 1. 7-(4-(Diisopropylamino)butyl)-13-((2-hexylnonanoyl)oxy)-7-hydroxytridecyl 2-hexyldecanoate (CL4F6) was synthesized using the method described in Synthesis Example 1 below. 7-Hydroxy-7-(4-((1-methylpiperidine)-4-carbonyl)oxy)butyl)tridecane-1,13-diyl bis(2-hexyldecanoate) (CL15F6) was synthesized using the method described in International Publication No. 2022 / 071582. ALC-0315 was manufactured by Ambeed, DLin-MC3-DMA (MC3) was manufactured by Selleck Biotech, SM-102 was manufactured by Cayman Chemical, and cholesterol (Chol) was a product of SIGMA Aldrich. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and methoxy polyethylene glycol 2000-modified 2-dimyristoyl-rac-glycerol (PEG-DMG) were products of NOF Corporation.

[0155] <Based on 31 Evaluation of lipid polymorphism by 31P NMR spectroscopy>

[0156] Lipid membrane based 31 The spectrum of P NMR spectroscopy differs depending on the phase. Therefore, the lipid membrane containing DOPC and the lipid to be evaluated was subjected to 31 The polymorphism of the phase state of the lipid membrane was evaluated based on the spectrum obtained by P NMR spectroscopy analysis, and the destructive ability of the lipid to be evaluated on the DOPC membrane was evaluated.

[0157] Specifically, DOPC and the lipid to be evaluated (DOPE or TOT-10) were mixed at a ratio of 1:1 (molar ratio) to obtain a lipid (total lipid 16 μmol) dissolved in 300 μL of chloroform, and the solvent was removed by a distillation device (Rotovap) to form a lipid film. The obtained lipid film was hydrated at 60°C and 500 μL of PBS (-) to serve as a sample for measurement.

[0158] Next, the sample was transferred to an NMR tube and proton separation was obtained using the JEOL ECA500 apparatus. 31 P NMR spectra. Collection parameters included: 60° pulse, spectral width of 280 kHz with 32,768 data points (in case of ECX 400P), and 1 second interpulse delay. Temperature was controlled at 25°C. Multiplication of the exponential function corresponding to the line broadening of 50 Hz was applied to the free induction decay before Fourier transformation. For chemical shifts, 85% phosphoric acid (H3PO4) was used as an external standard.

[0159] <Thin layer chromatography (TLC)>

[0160] In the following experiments, unless otherwise specified, the reactants of all reactions were monitored by TLC using pre-coated TLC plates (Millipore). Visualization of TLC was performed by UV light (254 nm), phosphomolybdic acid staining or p-methoxybenzaldehyde staining. The reaction products were purified using an automated combiflash (registered trademark) Rf chromatography system (Teledyne ISCO) or a Selekt system (Biotage).

[0161] <mrna>

[0162] As the mRNA encoding firefly luciferase (Fluc) (CleanCapFluc mRNA, 5 moU) contained in the nucleic acid of the lipid nanoparticle, a product manufactured by TriLink Biotechnologies was used.

[0163] <Preparation of lipid nanoparticles>

[0164] In the following experiments, unless otherwise specified, lipid nanoparticles were prepared by an alcohol dilution method using a flow path. As a flow path, a microfluidic device "iLiNP" (manufactured by Lilac Pharma) with a mixer was used. The flow rate of the device was controlled using a syringe pump (Harvard Apparatus).

[0165] Specifically, first, 80 μL of an ethanol solution containing each lipid component at a predetermined molar ratio and having a total lipid concentration adjusted to 8 mM and 240 μL of an acetate buffer (25 mM, pH 4.0) having a nucleic acid concentration adjusted to 55 μg / mL were fed into the microchannel, and the lipid nanoparticle solution discharged from the channel was recovered. The lipid nanoparticle solution was placed in a dialysis membrane (MWCO 12,000-14,000, manufactured by Spectrum Laboratories), the external aqueous phase was set to PBS (-) (pH 7.4), and after dialysis at 4°C for more than 2 hours, the lipid nanoparticle solution was recovered from the dialysis membrane.

[0166] <Measurement of Average Particle Size and Zeta Potential of Lipid Nanoparticles>

[0167] The average particle size (number average), ζ average particle size, and polydispersity index (PdI) of the lipid nanoparticles in PBS(-) (pH 7.4), and the zeta potential in 10 mM HEPES buffer (pH 7.4) were measured using a dynamic light scattering analysis device "Zetasizer Nano ZS ZEN3600" (manufactured by Malvern).

[0168] <Encapsulation efficiency of lipid nanoparticles>

[0169] The encapsulation rate of lipid nanoparticles was determined by measuring the amount of encapsulated nucleic acid (mRNA) using Ribogreen (manufactured by Life Technologies) as an RNA intercalator.

[0170] <Determination of apparent pKa of lipid nanoparticles>

[0171] The apparent pKa of the lipid nanoparticles was determined using p-toluenesulfonic acid (TNS). First, TNS (final concentration: 0.75 μM) and lipid nanoparticles (final concentration: 30 μM) were mixed in a buffer solution (200 mL) adjusted to each pH (pH 3.5 to 9.5). As the buffer solution, 20 mM citric acid buffer, 20 mM sodium phosphate buffer, or 20 mM Tris-HCl buffer containing 130 mM NaCl was used. Using a fluorescence spectrophotometer (Varioskan Lux, manufactured by Thermo Fisher Scientific), the fluorescence intensity of the prepared mixture was measured under the settings of λex = 321 nm and λem = 447 nm. The highest and lowest measured values were set as 100% and 0% charge rates, respectively, and the pH showing a 50% charge rate was calculated as the apparent pKa. The percentage of the cationically charged ionic neutral lipid was calculated using the Henderson-Hasselbalch equation.

[0172] <Measurement of Fluc activity>

[0173] First, BALB / c mice (4 weeks old, female) were administered lipid nanoparticles encapsulating Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) by intravenous injection, intramuscular injection, or intracerebral injection. The mRNA administration amounts (volumes) for intravenous injection, intramuscular injection, and intracerebral injection were 0.1 mg / 10 mL / kg, 1 μg / 50 μL / mouse, and 0.1 μg / 5 μL / mouse, respectively. Six hours after administration, the mice were euthanized, and each tissue (liver, quadriceps femoris, brain) was recovered, frozen with liquid nitrogen, and stored at -80°C. Using a bead mill cell disruption device (Micro Smash MS-100R, manufactured by TOMY Seiko), the tissues were homogenized with a passive lysis buffer (manufactured by Promega). Tissue debris was removed by centrifugation, and the supernatant was recovered. The Fluc activity in the supernatant was measured according to the manufacturer's protocol using a luciferase assay system (E1500, manufactured by Promega). Luminescence was measured using a luminometer (Luminescencer-PSN, manufactured by ATTO). The protein concentration was determined using a commercially available assay kit (BCA Protein Assay Kit: BCA protein quantification kit, manufactured by Pierce). The Fluc activity was expressed as relative light units (RLU) per 1 mg of protein.

[0174] <Hemolysis test>

[0175] In the hemolysis test, lipid nanoparticles are added to erythrocytes to measure hemolysis caused by membrane damage of erythrocytes. The pH in the endosome is reproduced by using a low pH buffer, thereby measuring the membrane fusion ability brought by the lipid nanoparticles and evaluating it as the endosomal escape ability. Specifically, a pH 7.4 buffer is used to schematically evaluate the blood conditions, a pH 6.0 buffer is used to schematically evaluate the early endosome, and a pH 5.0 buffer is used to schematically evaluate the late endosome.

[0176] As a buffer at pH 5.0, 10 mM malic acid buffer (120 mM NaCl) was used, as a buffer at pH 6.0, 10 mM HEPES buffer (120 mM NaCl) was used, and as a buffer at pH 7.4, 10 mM MES buffer (120 mM NaCl) was used.

[0177] A lipid nanoparticle solution with a total lipid concentration of 0.75 mM and a buffer solution of each pH were added to a transparent 96-well plate, and the lipid nanoparticle solution was diluted with a buffer solution of each pH so that the final lipid molar amount became 0.781 nmol to 12.5 nmol. The total liquid volume was 100 μL. It should be noted that, as a positive control, 90 μL of buffer solution of each pH was added to 10 μL of a physiological saline solution containing 10% TritonX-100 to make the total liquid volume 100 μL. In addition, as a negative control, 100 μL of buffer solution of each pH was added.

[0178] 100 μL of blood solution diluted with buffer of the same pH was added to these lipid nanoparticle dilution solutions, so that the total liquid volume of each well was 200 μL, and the well was shaken at 37°C and 900 rpm for 30 minutes. Then, the well plate was centrifuged at 4°C and 400 × g for 5 minutes. 100 μL of supernatant was taken out from each well of the well plate after centrifugation, and added to each well of the transparent 96-well plate for absorbance measurement. It should be noted that 100 μL of physiological saline was pre-added to the well of the positive control of the transparent 96-well plate for absorbance measurement, and 100 μL of 0.5% TritonX-100 was pre-added to the wells of the negative control and the sample added with lipid nanoparticles, so that the final concentration of TritonX-100 in each well was 0.25%. The absorbance at 545 nm was measured using a fluorescence spectrophotometer (VarioskanLux, manufactured by Thermo Fisher Scientific) for a well plate having a total liquid volume of 200 μL per well, and the hemolytic activity was calculated with the negative control as background (blank) and the positive control as 100%.

[0179] It should be noted that the blood solution was prepared from a fresh mouse erythrocyte suspension as follows.

[0180] First, with regard to the red blood cell suspension, BALB / c mice (4 weeks old, female) were euthanized, blood was collected from the inferior vena cava, and 1 mL of blood was added to a 1.5 mL tube with 1 μL of 10 mg / mL heparin. 1 mL of blood with heparin added was transferred to a 50 mL tube and diluted with 9 mL of normal saline. The diluted blood was centrifuged at 4 ° C and 400 × g for 5 minutes, and the supernatant was removed. After repeating this operation 5 times, 10 mL of normal saline was added to obtain a red blood cell suspension.

[0181] The erythrocyte concentration of the erythrocyte suspension is determined as described below. 10 μL of physiological saline solution containing 10% TritonX-100 and erythrocyte suspension or physiological saline are added to a transparent 96-well plate, and the erythrocyte suspension is diluted in a manner such that the total liquid volume becomes 200 μL and the final concentration of TritonX-100 becomes 0.25%. It should be noted that the erythrocyte suspension is diluted in a manner such that the dilution ratio is between 6.6 and 200 times. The well plate to which the solution is added is shaken at 37 ° C and 700 rpm for 1 minute, 100 μL of the shaken solution is taken out from each well, and 100 μL of physiological saline is added for 2-fold dilution. The absorbance at 545 nm is measured using a fluorescence spectrophotometer (VarioskanLux, Thermo Fisher Scientific) for a well plate with a total liquid volume of 200 μL per well, and a standard curve is prepared. According to the standard curve, the dilution ratio of the erythrocyte suspension at which the absorbance at 4-fold dilution becomes 1.0 is calculated. The red blood cell suspension was diluted with the buffer solution of each pH so as to achieve the dilution ratio, thereby preparing a blood solution.

[0182] [Synthesis Example 1] Synthesis of CL4F6

[0183] [Chemical formula 8]

[0184]

[0185] 7-(4-(diisopropylamino)butyl)tridecane-1,7,13-triol (1.0 mmol) synthesized by the method described in Patent Document 1 was dissolved in 5 mL of dichloromethane, and then 2-hexyldecanoic acid (2.20 mmol), DMAP (N,N-dimethyl-4-aminopyridine) (0.20 mmol) and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (3.0 mmol) were added and reacted at room temperature overnight. After the solvent was distilled off using a rotary evaporator, it was suspended in ethyl acetate and impurities were removed by filtration. The filtrate was separated and washed with 0.5N sodium hydroxide aqueous solution and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtering, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by subjecting it to silica gel chromatography [eluting solvent; dichloromethane: methanol (continuous gradient)] to obtain CL4F6.

[0186] [Example 1]

[0187] After condensing the hydroxyl group of Tris with the oleic acid residue, the amino group derived from Tris of the obtained esterified product was condensed with N-((9H-fluoren-9-ylmethoxy)carbonyl)-D-glutamic acid-5-tert-butyl ester hydrate to synthesize (S)-4-amino-5-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-5-oxopentanoic acid (TOT-9).

[0188] (1) Synthesis of tert-butyl (1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)carbamate (Compound 1)

[0189] [Chemical formula 9]

[0190]

[0191] To a solution obtained by dispersing Tris (10.0 g, 82.6 mmol) in a mixed solvent of methanol (75 mL) and tert-butyl alcohol (75 mL), di-tert-butyl dicarbonate (23.5 g, 107.7 mmol) dissolved in tert-butyl alcohol (75 mL) was added dropwise. After the obtained reaction mixture was stirred overnight at ambient temperature, the reaction mixture was evaporated. Purification was performed by precipitating the obtained residue with cold EtOAc. Compound 1 (16.7 g, yield 91%) was obtained as a white powder by vacuum filtration.

[0192] (2) Synthesis of 2-((tert-butoxycarbonyl)amino)-2-((oleoyloxy)methyl)propane-1,3-diyl dioleate (Compound 2)

[0193] [Chemical formula 10]

[0194]

[0195] Compound 1 (2.21 g, 10.0 mmol) was dissolved in anhydrous DCM (dichloromethane, 50 mL), and oleic acid (9.32 g, 33.0 mmol), DMAP (N, N-dimethyl-4-aminopyridine) (122 mg, 1.0 mmol), and EDCI-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (7.67 g, 40.0 mmol) were further dissolved in the obtained mixture. The obtained reaction mixture was stirred overnight at ambient temperature and then evaporated. The obtained residue was suspended in EtOAc, washed with a saturated citric acid solution and water, and dried over anhydrous Na2SO4. The solution was filtered and the filtrate was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HCD, manufactured by Biotage) and purified by flash chromatography using a gradient mobile phase of hexane and EtOAc. Thus, Compound 2 (8.50 g, yield 84%) was obtained as a colorless oil.

[0196] (3) Synthesis of 2-((oleoyloxy)methyl)propane-1,3-diyl dioleate (TOT-0)

[0197] [Chemical formula 11]

[0198]

[0199] Compound 2 (8.50 g, 8.38 mmol) was dissolved in anhydrous DCM (44 mL), and TFA (trifluoroacetic acid) (5 mL) and TIPS (triisopropylsilane) (1 mL) were added to the obtained mixture. The obtained reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was diluted with EtOAc, the organic phase was washed with 1N NaOH aqueous solution, water and brine, and dried over anhydrous Na2SO4. The solution was filtered and the filtrate was evaporated. The residue was loaded onto a normal phase chromatographic column (Sfaer Silica HCD, Biotage), and purified by flash chromatography using a gradient mobile phase of hexane and EtOAc. Thus, TOT-0 (5.00 g, yield 65%) was obtained in the form of a colorless oil.

[0200] (4) Synthesis of (S)-4-amino-5-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-5-oxopentanoic acid (TOT-9).

[0201] [Chemical formula 12]

[0202]

[0203] TOT-0 (366 mg, 0.40 mmol) was dissolved in ethanol (4 mL), and N-((9H-fluorene-9-ylmethoxy)carbonyl)-D-glutamic acid-5-tert-butyl ester hydrate (170 mg, 0.44 mmol) and DMT-MM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) (122 mg, 0.44 mmol) were added to the obtained mixture. The reaction mixture was stirred at ambient temperature overnight and then evaporated. The obtained residue was suspended in EtOAc (4 mL), washed with 0.5N NaOHaq (5 mL), and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated. The obtained solution was dissolved in anhydrous DCM (2.8 mL), and TFA (0.8 mL) and TIPS (0.4 mL) were added to the obtained mixture. The reaction mixture was stirred overnight at ambient temperature, diluted with EtOAc, and the obtained organic phase was washed with 1N NaOH aqueous solution, water and brine, and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated. After adding DMF (1.8mL) and piperidine (200μL) to the obtained solution, it was stirred overnight at ambient temperature and evaporated. The obtained residue was loaded onto a normal phase chromatographic column (Sfaer Silica HCD, Biotage Company), and purified by flash chromatography using a gradient mobile phase of hexane and EtOAc. Thus, TOT-9 (213mg, yield 51%) was obtained in the form of a colorless viscous oil.

[0204] [Example 2]

[0205] The amino group in TOT-0 synthesized in Example 1 was condensed with 4-(dimethylamino)butyric acid to synthesize 2-((4-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-4-oxobutyl)dimethylammonio)acetate (TOT-10).

[0206] [Chemical formula 13]

[0207]

[0208] TOT-0 (366 mg, 0.40 mmol) was dissolved in anhydrous DCM (4 mL), and 4-(dimethylamino)butyric acid hydrochloride (83.8 mg, 0.50 mmol), DMAP (4.9 mg, 0.04 mmol), TEA (83.6 μL, 0.60 mmol), and EDCI (115 mg, 0.60 mmol) were added to the obtained mixture. The reaction mixture was stirred at 35 ° C overnight and evaporated. The obtained residue was suspended in hexane / EtOAc (1 mL: 1.4 mL), washed with 0.5N NaOHaq (5 mL), and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated. The obtained residue was loaded onto a normal phase chromatographic column (Sfaer Silica HCD, Biotage) and purified by flash chromatography using a gradient mobile phase of DCM and methanol. Thus, 2-(4-(dimethylamino)butyramide)-2-((oleoyloxy)methyl)propane-1,3-diyl dioleate (Compound 3) (341 mg, yield 83%) was obtained as a colorless oil.

[0209] Compound 3 (154 mg, 0.15 mmol) was dissolved in anhydrous DCM (1 mL), and bromoacetic acid (41.7 mg, 0.30 mmol) and N,N-diisopropylamine (DIPEA) (102 μL, 0.60 mmol) were added to the obtained mixture. The reaction mixture was stirred at 50 ° C for 2 hours under an argon atmosphere and evaporated. The obtained residue was loaded onto a reverse phase chromatographic column (Sfaer C18, manufactured by Biotage) and purified by flash chromatography using a gradient mobile phase of water and 2-propanol. Thus, TOT-10 (55.5 mg, yield 34%) was obtained in the form of a colorless viscous oil.

[0210] [Example 3]

[0211] The amino group in TOT-0 synthesized in Example 1 was condensed with 3-(dimethylamino)propionic acid to synthesize 2-((3-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-3-oxopropyl)dimethylammonio)acetate (TOT-11).

[0212] [Chemical formula 14]

[0213]

[0214] 2-(3-(dimethylamino)propaneamide)-2-((oleoyloxy)methyl)propane-1,3-diyl dioleate (Compound 4) (245 mg, yield 60%) was obtained in the same manner as in the synthesis of Compound 3 in Example 2, except that 3-(dimethylamino)propanoic acid hydrochloride (76.8 mg, 0.50 mmol) was used instead of 4-(dimethylamino)butyric acid hydrochloride.

[0215] Compound 4 (152 mg, 0.15 mmol) was dissolved in anhydrous DCM (1 mL), and bromoacetic acid (31.3 mg, 0.225 mmol) and DIPEA (76.5 μL, 0.45 mmol) were added to the obtained mixture. The reaction mixture was stirred overnight at 40 ° C under an argon atmosphere and evaporated. The obtained residue was loaded onto a reverse phase chromatographic column (Sfaer C18, Biotage), and purified by flash chromatography using a gradient mobile phase of water and 2-propanol. Thus, TOT-11 (85.8 mg, yield 53%) was obtained in the form of a colorless viscous oil.

[0216] [Example 4]

[0217] The amino group in TOT-0 synthesized in Example 1 was condensed with Fmoc-N-methyl-β-alanine to synthesize 2-((3-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-3-oxopropyl)(methyl)ammonio)acetate (TOT-12).

[0218] [Chemical formula 15]

[0219]

[0220] TOT-0 (914 mg, 1.00 mmol) was dissolved in ethanol (10 mL), and Fmoc-N-methyl-β-alanine (358 mg, 1.10 mmol) and DMT-MM (304 mg, 1.10 mmol) were added to the obtained mixture. The reaction mixture was stirred at 40°C overnight and then evaporated. The obtained residue was suspended in hexane / EtOAc (1 mL: 11.20 mL), washed with 0.5N NaOHaq (5 mL), brine, and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated.

[0221] The crude residue (colorless oil) was dissolved in anhydrous DCM (4 mL), and piperidine (1 mL) was added to the resulting mixture. The reaction mixture was stirred at ambient temperature for 10 minutes and then evaporated. The resulting residue was loaded onto a normal phase chromatographic column (Sfaer Silica HCD, Biotage) and purified by flash chromatography using a gradient mobile phase of DCM and methanol. Thus, a colorless viscous oil (388 mg, 39% yield) was obtained.

[0222] The obtained colorless oil (388 mg, 0.388 mmol) was dissolved in anhydrous DCM (5 mL), and bromoacetic acid (80.9 mg, 0.582 mmol) and DIPEA (198 μL, 1.16 mmol) were added to the obtained mixture. The reaction mixture was stirred overnight at 35 ° C under an argon atmosphere and evaporated. The obtained residue was loaded onto a reverse phase chromatographic column (Sfaer C18, Biotage), and purified by flash chromatography using a gradient mobile phase of water and 2-propanol. Thus, TOT-12 (152.4 mg, yield 37%) was obtained in the form of a colorless viscous oil.

[0223] [Example 5]

[0224] The amino group in TOT-0 synthesized in Example 1 was condensed with 4-tert-butyl-N-(tert-butoxy-carbonyl)-L-asparagine to synthesize (S)-3-amino-4-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-4-oxobutanoic acid (TOT-13).

[0225] [Chemical formula 16]

[0226]

[0227] TOT-0 (366 mg, 0.40 mmol) was dissolved in ethanol (10 mL), and 4-tert-butyl-N-(tert-butoxy-carbonyl)-L-asparagine (173 mg, 0.44 mmol) and DMT-MM (122 mg, 0.44 mmol) were added to the obtained mixture. The reaction mixture was stirred overnight at ambient temperature and then evaporated. The obtained residue was suspended in EtOAc (4 mL), washed with 0.5 N NaOHaq (5 mL), and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated. The residue was dissolved in anhydrous DCM (2.8 mL), TFA (0.8 mL) and TIPS (0.4 mL) were added, and stirred overnight at ambient temperature. The obtained reaction mixture was diluted with EtOAc, and the organic phase was washed with 1N NaOH aqueous solution, water and brine, and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated. The obtained residue was loaded onto a normal phase chromatography column (Sfaer Silica HCD, manufactured by Biotage) and purified by flash chromatography using a gradient mobile phase of hexane and EtOAc. Thus, TOT-13 (180 mg, yield 44%) was obtained as a colorless viscous oil.

[0228] [Example 6]

[0229] The amino group in TOT-0 synthesized in Example 1 was condensed with 1-tert-butyl-N-(tert-butoxy-carbonyl)-L-asparagine to synthesize N4-(1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)-L-asparagine (TOT-14).

[0230] [Chemical formula 17]

[0231]

[0232] TOT-0 (366 mg, 0.40 mmol) was dissolved in ethanol (4 mL), and 1-tert-butyl-N-(tert-butoxy-carbonyl)-L-asparagine (127 mg, 0.44 mmol) and DMT-MM (122 mg, 0.44 mmol) were added to the obtained mixture. The reaction mixture was stirred at ambient temperature overnight and then evaporated. The obtained residue was suspended in EtOAc (4 mL), washed with 0.5 N NaOH aq (5 mL), and dried over anhydrous Na2SO4. The residue was dissolved in anhydrous DCM (2.8 mL), and TFA (0.8 mL) and TIPS (0.4 mL) were added. The obtained reaction mixture was diluted with EtOAc, and the organic phase was washed with 1N NaOH aqueous solution, water and brine, and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated. The product was loaded onto a normal phase chromatography column (Sfaer Silica HCD, manufactured by Biotage) and purified by flash chromatography using a gradient mobile phase of hexane and EtOAc. Thus, TOT-14 (199 mg, yield 48%) was obtained as a colorless viscous oil.

[0233] [Example 7]

[0234] The amino group in TOT-0 synthesized in Example 1 was condensed with 1-tert-butyl-N-(tert-butoxy-carbonyl)-L-glutamic acid to synthesize N5-(1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)-L-glutamine (TOT-15).

[0235] [Chemical formula 18]

[0236]

[0237] TOT-0 (366 mg, 0.40 mmol) was dissolved in ethanol (4 mL), and 1-tert-butyl-N-(tert-butoxy-carbonyl)-L-glutamic acid (133.4 mg, 0.44 mmol) and DMT-MM (122 mg, 0.44 mmol) were added to the obtained mixture. The obtained reaction mixture was stirred overnight at ambient temperature and then evaporated. The obtained residue was suspended in EtOAc (4 mL), washed with 0.5 N NaOHaq (5 mL), and dried over anhydrous Na2SO4. The obtained solution was filtered and the filtrate was evaporated, then dissolved in anhydrous DCM (2.8 mL), and TFA (0.8 mL) and TIPS (0.4 mL) were added. The obtained reaction mixture was stirred overnight at ambient temperature and then diluted with EtOAc. The organic phase was washed with 1N NaOH aqueous solution, water and brine, and then dried over anhydrous Na2SO4. The obtained residue was loaded onto a normal phase chromatography column (Sfaer Silica HCD, manufactured by Biotage) and purified by flash chromatography using a gradient mobile phase of hexane and EtOAc. Thus, TOT-15 (141 mg, yield 34%) was obtained as a colorless viscous oil.

[0238] [Example 8]

[0239] The effect of TOT-10 synthesized in Example 2 on the stability of DOPC membrane was investigated. Specifically, a lipid membrane with a lipid composition of DOPC / TOT-10 = 50 / 50 (mol%) and a lipid membrane with a lipid composition of DOPC / DOPE = 50 / 50 (mol%) were prepared. 31 P NMR spectroscopy to obtain proton separation 31 P NMR spectrum.

[0240] Figure 1 (A) shows the DOPC / DOPE membrane 31 P NMR spectrum, Figure 1 (B) shows the DOPC / TOT-10 membrane 31 PNMR spectrum. In the DOPC / DOPE film, peaks from the lamellar phase were observed ( Figure 1 In contrast, in the DOPC / TOT-10 film, no peaks derived from the lamellar phase were observed, but peaks derived from the hexagonal phase and the isotropic phase were observed ( Figure 1 (B)). These results show that TOT-10 can reduce and destroy the membrane stability of lipid membranes containing phosphatidylcholine.

[0241] [Example 9]

[0242] Lipid nanoparticles were prepared using TOT-9 to TOT-15 synthesized in Examples 1 to 7 as constituent lipids.

[0243] pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than TOT-9 to TOT-15. CL4F6 was used as a pH-sensitive cationic lipid. In addition, DSPC was used instead of TOT.

[0244] Specifically, CL4F6, TOT-9 to TOT-15, cholesterol and PEG-DMG were used in a composition of CL4F6 / TOT / chol / PEG-DMG = 50 / 10 / 40 / 1 (mol%), and lipid nanoparticles loaded with Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) were prepared by alcohol dilution method. In addition, DSPC was used instead of TOT, and the same operation was performed except that CL4F6 / DSPC / chol / PEG-DMG = 50 / 10 / 40 / 1 (mol%) was used to prepare lipid nanoparticles loaded with Fluc mRNA.

[0245] Next, the prepared lipid nanoparticles loaded with Fluc mRNA were administered to Balb / c mice, and the Fluc activity (RLU / mg protein) was measured. The results are shown in Figure 2 . In mice administered with any lipid nanoparticles, Fluc activity was confirmed in the liver and spleen. This result confirms that lipid nanoparticles with TOT-9 to TOT-15 as constituent lipids are useful as gene carriers targeting the liver and spleen. In addition, all lipid nanoparticles loaded with Fluc mRNA containing TOT showed higher Fluc activity in the liver than lipid nanoparticles loaded with Fluc mRNA containing DSPC.

[0246] [Example 10]

[0247] The gene expression efficiency in the liver when lipid nanoparticles containing TOT-15 synthesized in Example 7 and pH-sensitive cationic lipids as constituent lipids were administered by intravenous injection was investigated.

[0248] Specifically, pH-sensitive cationic lipids, TOT-15, cholesterol and PEG-DMG were used in a composition of pH-sensitive cationic lipids / TOT-15 / chol / PEG-DMG = 50 / 10 / 40 / 1.5 (mol%), and lipid nanoparticles loaded with Fluc mRNA were prepared by alcohol dilution method. CL4F6, CL4H6, DLin-MC3-DMA, or ALC-0315 were used as pH-sensitive cationic lipids. In addition, DSPC was used instead of TOT, and the same operation was performed to prepare lipid nanoparticles loaded with Fluc mRNA in a composition of pH-sensitive cationic lipids / DSPC / chol / PEG-DM G = 50 / 10 / 40 / 1.5 (mol%).

[0249] Next, the prepared lipid nanoparticles loaded with Fluc mRNA were administered to Balb / c mice by intravenous injection, and the Fluc activity (RLU / mg protein) was measured. The results are shown in Figure 3 . For lipid nanoparticles loaded with Fluc mRNA containing CL4F6, CL4H6, or DLin-MC3-DMA, the Fluc activity in the liver of lipid nanoparticles containing TOT-15 was significantly higher than that of lipid nanoparticles containing DSPC. In addition, for lipid nanoparticles loaded with Fluc mRNA containing ALC-0315, it was also observed that the lipid nanoparticles containing TOT-15 had a better Fluc activity in the liver than the lipid nanoparticles containing DSPC.

[0250] [Example 11]

[0251] The gene expression efficiency in the quadriceps femoris muscle was investigated when lipid nanoparticles containing TOT-15 synthesized in Example 7 and pH-sensitive cationic lipids as constituent lipids were administered by intramuscular injection.

[0252] Specifically, the lipid nanoparticles loaded with Fluc mRNA containing CL4F6 or ALC-0315 among the lipid nanoparticles loaded with Fluc mRNA prepared in Example 10 were administered to Balb / c mice by intramuscular injection, and the Fluc activity (RLU / mg protein) was measured. The results are shown in Figure 4 Regarding the Fluc mRNA-loaded lipid nanoparticles containing CL4F6 or ALC-0315, when administered by intramuscular injection, the Fluc activity in the quadriceps muscle of the lipid nanoparticles containing TOT-15 was also significantly higher than that of the lipid nanoparticles containing DSPC at the same time.

[0253] [Example 12]

[0254] The gene expression efficiency in the brain was investigated when lipid nanoparticles containing TOT-15 synthesized in Example 7 and a pH-sensitive cationic lipid as constituent lipids were administered by intracerebral injection.

[0255] Specifically, the lipid nanoparticles loaded with Fluc mRNA containing CL4F6 among the lipid nanoparticles loaded with Fluc mRNA prepared in Example 10 were administered to Balb / c mice by intracerebral injection, and the Fluc activity (RLU / mg protein) was measured. The results are shown in Figure 5 Regarding the lipid nanoparticles containing CL4F6 and loaded with Fluc mRNA, when administered by intracerebral injection, the lipid nanoparticles containing TOT-15 also showed significantly higher Fluc activity in the brain than the lipid nanoparticles containing DSPC at the same time.

[0256] [Example 13]

[0257] The effect of the proportion of ionic neutral lipids in the constituent lipids of lipid nanoparticles on the delivery efficiency of the lipid nanoparticles to target cells in the body was investigated. TOT-10 synthesized in Example 2 was used as the ionic neutral lipid. pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than ionic neutral lipids. CL4F6 was used as a pH-sensitive cationic lipid. In addition, DSPC was used instead of TOT-10.

[0258] Specifically, CL4F6, TOT-10, cholesterol and PEG-DMG were used in a composition of CL4F6 / TOT-10 / chol / PEG-DMG=x / y / 100-xy / z (mol%), and lipid nanoparticles (lipid nanoparticles loaded with FlucmRNA) (LNP-1~9) were prepared by alcohol dilution method. The average particle size (number average), ζ average particle size, PdI, and encapsulation efficiency of the prepared lipid nanoparticles were measured. The results are shown in Table 1. In addition, DSPC was used instead of TOT, and the same operation was performed, and the composition of CL4F6 / DSPC / chol / PEG-DMG=50 / 10 / 40 / 1 (mol%) was used to prepare lipid nanoparticles (LNP-10) loaded with FlucmRNA.

[0259] [Table 1]

[0260]

[0261] Next, the prepared lipid nanoparticles loaded with Fluc mRNA (0.1 mg RNA / kg) were administered to Balb / c mice (4-week-old females), and the liver and spleen of the mice were collected 6 hours after administration to measure Fluc activity (expression level of Fluc protein) (RLU / mg protein) (N=1). The results are shown in Figure 6 The activity in the liver increases in proportion to the content of CL4F6. When the content of CL4F6 is equal, if the content of TOT-10 is high, the Fluc activity decreases. In particular, considering the expression efficiency of Fluc in the liver, lipid nanoparticles loaded with Fluc mRNA having a TOT-10 content of 30 mol% or less are preferred, and lipid nanoparticles loaded with Fluc mRNA having a TOT-10 content of about 10 mol% are most preferred.

[0262] [Example 14]

[0263] The membrane-destructive ability of lipid nanoparticles prepared using TOT-9 to TOT-15 synthesized in Examples 1 to 7 as constituent lipids was investigated by a hemolysis test.

[0264] pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than TOT-9 to TOT-15. CL4F6 was used as a pH-sensitive cationic lipid. In addition, DSPC was used instead of TOT.

[0265] <Lipid Nanoparticles>

[0266] Specifically, lipid nanoparticles were prepared using CL4F6, TOT-9 to TOT-15, cholesterol and PEG-DMG in a composition of CL4F6 / TOT / chol / PEG-DMG=50 / 10 / 40 / 1.5 (mol%) by the alcohol dilution method. In addition, lipid nanoparticles were prepared in the same manner except that DSPC was used instead of TOT and a composition of CL4F6 / DSPC / chol / PEG-DMG=50 / 10 / 40 / 1.5 (mol%) was used. The average particle size (number average), ζ average particle size, PdI, and charge rate and apparent pKa under various pH conditions of the prepared lipid nanoparticles were measured. The measurement results are shown in Tables 2 and 3. Figure 7 .

[0267] [Table 2]

[0268] Neutral lipids Average particle size [nm] Number average particle size [nm] PdI pKa DSPC 128 67 0.21 6.12 TOT-9 157 109 0.15 6.27 TOT-10 141 73 0.28 6.39 TOT-11 131 83 0.17 6.41 TOT-12 135 86 0.18 6.34 TOT-13 125 81 0.12 6.19 TOT-14 141 76 0.23 6.24 TOT-15 119 61 0.24 6.29

[0269] Hemolysis tests were performed on each lipid nanoparticle to measure the hemolytic activity at pH 5.0, 6.0, and 7.4. Under pH 5.0, all lipid nanoparticles caused membrane damage and showed the same degree of hemolytic activity ( Figure 8 In addition, under the condition of pH 7.4, none of the lipid nanoparticles caused membrane damage ( Figure 8 (C)). Under the condition of pH 6.0, the lipid nanoparticles containing DSPC did not cause membrane damage, but the lipid nanoparticles containing TOT-9 to TOT-15 all showed a maximum hemolytic activity of about 30% ( Figure 8 (B)). These results show that TOT-9 to TOT-15 promote membrane fusion of early endosomes, indicating that high delivery efficiency to target cells can be achieved through the membrane fusion promoting ability.

[0270] Lipid nanoparticles were prepared in the same manner as above using CL4F6, ALC-0315, MC3, SM-102, CL15F6 or CL15H6 as cationic lipids and TOT-15 or DSPC as ionic neutral lipids, and the charge rate and apparent pKa under various pH conditions were measured. The measurement results are shown in Table 3 and Fig. 9 . Fig. 9 (A) is the one using CL4F6, Fig. 9 (B) uses MC3, Fig. 9 (C) uses CL15F6, Fig. 9 (D) is the one using ALC-0315, Fig. 9 (E) is the one using SM-102, Fig. 9 (F) is the result of lipid nanoparticles using CL15H6. For lipid nanoparticles containing any cationic lipid, the pKa of the case containing TOT-15 and the case containing DSPC were similar (Table 3), but in the high pH region in the latter half of the S-shaped curve, the pKa of the case containing DSPC was different (e.g., Fig. 9 Compared with the case containing TOT-15 (e.g., in Fig. 9 The charge rate of "CL4F6-TOT15" in (A) is high ( Fig. 9 ).

[0271] [Table 3]

[0272]

[0273] Hemolysis tests were performed on each lipid nanoparticle to measure the hemolytic activity under the conditions of pH 5.0, 6.0, and 7.4. Under the condition of pH 5.0, regardless of the type of cationic lipid, lipid nanoparticles containing TOT-15 and lipid nanoparticles containing DSPC showed the same degree of hemolytic activity ( Fig.10 On the other hand, under the condition of pH 6.0, when CL4F6 and ALC were used as cationic lipids, lipid nanoparticles containing DSPC did not cause membrane damage, while lipid nanoparticles containing TOT-15 showed hemolytic activity ( Fig.10 When MC3, SM, CL15F6, or CL15H6 was used as the cationic lipid, the lipid nanoparticles containing TOT-15 showed hemolytic activity equal to or less than that of the lipid nanoparticles containing DSPC ( Fig.10 (B)). Under pH 7.4, lipid nanoparticles containing CL15F6 and CL15H6 caused membrane damage, but those containing TOT-15 showed higher hemolytic activity than those containing DSPC ( Fig.10 (C).

[0274] [Example 15]

[0275] The delivery efficiency of lipid nanoparticles using TOT-15 synthesized in Example 7 to the liver was compared with that of lipid nanoparticles using DSPC. pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than TOT-15 and DSPC. CL4F6 was used as a pH-sensitive cationic lipid.

[0276] Specifically, CL4F6, TOT-15, cholesterol and PEG-DMG were used in a composition of CL4F6 / TOT-15 / chol / PEG-DMG = 50 / 10 / 40 / 1.5 (mol%), and lipid nanoparticles loaded with Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) were prepared by alcohol dilution method. In addition, DSPC was used instead of TOT, and the same operation was performed to prepare lipid nanoparticles loaded with Fluc mRNA in a composition of CL4F6 / DSPC / chol / PEG-DMG = 50 / 10 / 40 / 1.5 (mol%).

[0277] Next, the prepared lipid nanoparticles loaded with Fluc mRNA (0.01 mg RNA / kg) were administered to Balb / c mice (4-week-old females), and the livers of the mice were collected 0.5 hours or 6 hours after administration, and the amount of Fluc mRNA taken up in the liver was measured by qRT-PCR. The measurement results are shown in Fig.11 . 0.5 hours after administration ( Fig.11 (A)) and 6 hours later ( Fig.11 In any of (B)), the lipid nanoparticles containing TOT-15 were taken up by the liver about twice as much mRNA as the lipid nanoparticles containing DSPC. These results confirm that the lipid nanoparticles containing TOT-15 deliver more mRNA than the lipid nanoparticles containing DSPC.

[0278] In addition, the Fluc activity (expression level of Fluc protein) (RLU / mg protein) was measured 6 hours after administration of lipid nanoparticles loaded with Fluc mRNA. The results are shown in Fig.12 In the liver, TOT-15-containing lipid nanoparticles showed about 6.1 times higher mRNA Fluc activity than DSPC-containing lipid nanoparticles. The difference in the amount of mRNA transferred to the liver between TOT-15-containing lipid nanoparticles and DSPC-containing lipid nanoparticles was smaller than the difference in Fluc activity between the two, and it is believed that the increased activity brought about by TOT-15-containing lipid nanoparticles is not only caused by the increase in the amount transferred to target cells, but also by the high escape efficiency from endosomes.

[0279] [Example 16]

[0280] The lipid nanoparticles using TOT-15 synthesized in Example 7 were used as DDS carriers for genome editing. Lipid nanoparticles using DSPC were used as controls. pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than TOT-15 and DSPC. CL4F6 was used as a pH-sensitive cationic lipid.

[0281] Specifically, first, an RNA solution was prepared with Cas9-mRNA (TriLink, 5moU modified) and a gRNA targeting transthyretin (TTR) in a mass ratio of 1:2. Together with the RNA solution, CL4F6, TOT-15, cholesterol and PEG-DMG were used in a composition of CL4F6 / TOT-15 / chol / PEG-DMG = 50 / 10 / 40 / 1.5 (mol%) to prepare lipid nanoparticles (lipid nanoparticles for TTR target genome editing) loaded with Cas9-mRNA and TTR-gRNA using an alcohol dilution method. DSPC was used instead of TOT-15 to prepare lipid nanoparticles (lipid nanoparticles for TTR target genome editing) in the same manner as a control. The average particle size (number average), ζ average particle size, PdI, and encapsulation efficiency of the prepared lipid nanoparticles were measured. The results are shown in Table 4.

[0282] [Table 4]

[0283] Ionic neutral lipids Average particle size [nm] Number average particle size [nm] Encapsulation rate [%] PdI TOT-15 193 136 100.7 0.12 DSPC 168 61 91.1 0.18

[0284] Next, Balb / c mice (4-week-old females) were administered lipid nanoparticles (1.0 mg mRNA / kg) for editing each TTR target genome through the tail vein, and euthanized one week after administration, and blood was collected from the inferior vena cava. The blood was allowed to stand at room temperature for 1 hour, centrifuged at 25°C and 1,000×g for 15 minutes, and the supernatant was recovered to obtain serum. The TTR concentration in serum was determined using a commercially available ELISA kit (mouse prealbumin ELISA kit, manufactured by Aviva Systems Biology). The standard curve sample was prepared by gradient dilution from 500ng / mL to prepare 300μL of 250, 125, 62.5, 31.25, and 15.63ng / mL solutions each. The serum sample was obtained by diluting the serum 10,000 times. 100μL of the standard curve sample and serum sample were added to the well plate and incubated at 25°C for 30 minutes. The wells were washed 4 times, 100 μL of enzyme-antibody conjugate was added to each well, the plate was shielded with aluminum foil, and then incubated at 25° C. for 20 minutes. The wells were washed 4 times again, 100 μL of TMB substrate solution (manufactured by Aviva Systems Biology) was added to each well, the plate was shielded with aluminum foil, and then incubated at 25° C. for 10 minutes, 100 μL of stop solution (manufactured by Aviva Systems Biology) was added, and the absorbance at 450 nm was measured using a fluorescence spectrophotometer (VarioskanLux, manufactured by Thermo Fisher Scientific).

[0285] Fig.13 The TTR level in serum is shown in Fig.13 (A)) and TTR knockdown efficiency ( Fig.13 (B) The results of the assay. Compared with lipid nanoparticles containing DSPC, lipid nanoparticles containing TOT-15 had a TTR knockdown efficiency that was approximately 3.4 times higher.

[0286] [Example 17]

[0287] The storage stability of lipid nanoparticles using TOT-15 synthesized in Example 7 was investigated. Lipid nanoparticles using DSPC were used as a control. pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than TOT-15 and DSPC. CL4F6 was used as a pH-sensitive cationic lipid.

[0288] Specifically, CL4F6, TOT-15, cholesterol and PEG-DMG were used in a composition of CL4F6 / TOT / chol / PEG-DMG=50 / 10 / 40 / 1.5 (mol%), and lipid nanoparticles loaded with Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) were prepared by the alcohol dilution method. In addition, DSPC was used instead of TOT-15, and the same operation was performed, and lipid nanoparticles loaded with Fluc mRNA (lipid nanoparticles loaded with Fluc mRNA) were prepared using a composition of CL4F6 / DSPC / chol / PEG-DMG=50 / 10 / 40 / 1.5 (mol%), and stored at 4°C for 4 weeks. The average particle size (number average), PdI, and encapsulation efficiency of the prepared lipid nanoparticles were measured over time. The measurement results during preparation are shown in Table 5. In addition, the results of the time-dependent measurements of the average particle size (number average) and the encapsulation efficiency are shown in Table 5. Fig.14 .like Fig.14 As shown, even storage at 4°C for 4 weeks had no effect on the particle size and encapsulation efficiency of lipid nanoparticles containing TOT-15.

[0289] [Table 5]

[0290] Ionic neutral lipids Number average particle size [nm] PdI Encapsulation rate [%] TOT-15 58 0.23 95.1 DSPC 80 0.21 90.5

[0291] In addition, lipid nanoparticles (0.01 mg RNA / kg) loaded with Fluc mRNA were administered to Balb / c mice (4-week-old females) immediately after manufacture and after 4 weeks of storage, and the liver of the mice was recovered 6 hours after administration to measure Fluc activity (expression level of Fluc protein) (RLU / mg protein). The results are shown in Fig.15 .like Fig.15 As shown, even if stored at 4°C for 4 weeks, there was no effect on the Flu activity of the lipid nanoparticles containing TOT-15, indicating that the lipid nanoparticles can be stably stored for at least 1 month.

[0292] [Example 18]

[0293] The lipid nanoparticles using TOT-15 synthesized in Example 7 were used as DDS carriers for GFP expression to evaluate GFP expression in living tissues. Lipid nanoparticles using DSPC were used as controls. pH-sensitive cationic lipids, cholesterol, and PEG-DMG were used as lipids other than TOT-15 and DSPC. CL4F6 was used as a pH-sensitive cationic lipid.

[0294] Lipid nanoparticles loaded with EGFP-mRNA (manufactured by TriLink, 5moU modified) were prepared by alcohol dilution method using CL4F6, TOT / chol / PEG-DMG=50 / 10 / 40 / 1.5 (mol%), 50 / 20 / 30 / 1.5 (mol%), or 50 / 30 / 20 / 1.5 (mol%). Lipid nanoparticles loaded with EGFP-mRNA (lipid nanoparticles loaded with EGFPmRNA) were prepared by using CL4F6 / DSPC / chol / PEG-DMG=50 / 10 / 40 / 1.5 (mol%) instead of TOT-15. Lipid nanoparticles loaded with EGFP-mRNA (lipid nanoparticles loaded with EGFPmRNA) were prepared by the same operation except that DSPC was used instead of TOT-15.

[0295] Balb / c mice (4-week-old females) were administered lipid nanoparticles loaded with EGFP mRNA (1.00 mg RNA / kg) through the tail vein. 10 minutes before tissue recovery, DyLight-649 Lycopersicon (Tomato) Lectin (DyLight 649 labeled tomato lectin) was administered through the tail vein at 40 μg / mouse to fluorescently label vascular endothelial cells. Euthanasia was performed 24 hours after the administration of lipid nanoparticles loaded with EGFP mRNA, and the tissue was recovered. When staining the nucleus, a portion of the tissue was immersed in PBS (-) supplemented with 1 μg / mL Hoechst 33342, cooled with ice, and incubated for 30 minutes in the dark. After washing the stained tissue with PBS (-), it was observed using a confocal laser scanning microscope. The microscope image is shown in Fig.16 The results showed a tendency that the EGFP expression activity in cells other than hepatocytes increased as the ratio of TOT-15 in the constituent lipids of the lipid nanoparticles increased.< / mrna>

Claims

1. An ionic neutral lipid comprising a compound represented by the following formula (I): [Chemical formula 1] In formula (I), R 1 is a hydrocarbon group having 1 to 22 carbon atoms; the three R 1 They may be the same group or different groups; a1 and a2 are each independently an integer of 0 to 4; b1 and b2 are 0 or 1 satisfying b1+b2=1; R 2 and R 3 Each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R 4 It is an anionic group.

2. The ionic neutral lipid according to claim 1, wherein In the general formula (I), R 4 It is a carboxylic acid group, a sulfonic acid group, a sulfate group or a phosphoric acid group.

3. The ionic neutral lipid according to claim 1, wherein There are 3 R in one molecule 1 All are the same group.

4. The ionic neutral lipid according to claim 1, wherein The R 1 It is a hydrocarbon group having 15 to 22 carbon atoms.

5. The ionic neutral lipid according to claim 1, which is a compound represented by any one of the following general formulas (Z-9) to (Z-15): [Chemical formula 2] [Chemical formula 3] In the formula, R 1 Same as general formula (I).

6. The ionic neutral lipid according to claim 1, which is (S)-4-amino-5-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-5-oxopentanoic acid, 2-((4-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-4-oxobutyl)dimethylammonio)acetate, 2-((3-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-3-oxopropyl)dimethylammonio)acetate, 2-(( 3-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-3-oxopropyl)(methyl)ammonio)acetate, (S)-3-amino-4-((1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)amino)-4-oxobutanoic acid, N4-(1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)-L-asparagine, or N5-(1,3-bis(oleoyloxy)-2-((oleoyloxy)methyl)propan-2-yl)-L-glutamine.

7. Ionic neutral lipids with a Tris backbone, A fatty acid residue is bonded to each of the three oxygen atoms of the Tris skeleton. The number of carbon atoms in the hydrocarbon chain of the fatty acid residue is 1 to 22, and the fatty acid residue may have one or more unsaturated bonds. The three fatty acid residues in one molecule may be the same group or two or more different groups. An anionic group is linked to one nitrogen atom of the Tris skeleton via a divalent linking group having an ammonium cationic group or a quaternary ammonium cationic nitrogen atom. 8 . Lipid nanoparticles comprising the ionic neutral lipid according to claim 1 .

9. The lipid nanoparticle of claim 8, further comprising sterol and polyalkylene glycol modified lipids.

10. The lipid nanoparticle according to claim 8, further comprising a pH-sensitive cationic lipid having a pKa of 6.6 or less. The lipid nanoparticle of claim 8 , comprising a nucleic acid.

12. The lipid nanoparticle of claim 11, wherein The nucleic acid is siRNA, mRNA or plasmid DNA.

13. A pharmaceutical composition comprising lipid nanoparticles containing the ionic neutral lipid according to any one of claims 1 to 7 as an active ingredient.

14. A pharmaceutical composition comprising lipid nanoparticles comprising the ionic neutral lipid according to any one of claims 1 to 7 and a nucleic acid as an active ingredient.

15. The pharmaceutical composition according to claim 14, which is used for gene therapy.

16. A method for expressing a foreign gene, wherein: The lipid nanoparticles according to claim 11 encapsulating a desired foreign gene that can be expressed in target cells are administered to a subject animal (excluding humans) to allow the foreign gene to be expressed in the target cells of the subject animal.

Images