Aminopolyester and lipid nanoparticles

Abstract

To provide a lipid nanoparticle that serves as a gene delivery carrier and an amino polyester that forms a constituent lipid of the lipid nanoparticle.SOLUTION: Provided is an aminopolyester having a structure represented by general formula (P-1) [in the formula, Z1 is an alkylene group; one hydrogen atom in the alkylene group of Z1 may be substituted with -O-[D1]n2-H; D1 is a divalent group represented by the following general formula (P) (in the formula, a carbon atom in the carbonyl group is bonded to the above Z1 via an oxygen atom; one of R11 to R13 is a C1-C12 alkyl group, and the rest are hydrogen atoms; R14 is a hydrogen atom or a methyl group; Z2 is a 1,4-cyclohexylene group; n11 to n13 are each independently 0 or 1); n1 is an integer of 1 or more; and n2 is an integer of 0 or more].SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to lipid nanoparticles useful as gene delivery carriers.

Background Art

[0002] In recent years, lipid nanoparticles (LNPs) have been used as carriers for encapsulating lipophilic drugs and nucleic acids such as siRNA (short interfering RNA) or mRNA and delivering them to target cells. For example, lipid nanoparticles containing a pH-sensitive cationic lipid as a constituent lipid have been reported as carriers for efficiently delivering nucleic acids such as siRNA into target cells (Patent Document 1). In addition, lipid nanoparticles that can be taken into target cells via receptors by modifying the surface of the lipid nanoparticles with a target ligand have also been developed.

[0003] Due to its vascular structure and physiological properties, the liver is easily accessible to carriers administered systemically. Therefore, there are many reports on gene delivery carriers targeting the liver. On the other hand, carriers that specifically achieve gene expression in organs other than the liver are still few. So far, lipid nanoparticles that can be taken into target cells via receptors by modifying the surface of the lipid nanoparticles with a target ligand have been developed.

[0004] Recently, it has been reported that nanoparticles composed of aminopolyester (APE) are delivered to various organs such as the liver, lung, and spleen (Patent Document 2, Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006] [Non-Patent Document 1] Kowalski et al., Advanced Materials, 2018, vol.30, 1801151. [Non-Patent Document 2] Hu et al., Journal of Polymer Science Part A: Polymer Chemistry, 2019, vol.57, p.752-757. [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] Even lipid nanoparticles modified with a target ligand tend to migrate to the liver. Therefore, the development of a carrier that selectively delivers to a target tissue while suppressing migration to the liver is important for realizing systemic gene therapy.

[0008] An object of the present invention is to provide lipid nanoparticles that can be selectively delivered to the spleen and lungs without using a target ligand or the like, and an amino polyester that is a constituent lipid of the lipid nanoparticles. [Means for Solving the Problems]

[0009] The present inventors have found that lipid nanoparticles containing an amino polyester synthesized by ring-opening polymerization of ε-decalactone are useful as gene delivery carriers that specifically highly express in spleen tissue and lung tissue, and have completed the present invention.

[0010] That is, the present invention provides the following amino polyesters and the like. [1] The following general formula (P-1)

[0011] [Chemical Formula]

[0012] [Wherein, Z 1 is an alkylene group having 1 to 6 carbon atoms; in the alkylene group of said Z 1 , one hydrogen atom bonded to a carbon atom not bonded to either an oxygen atom or a nitrogen atom may be substituted with -O-[D 1 n2-H; D 1 is a divalent group represented by the following general formula (P)

[0013] [Chemical formula]

[0014] (In the formula, the carbon atom of the carbonyl group is bonded to said Z 1 via an oxygen atom; R 11 ~R 13 , one of which is an alkyl group having 1 to 12 carbon atoms and the rest are hydrogen atoms; R 14 is a hydrogen atom or a methyl group; Z 2 is a 1,4-cyclohexylene group; when R 11 is an alkyl group having 1 to 12 carbon atoms, n11 is 0 or 1, and when R 11 is a hydrogen atom, n11 is 0; when R 12 is an alkyl group having 1 to 12 carbon atoms, n12 is 0 or 1, and when R 12 is a hydrogen atom, n12 is 0; when R 13 is an alkyl group having 1 to 12 carbon atoms, n13 is 0 or 1, and when R 13 is a hydrogen atom, n13 is 0; a black circle represents a bond to another group) is a divalent group represented by; n1 is an integer of 1 or more; n2 is an integer of 0 or more: a black circle represents a bond to another group] An aminopolyester having a structure represented by [2] The aminopolyester according to [1], wherein the number of D 1 present in one molecule is 5 to 200 [3] The following general formula (A-1) or (A-2)

[0015] [Chemical formula]

[0016] [In the formula, R 0H is a group represented by the following general formula (H-1) or (H-2)

[0017] [Chemical formula]

[0018] (wherein, a1 is an integer of 1 to 6; a2 and a3 are integers of 1 or more, and the sum of both is an integer of 2 to 6; the black circle represents a bond with another group) and is a group represented by; R a is an alkyl group having 1 to 6 carbon atoms which may be substituted with a dialkylamino group, a 1-pyrrolidyl group, or a 1-imidazolyl group; R 1 is a group represented by the following general formula (H-3) or (H-4)

[0019] [Chemical formula]

[0020] (wherein, R 0H and R a are the same as described above; c1 is an integer of 1 to 6; c2 is an integer of 1 to 6; the black circle represents a bond with another group) and is a group represented by; p is an integer of 0 or more and 3 or less, q is an integer of 0 or more and 3 or less, r is an integer of 0 or more and 2 or less, provided that p + q + r = 3; R 2 is R 0H or R 1 ; R 3 is R 0H , R a , or R 1 ; A is a 5- to 7-membered ring having two nitrogen atoms) Any of the amino alcohols represented by the formula, and the following general formula (La), (Lg), or (Le)

[0021] [Chemical formula]

[0022] [wherein, R 11 ~R 13 are each independently an alkyl group having 1 to 12 carbon atoms; Z 2 is a 1,4-cyclohexylene group; when R 11 is an alkyl group having 1 to 12 carbon atoms, n11 is 0 or 1, and when R 11 is a hydrogen atom, n11 is 0; when R 12 is an alkyl group having 1 to 12 carbon atoms, n12 is 0 or 1, and when R 12 is a hydrogen atom, n12 is 0; when R 13 is an alkyl group having 1 to 12 carbon atoms, n13 is 0 or 1, and when R 13 is a hydrogen atom, n13 is 0)] The amino polyester according to [1] or [2] above, which is obtained by subjecting a caprolactone derivative represented by the formula to ring-opening polymerization. [4] The amino alcohol is represented by the following general formulas (A-1-1) to (A-1-9) and (A-2-1) to (A-2-4)

[0023] [Chemical formula]

[0024] [wherein, R 0H , R a , and R 1 are the same as those in the general formula (A-1) above] The amino polyester according to [3] above, which is an amino alcohol represented by any of the above. [5] The amino alcohol is represented by the following formulas (AA01) to (AA17)

[0025] [Chemical formula]

[0026] [Chemical formula]

[0027] The amino polyester of [3] above, which is an amino alcohol represented by any of them. [6] The caprolactone derivative of the amino polyester of any of [3] to [5] above, wherein the caprolactone derivative is a caprolactone derivative represented by any of the following formulas (E1), (E3), (E4), (E6), (E4sec), (G4), (G8), (A4), or (B1E3)

[0028] [Chemical formula]

[0029] The caprolactone derivative of the amino polyester of any of [3] to [5] above, wherein the caprolactone derivative is a caprolactone derivative represented by any of them. [7] The amino polyester of [3] above, wherein the amino alcohol is an amino alcohol represented by any of the formulas (AA01) to (AA17), and the caprolactone derivative is a caprolactone derivative represented by any of the formulas (E1), (E3), (E4), (E6), (E4sec), (G4), (G8), (A4), or (B1E3). [8] The following general formulas (P-1-1) to (P-1-9) and (P-2-1) to (P-2-4)

[0030] [Chemical formula]

[0031] [In the formula, R OP is the R OH , or a group in which at least one hydroxy group in the R OH is replaced with -O-[D 1 n1-H; R 1P is the R 1 , or a group in which at least one hydroxy group in the R 1 is replaced with -O-[D 1 n1-H; D 1 and n1 are the same as those in the general formula (P-1); R ais the same as in the general formula (A-1); in one molecule, at least 5 Ds 1 are contained] The aminopolyester of [3] represented by any of the following. [9] The general formula (A-1) or (A-2) [wherein, R 0H is a group represented by the general formula (H-1) or (H-2) (wherein, a1 is an integer of 1 to 6; a2 and a3 are 1 or more, and the sum of both is an integer of 2 to 6; a black circle represents a bond to another group); R a is an alkyl group having 1 to 6 carbon atoms which may be substituted with a dialkylamino group, a 1-pyrrolidyl group, or a 1-imidazolyl group; R 1 is a group represented by the general formula (H-3) or (H-4) (wherein, R 0H and R a are the same as described above; c1 is an integer of 1 to 6; c2 is an integer of 1 to 6; a black circle represents a bond to another group); p is an integer of 0 or more and 3 or less, q is an integer of 0 or more and 3 or less, r is an integer of 0 or more and 2 or less, provided that p + q + r = 3; R 2 is R 0H or R 1 ; R 3 is R 0H , R a , or R 1 ; A is a 5- to 7-membered ring having 2 nitrogen atoms] Any of the amino alcohols represented by the following and the general formula (La), (Lg), or (Le) [wherein, R 11 to R 13 are each independently an alkyl group having 1 to 12 carbon atoms; Z 2 is a 1,4-cyclohexylene group; when R 11 is an alkyl group having 1 to 12 carbon atoms, n11 is 0 or 1, and when R 11 is a hydrogen atom, n11 is 0; when R 12 is an alkyl group having 1 to 12 carbon atoms, n12 is 0 or 1, and when R 12 is a hydrogen atom, n12 is 0; R 13When it is an alkyl group having 1 to 12 carbon atoms, n13 is 0 or 1, and R 13 When it is a hydrogen atom, n13 is 0] A method for producing an aminopolyester, which comprises subjecting a caprolactone derivative represented by the formula to ring-opening polymerization to produce an aminopolyester.

[10] A lipid nanoparticle containing any one of the aminopolyester of [1] to [8].

[11] The lipid nanoparticle of

[10] , further containing a polyalkylene glycol-modified lipid.

[12] The lipid nanoparticle of

[10] or

[11] , containing a nucleic acid.

[13] The lipid nanoparticle of

[12] , wherein the nucleic acid is a gene to be expressed in splenocytes or lung tissue cells.

[14] A pharmaceutical composition comprising, as an active ingredient, a lipid nanoparticle containing any one of the aminopolyester of [1] to [8].

[15] The pharmaceutical composition of

[14] , which is used for the treatment of spleen diseases or lung diseases.

[16] A lipid nanoparticle containing any one of the aminopolyester of [1] to [8], which encapsulates a foreign gene for the purpose of expression in cells of spleen tissue or lung tissue, is administered to a test animal (excluding humans), and the foreign gene is expressed inside the spleen tissue or lung tissue of the test animal. A method for expressing a foreign gene. [Effect of the Invention]

[0032] The lipid nanoparticle according to the present invention can highly express the encapsulated gene in lung tissue or spleen tissue. Therefore, the lipid nanoparticle is useful as a gene delivery carrier to lung tissue or spleen tissue, which is used for immunotherapy and gene therapy. [Embodiments for Carrying Out the Invention]

[0033] Hereinafter, embodiments of the present invention will be specifically described. In the specification of the present application, "X1 to X2 (X1 and X2 are real numbers satisfying X1 < X2)" means "X1 or more and X2 or less".

[0034] <Amino polyester> The lipid nanoparticles according to the present invention contain an amino polyester having a structure represented by the following general formula (P-1) (hereinafter sometimes referred to as "structure (P-1)"). The amino polyester according to the present invention is characterized by containing the following. Structure (P-1) has a polymerization unit consisting of D 1 . This polymerization unit D 1 is a divalent group represented by the following general formula (P). In general formula (P-1) and general formula (P), the black circle represents a bond with another group. In general formula (P), the carbon atom of the carbonyl group is bonded to the Z 1 via an oxygen atom.

[0035] [Chemical formula]

[0036] [Chemical formula]

[0037] In general formula (P), R 11 ~R 13 One of these is an alkyl group having 1 to 12 carbon atoms (C 1-12 alkyl group), and the rest are hydrogen atoms. The polymerization unit D 1 has a structure having an alkyl chain at the α-position carbon (R 11 is a C 1-12 alkyl group, and R 12 and R 13 are hydrogen atoms: hereinafter sometimes referred to as "polymerization unit D 1 (A)"), and a structure having an alkyl chain at the γ-position carbon (R 12 is a C 1-12 alkyl group, and R 11 and R 13 are hydrogen atoms: hereinafter sometimes referred to as "polymerization unit D 1 (G)"), and a structure having an alkyl chain at the ε-position carbon (R 13 is a C 1-12 alkyl group, and R11 and R 12 a structure in which is a hydrogen atom: hereinafter, "polymerization unit D 1 (E)") and the like.

[0038] In the general formula (P), R 11 ~R 13 Any of the alkyl groups which is is not particularly limited as long as it is an alkyl group having 1 to 12 carbon atoms, and may be a linear alkyl group or a branched alkyl group. Examples of the alkyl group having 1 to 12 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, sec-pentyl group, tert-pentyl group, n-hexyl group, isohexyl group, neohexyl group, sec-hexyl group, tert-hexyl group, n-heptyl group, isoheptyl group, sec-heptyl group, tert-heptyl group, neoheptyl group, n-octyl group, isooctyl group, sec-octyl group, tert-octyl group, neooctyl group, n-nonyl group, isononyl group, sec-nonyl group, tert-nonyl group, neononyl group, n-decyl group, isodecyl group, sec-decyl group, tert-decyl group, neodecyl group, n-undecyl group, isoundecyl group, sec-undecyl group, tert-undecyl group, neoundecyl group, n-dodecyl group, isododecyl group, sec-dodecyl group, tert-dodecyl group, neododecyl group and the like. The polymerization unit D in the aminopolyester according to the present invention 1 As, R 11 ~R 13 Any of the alkyl groups which is is preferably an alkyl group having 1 to 9 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms.

[0039] In the general formula (P), Z 2 is a 1,4-cyclohexylene group, and n11 to n13 are each independently 0 or 1. When n11 is 0, -(Z 2 )n11- means a single bond. The same applies to n12 and n13. R 11 is C 1-12In the case of an alkyl group, n11 is 0 or 1, and R 11 is a hydrogen atom, n11 is 0. Similarly, R 12 is C 1-12 In the case of an alkyl group, n12 is 0 or 1, and R 12 is a hydrogen atom, n12 is 0, and also, R 13 is C 1-12 In the case of an alkyl group, n13 is 0 or 1, and R 13 is a hydrogen atom, n13 is 0.

[0040] In general formula (P), R 14 is a hydrogen atom or a methyl group.

[0041] In general formula (P-1), n1 is the repeating number (degree of polymerization) of polymerization unit D 1 and is an integer of 1 or more. As the aminopolyester according to the present invention, n1 is not particularly limited as long as it is an integer of 1 or more, and for example, it can be 1 to 200, preferably 1 to 150, more preferably 1 to 100, still more preferably 1 to 50, and even more preferably 1 to 20.

[0042] In general formula (P-1), Z 1 is an alkylene group having 1 to 6 carbon atoms (C 1-6 alkylene group). The C 1-6 alkylene group may be a linear alkylene group or a branched alkylene group. Examples of the C 1-6 alkylene group include a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, a sec-butylene group, a tert-butylene group, an n-pentylene group, an isopentylene group, a neopentylene group, a sec-pentylene group, a tert-pentylene group, an n-hexylene group, an isohexylene group, a neohexylene group, a sec-hexylene group, a tert-hexylene group, etc. As the structure (P-1) in the aminopolyester according to the present invention, Z 1 is preferably a linear C 1-6 alkylene group, and a linear C 1-3An alkylene group is more preferable, and an ethylene group or a propylene group is even more preferable.

[0043] Z 1 When Z is an alkylene group having 2 or more carbon atoms, one hydrogen atom bonded to a carbon atom in the alkylene group that is not bonded to either an oxygen atom or a nitrogen atom is -O-[D 1 n2-H may be substituted. The D 1 is the same as described above, and n2 is an integer of 0 or more. When n2 is 0, -O-[D 1 n2-H is a hydroxy group (-OH).

[0044] Z 1 has -O-[D 1 n2-H as a substituent, the structure (P-1) has n1 + n2 polymerization units D 1 . As the aminopolyester according to the present invention, n1 + n2 is not particularly limited as long as it is an integer of 1 or more, and for example, it can be 1 to 200, preferably 1 to 150, more preferably 1 to 100, even more preferably 1 to 50, and even more preferably 1 to 20.

[0045] The aminopolyester according to the present invention may have at least one structure (P-1), and may have two or more. When a molecule has a plurality of structures (P-1), the number of polymerization units D 1 contained in the aminopolyester is the total number of D 1 contained in each structure (P-1). In an aminopolyester having a plurality of structures (P-1) per molecule, the number (n1) of polymerization units D 1 in the structure (P-1) may be different for each structure (P-1), or all the structures (P-1) may be the same. In the present invention and the specification of the present application, the number of polymerization units D 1 present in one molecule of the aminopolyester may be referred to as the "degree of polymerization (of the aminopolyester)".

[0046] The degree of polymerization of the aminopolyester according to the present invention can be calculated from the size of polymerization unit D 1 The size (number average molecular weight) of the aminopolyester and polymerization unit D 1 can be measured by size exclusion chromatography (SEC) analysis by the method described in the following examples. The degree of polymerization of the aminopolyester according to the present invention can be 1 to 200, preferably 5 to 200, more preferably 5 to 150, still more preferably 5 to 100, even more preferably 5 to 50, and particularly preferably 5 to 35. The aminopolyester with the desired degree of polymerization can be controlled by adjusting the molar ratio of the polymerization initiator (amino alcohol) and monomer (caprolactone derivative) used in the reaction.

[0047] The lipid nanoparticles according to the present invention contain the aminopolyester according to the present invention as a lipid component constituting the lipid membrane. The polyester moiety in the aminopolyester according to the present invention has high specificity for spleen tissue and lung tissue. Therefore, by containing the aminopolyester as a lipid component, the lipid nanoparticles according to the present invention are useful as a spleen-specific or lung-specific delivery carrier without modifying a target ligand or the like on the surface of the lipid nanoparticles. In addition, since the aminopolyester according to the present invention has an amino group, it can efficiently encapsulate basic substances such as nucleic acids. Therefore, by containing the aminopolyester as a lipid component, the lipid nanoparticles according to the present invention are useful as a carrier for specifically delivering a basic substance to spleen tissue or lung tissue, and particularly as a gene delivery carrier for encapsulating a foreign gene to be expressed inside spleen tissue or lung tissue, and is very excellent.

[0048] The aminopolyester having structure (P-1) can be synthesized, for example, by ring-opening polymerization of a 7-membered lactone using an amino alcohol as an initiator. This ring-opening polymerization reaction can be efficiently carried out at a temperature of about room temperature by using a basic catalyst in a relatively low-polar aprotic organic solvent such as toluene. As the basic catalyst, an inorganic base catalyst such as a metal base may be used, but an organic base catalyst is preferred. Among them, Bronsted strong basic organic catalysts such as phosphazene bases, organolithium compounds, and bicyclic guanidine-type organic bases are preferred, and phosphazene bases are more preferred. As the bicyclic guanidine-type organic base, TBD (triazabicyclodecene, CAS No.: 5807-14-7), MTBD (methyltriazabicyclodecene, CAS No.: 84030-20-6) are preferred. As the phosphazene base, t-Bu-P4 base (CAS No.: 111324-04-0) is particularly preferred.

[0049] As the 7-membered lactone used in the synthesis of the aminopolyester according to the present invention, a caprolactone derivative represented by the following general formula (La), (Lg), or (Le) is preferred.

[0050] [Chemical formula]

[0051] In the general formulas (La), (Lg), and (Le), R 11 ~R 13 , Z 2 , and n11 to n13 are all the same as those in the general formula (P). By using the caprolactone derivative represented by the general formula (La), an aminopolyester having the polymerization unit D 1 (A) is synthesized. By using the caprolactone derivative represented by the general formula (Lg), an aminopolyester having the polymerization unit D 1 (G) is synthesized, and by using the caprolactone derivative represented by the general formula (Le), an aminopolyester having the polymerization unit D 1 (E) is synthesized.

[0052] Examples of the caprolactone derivative represented by the general formula (La), (Lg), or (Le) include compounds of the following formulas (E1), (E3), (E4), (E6), (E4sec), (G4), (G8), (A4), or (B1E3).

[0053]

Chemical formula

[0054] The amino alcohol used for the synthesis of the amino polyester according to the present invention is preferably an amino alcohol represented by any of the following general formulas (A-1) or (A-2).

[0055]

Chemical formula

[0056] In the general formula (A-1), R 0H is a group represented by the following general formula (H-1) or (H-2). The amino alcohol represented by the general formula (A-1) starts polymerization with the hydroxy group in R 0H and the caprolactone derivative.

[0057]

Chemical formula

[0058] In the general formula (H-1), a1 is an integer of 1 to 6. In the general formula (H-2), a2 and a3 are integers of 1 or more, and the sum of both is an integer of 2 to 6. In the general formulas (H-1) and (H-2), the black circle represents a bond with another group.

[0059] In the general formula (A-1), R a is a C 1-6 alkyl group which may be substituted with a dialkylamino group, a 1-pyrrolidyl group, or a 1-imidazolyl group. The C 1-6The alkyl group may be a linear alkyl group or a branched alkyl group. C 1-6 Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a sec-pentyl group, a tert-pentyl group, an n-hexyl group, an isohexyl group, a neohexyl group, a sec-hexyl group, a tert-hexyl group, and the like.

[0060] R a is, when one of the hydrogen atoms of the C 1-6 alkyl group is a group substituted with a dialkylamino group, a 1-pyrrolidyl group, or a 1-imidazolyl group, the hydrogen atom substituted with these substituents may be any of the hydrogen atoms of the C 1-6 alkyl group. For the synthesis of the aminopolyester according to the present invention, a group in which the hydrogen atom of the methyl group at the chain end of the C 1-6 alkyl group is substituted is preferable, a group in which the hydrogen atom of the terminal methyl group of the linear C 1-6 alkyl group is substituted is more preferable, and a group in which the hydrogen atom of the terminal methyl group of an ethyl group or an n-propyl group is substituted is even more preferable.

[0061] In the general formula (A-1), R 1 is a group represented by the following general formula (H-3) or (H-4). In the general formulas (H-3) and (H-4), the black circle represents a bond with another group.

[0062]

Chemical formula

[0063] In the general formulas (H-3) and (H-4), R 0H and R a are the same as those in the general formula (A-1). In the general formula (H-3), c1 is an integer of 1 to 6. In the general formula (H-4), c2 is an integer of 1 to 6. The amino alcohol represented by the general formula (A-1) is R 1 in R0H The hydroxy group of

[0064] In general formula (A-1), p is an integer of 0 or more and 3 or less, q is an integer of 0 or more and 3 or less, and r is an integer of 0 or more and 2 or less. However, p + q + r = 3. The amino alcohol represented by general formula (A-1) is R 0H and R 1 has at least one of either. The larger the number of R 0H (including those present in R 1 ) in one molecule, the more starting points for polymerization with the caprolactone derivative, and the easier it is to obtain an amino polyester with a branched-chain structure. On the other hand, when the number of R 0H in one molecule is small, or in the case of an amino alcohol in which a plurality of R 0H are present substantially on the same straight line, a linear-structured amino polyester can be obtained.

[0065] In general formula (A-2), R 2 is R 0H or R 1 . Also, in general formula (A-2), R 3 is R 0H , R a , or R 1 . The said R 0H , R a , and R 1 are all the same as in the said general formula (A-1).

[0066] In general formula (A-2), A is a 5- to 7-membered ring having two nitrogen atoms. Examples of A include a piperazinyl group, a diazepanyl group, an imidazolidinyl group, etc. As the amino alcohol represented by general formula (A-2), a compound in which A is a piperazinyl group or a diazepanyl group is preferable, and a compound in which A is a piperazinyl group is more preferable.

[0067] Examples of the amino alcohol represented by the general formula (A-1) include compounds represented by the following general formulas (A-1-1) to (A-1-9). Examples of the amino alcohol represented by the general formula (A-2) include compounds represented by the following general formulas (A-2-1) to (A-2-4). In each formula, R 0H 、R a 、and R 1 are the same as those in the general formula (A-1).

[0068]

Chemical formula

[0069] By subjecting these amino alcohols to ring-opening polymerization with a caprolactone derivative represented by the general formula (La), (Lg), or (Le), amino polyesters represented by the following general formulas (P-1-1) to (P-1-9) and (P-2-1) to (P-2-4) can be synthesized.

[0070]

Chemical formula

[0071] In the general formulas (P-1-1) to (P-1-9) and (P-2-1) to (P-2-4), R OP represents a group in which at least one hydroxy group in the above R OH , or the above R OH is substituted with -O-[D 1 n1-H. Further, R 1P represents a group in which at least one hydroxy group in the above R 1 , or the above R 1 is substituted with -O-[D 1 n1-H. Note that D 1 and n1 are the same as those in the general formula (P-1). R a is the same as that in the general formula (A-1). In one molecule of the amino polyesters represented by the general formulas (P-1-1) to (P-1-9) and (P-2-1) to (P-2-4), a plurality of R OPmay be the same group as each other or different groups. Similarly, a plurality of R's in one molecule 1P and R a may also be the same group as each other or different groups, respectively.

[0072] The aminopolyester according to the present invention does not require all the hydroxy groups in R OH to undergo ring-opening polymerization with the caprolactone derivative, and a part of them may remain as hydroxy groups. Also, the polymer chains extending from a plurality of hydroxy groups in one molecule of the aminopolyester may all have the same degree of polymerization or may each have a different degree of polymerization. The degree of polymerization of the aminopolyester according to the present invention is 5 or more, that is, it preferably has at least 5 D's in one molecule 1 in it.

[0073] Specific examples of the amino alcohol represented by the general formula (A-1) or (A-2) include the compounds of the following formulas (AA01) to (AA17).

[0074]

Chemical formula

[0075]

Chemical formula

[0076] For example, by subjecting the amino alcohol represented by any of the formulas (AA01) to (AA17) and the caprolactone derivative represented by any of the formulas (E1), (E3), (E4), (E6), (E4sec), (G4), (G8), (A4), or (B1E3) to ring-opening polymerization, an aminopolyester useful as a constituent lipid of lipid nanoparticles suitable as a gene carrier can be synthesized.

[0077] <Lipid nanoparticles> As for the lipid nanoparticles according to the present invention, all the lipids constituting the lipid nanoparticles may be the amino polyesters according to the present invention, or may contain amino polyesters and other lipids. Since the selectivity for the target tissue can be made sufficiently high, the content of the amino polyesters according to the present invention with respect to the total lipid amount constituting the lipid nanoparticles according to the present invention is preferably 30 to 100% (mol), more preferably 30 to 70% (mol), and even more preferably 40 to 60% (mol).

[0078] Among the constituent lipids of the lipid nanoparticles according to the present invention, as the lipids other than the amino polyesters according to the present invention, lipids generally used when forming liposomes can be used. Examples of such lipids include phospholipids, sterols, or saturated or unsaturated fatty acids. These can be used alone or in combination of two or more.

[0079] Examples of phospholipids include phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, ceramide phosphoryl glycerol phosphate, phosphatidic acid and the like. Examples of sterols include animal-derived sterols such as cholesterol, cholesteryl succinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, brassicasterol; and microorganism-derived sterols such as thymosterol, ergosterol. The lipid nanoparticles according to the present invention preferably contain sterols, and more preferably contain cholesterol.

[0080] The lipid nanoparticles according to the present invention preferably contain a polyalkylene glycol-modified lipid as a lipid component. Polyalkylene glycol is a hydrophilic polymer. By constructing lipid nanoparticles using a polyalkylene glycol-modified lipid as a lipid membrane-forming lipid, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Modifying the surface with polyalkylene glycol may enhance the stability such as the blood retention of the lipid nanoparticles.

[0081] As the polyalkylene glycol, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc. can be used. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, more preferably about 1,000 to 5,000.

[0082] For example, for the modification of lipids with polyethylene glycol, stearylated polyethylene glycol (such as PEG45 stearate (STR-PEG45), etc.) can be used. In addition, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[methoxypolyethylene glycol-2000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, polyethylene glycol derivatives such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG 2k) can also be used, but the polyalkylene glycolated lipids are not limited to these.

[0083] The ratio of the polyalkylene glycol-modified lipid to the total lipid amount constituting the lipid nanoparticles according to the present invention is not particularly limited as long as it does not impair the tissue-specific gene expression activity by the aminopolyester according to the present invention. For example, the ratio of the polyalkylene glycol-modified lipid to the total lipid amount constituting the lipid nanoparticles is preferably 1 to 3 mol%.

[0084] For the lipid nanoparticles according to the present invention, appropriate surface modification or the like can be carried out as needed. The lipid nanoparticles according to the present invention can enhance the blood retention property by modifying the surface with a hydrophilic polymer or the like. In some cases, surface modification can be carried out by using a lipid modified with these modifying groups as a constituent lipid of the lipid nanoparticles.

[0085] In the production of the lipid nanoparticles according to the present invention, as lipid derivatives for enhancing blood retention, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, etc. can also be used. Further, as hydrophilic polymers for enhancing blood retention, in addition to polyalkylene glycols, dextran, pullulan, 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.

[0086] Further, in order to promote nuclear translocation of the lipid nanoparticles according to the present invention, for example, the lipid nanoparticles can be surface-modified with an oligosaccharide compound having 3 or more saccharides. The type of the oligosaccharide compound having 3 or more saccharides is not particularly limited. For example, an oligosaccharide compound having about 3 to 10 sugar units bonded thereto can be used, and preferably an oligosaccharide compound having about 3 to 6 sugar units bonded thereto can be used. Among them, preferably an oligosaccharide compound which is a trimer to hexamer of glucose can be used, and more preferably an oligosaccharide compound which is a trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose, etc. can be preferably used. Among these, maltotriose, maltotetraose, maltopentaose, or maltohexaose in which glucose is α1-4-bonded is more preferable. Particularly preferable is maltotriose or maltotetraose, and most preferable is maltotriose. The amount of surface modification of the lipid nanoparticles with the oligosaccharide compound is not particularly limited. For example, it is about 1 to 30 mol%, preferably about 2 to 20 mol%, more preferably about 5 to 10 mol% with respect to the total lipid amount.

[0087] The method for surface-modifying lipid nanoparticles with oligosaccharide compounds is not particularly limited. For example, liposomes in which lipid nanoparticles are surface-modified with monosaccharides such as galactose or mannose (International Publication No. WO2007 / 102481) are known, so the surface-modifying method described in this publication can be adopted. All of the disclosures of the above publication are incorporated herein by reference as the disclosure of this specification.

[0088] In addition, the lipid nanoparticles according to the present invention can be imparted with any one or two or more functions such as, for example, a temperature change sensitivity function, a membrane permeation function, a gene expression function, and a pH sensitivity function. By appropriately adding these functions, the retention of lipid nanoparticles in the blood can be improved, and after endocytosis in target cells, the lipid nanoparticles can be efficiently escaped from endosomes, and the encapsulated nucleic acid can be more efficiently expressed in cells of lung tissue and spleen tissue.

[0089] The lipid nanoparticles according to the present invention may contain one or more substances selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides. Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and the like. Examples of the membrane polypeptide include a membrane surface polypeptide or a membrane-intrinsic polypeptide. The blending amounts of these substances are not particularly limited and can be appropriately selected according to the purpose.

[0090] Since the lipid nanoparticles according to the present invention are likely to achieve high delivery efficiency to target tissue cells existing in vivo, the average particle diameter thereof is preferably 500 nm or less, more preferably 50 to 450 nm, and even more preferably 100 to 350 nm. The average particle diameter of the lipid nanoparticles means the number average particle diameter measured by the dynamic light scattering method (Dynamic light scattering: DLS). The measurement by the dynamic light scattering method can be carried out by a conventional method using a commercially available DLS device or the like.

[0091] The polydispersity index (PdI) of the lipid nanoparticles according to the present invention is about 0.01 to 0.7, preferably about 0.05 to 0.6, and more preferably about 0.05 to 0.3. The zeta potential can be in the range of -50 mV to 20 mV, preferably in the range of -25 mV to 15 mV.

[0092] The form of the lipid nanoparticles according to the present invention is not particularly limited, but it is preferably in a form dispersed in an aqueous solvent. Examples of such a form include nanoparticles in which a monolayer composed of a hydrophilic substance is formed on the surface of a core containing lipid. Also, unilamellar liposomes, multilamellar liposomes, spherical micelles, etc. can be mentioned, and it may be an amorphous layered structure composed of amphiphilic lipid molecules. For example, the lipid nanoparticles composed of an aminopolyester, a polyalkylene glycol-modified lipid, and a nucleic acid according to the present invention are nanoparticles having a core composed of an aminopolyester and a nucleic acid and a polyalkylene glycol layer on the nanoparticle surface.

[0093] The lipid nanoparticles according to the present invention preferably contain a component intended to be delivered into target cells inside the particles covered with a lipid membrane. The component contained inside the lipid nanoparticles according to the present invention is not particularly limited as long as it is of an encapsulable size, and any substance such as nucleic acids, saccharides, peptides, low molecular weight compounds, and metal compounds can be encapsulated in the lipid nanoparticles according to the present invention.

[0094] As a component to be encapsulated in the lipid nanoparticles according to the present invention, a nucleic acid is preferable. The nucleic acid may be DNA, RNA, or their analogs or derivatives (for example, peptide nucleic acid (PNA), phosphorothioate DNA, etc.). The nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention may be a single-stranded nucleic acid, a double-stranded nucleic acid, linear, or circular.

[0095] The nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention preferably contains a foreign gene for expression in target cells, and more preferably is a nucleic acid that functions to express the foreign gene intracellularly by being taken up into cells. The foreign gene may be a gene originally contained in the genomic DNA of target cells (preferably spleen tissue cells or lung tissue cells), or a gene not contained in the genomic DNA. Examples of such nucleic acids include gene expression vectors containing a nucleic acid consisting of a base sequence encoding the gene to be expressed. The gene expression vector may exist as an episomal gene in the introduced cells, or may be incorporated into the genomic DNA by homologous recombination.

[0096] The gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is not particularly limited, and generally, vectors used in gene therapy and the like can be used. As the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention, a nucleic acid vector such as a plasmid vector is preferable. The plasmid vector may remain circular or may be encapsulated in the lipid nanoparticles according to the present invention in a state of being previously linearized. The gene expression vector can be designed by a conventional method using generally used molecular biology tools based on the base sequence information of the gene to be expressed, and can be produced by various known methods.

[0097] The nucleic acid encapsulated in the lipid nanoparticles according to the present invention is preferably a functional nucleic acid that controls the expression of a target gene present in target cells. Examples of such functional nucleic acids include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, and the like. Further, it may be an siRNA expression vector that expresses siRNA in cells. The siRNA expression vector can be prepared from a commercially available siRNA expression vector, or it may be appropriately modified.

[0098] The method for producing the lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be adopted. For example, all lipid components are dissolved in an organic solvent such as chloroform, and a lipid film is formed by performing vacuum drying with an evaporator or spray drying with a spray dryer. Then, a component to be encapsulated in the lipid nanoparticles, for example, an aqueous solvent containing a nucleic acid or the like, is added to the above-mentioned dried mixture, and further emulsified by an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier. It can also be produced by a method well known as a method for producing liposomes, for example, the reverse phase evaporation method. When it is desired to control the size of the lipid nanoparticles, extrusion (pushing and filtering) can be performed under high pressure using a membrane filter with uniform pore size.

[0099] The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include buffer solutions such as phosphate buffer, citrate buffer, phosphate buffered saline, physiological saline, and media for cell culture. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles. Furthermore, sugars (aqueous solutions) such as monosaccharides of glucose, galactose, mannose, fructose, inositol, ribose, xylose, disaccharides of lactose, sucrose, cellobiose, trehalose, maltose, trisaccharides of raffinose, melibiose, polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol, and polyhydric alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol may be added. To stably store the lipid nanoparticles dispersed in this aqueous solvent for a long period, it is desirable to eliminate electrolytes in the aqueous solvent as much as possible from the aspect of physical stability such as aggregation inhibition. Also, from the aspect of chemical stability of the lipid, it is desirable to set the pH of the aqueous solvent from weakly acidic to near neutral (about pH 3.0 to 8.0) and / or remove dissolved oxygen by nitrogen bubbling or the like.

[0100] When lyophilizing or spray-drying the aqueous dispersion of the obtained lipid nanoparticles, for example, sugars such as monosaccharides of glucose, galactose, mannose, fructose, inositol, ribose, xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, maltose, trisaccharides such as raffinose, melezitose, polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol, etc. (aqueous solution) may be used to improve stability. Also, when freezing the above aqueous dispersion, for example, the above-mentioned saccharides, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, polyhydric alcohols such as 1,3-butylene glycol (aqueous solution) may be used to improve stability.

[0101] When the lipid nanoparticles according to the present invention encapsulating a gene expression vector are administered to an animal individual, the gene expression vector encapsulated in the lipid nanoparticles is selectively expressed in spleen tissue or lung tissue rather than in other organs. Similarly, when the lipid nanoparticles according to the present invention encapsulating an siRNA expression vector are administered to an animal individual, the siRNA expression vector encapsulated in the lipid nanoparticles is selectively expressed in spleen tissue or lung tissue rather than in other organs, and the expression of the gene targeted by the expression vector is suppressed. For example, when the lipid nanoparticles according to the present invention encapsulating a foreign gene to be expressed in cells of spleen tissue or lung tissue are administered to a test animal, the foreign gene can be expressed inside the spleen tissue or lung tissue of the test animal.

[0102] Due to this highly selective uptake into the spleen tissue or lung tissue, the lipid nanoparticles according to the present invention function as a delivery carrier targeting the spleen tissue or lung tissue. Therefore, the lipid nanoparticles according to the present invention are useful as a carrier for delivering a basic medicinal ingredient for the treatment of spleen diseases or lung diseases to the spleen tissue or lung tissue, and in particular, are useful as an active ingredient of a pharmaceutical composition used in immunotherapy or gene therapy targeting the spleen tissue or lung tissue. Examples of lung diseases include viral infections such as pneumonia and SARS, acute respiratory distress syndrome, lung cancer, pulmonary hypertension, pulmonary fibrosis, cystic fibrosis, and the like. Examples of spleen diseases include spleen cancer and splenomegaly.

[0103] In particular, the spleen contains a large number of immune cells such as T cells, B cells, macrophages, and dendritic cells. Therefore, the spleen is an important target tissue in vaccine development. For example, the lipid nanoparticles according to the present invention are very effective as a carrier for delivering a nucleic acid vaccine against pathogenic bacteria of infectious diseases, cancer (tumor antigen), biomolecules that cause the onset and progression of diseases, etc. to the spleen tissue. Examples of biomolecules that cause the onset and progression of diseases include amyloid-β protein in Alzheimer's disease.

[0104] The animal to which the lipid nanoparticles according to the present invention are administered is not particularly limited and may be a human or a non-human animal. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, and birds such as chickens, quails, and ducks. In addition, the administration route when administering the lipid nanoparticles according to the present invention to an animal is not particularly limited, but parenteral administration such as intravenous administration, enteral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, and pulmonary administration is preferred.

Examples

[0105] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Also, in the subsequent experiments, unless otherwise specified, "%" means "mass %".

[0106] [Reagents, etc.] Dehydrated tetrahydrofuran (THF) (>99.5%, water content <0.001%, manufactured by Kanto Chemical Co., Inc.), dehydrated dichloromethane (manufactured by Kanto Chemical Co., Inc., >99.5%, water content <0.001%), and dehydrated toluene (manufactured by Kanto Chemical Co., Inc., >99.5%, water content <0.001%) were used after purification with a solvent purification apparatus (manufactured by MBRAUN, MS-SPS compact, columns: MB-KOL-C, MB-KOL-A). Acetate buffer (25 mM, pH 4.0) was prepared by mixing sodium acetate (final concentration 40 mM), acetic acid (final concentration 94.1 mM), and potassium chloride (final concentration 5.90 mM), and making up to 1000 mL with ultrapure water (DDW). m-Chloroperbenzoic acid (mCPBA) (moisture content, 69.0 - 75.0%, manufactured by FUJIFILM Wako Pure Chemical Corporation), silica gel 60N (spherical, neutral, particle size: 63 - 210 μm, manufactured by Kanto Chemical Co., Inc.), Celite (registered trademark) 545 (manufactured by Kanto Chemical Co., Inc.), phosphazene base P4-t-Bu solution (0.8 M hexane solution, manufactured by Sigma-Aldrich), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) (manufactured by Tokyo Chemical Industry (TCI), >98.0%), palladium / carbon (Pd 10%) (manufactured by TCI, approximately 55% water-wetted product) were used as purchased.

[0107] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (manufactured by Avanti POLAR LIPIDS), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) (manufactured by NOF AMERICA CORPORATION), cholesterol (≧99%, manufactured by Sigma-Aldrich) were used as purchased. The mRNA encoding luciferase (CleanCap® Firefly Luciferase mRNA (5-methoxyuridine)) was purchased from TriLink Biotechnologies.

[0108] [Experimental animals] The breeding and experiments of ICR mice (purchased from Japan SLC, Inc.) were conducted in accordance with the animal experiment protocol reviewed and approved by the Hokkaido University Animal Experiment Committee based on the guidelines for the care and use of experimental animals.

[0109] [Nuclear magnetic resonance (NMR)] For the 1 1H NMR measurement, 13 13C NMR measurement, 1 1H- 1 1H COSY and 1 1H- 13 1H-13C HSQC were performed under the following conditions.

[0110] Instrument: JEOL JNM-ECS 400 (400 MHz) and JEOL JNM-ECX 400 (400 MHz) Solvent: deuterated chloroform Internal standard substance: tetramethylsilane (TMS) (0.00 ppm) Measurement temperature: 25 °C Number of integrations: 1 For 1H NMR, 16 or 32 times, 13 for 13C NMR, 128, 256 or 1024 times, 1 1H- 1 1H COSY, 4 times, 1 1H- 13 1H-13C HSQC, 3 times.

[0111] [Glove box] The polymerization charge was carried out inside a glove box (UNILAB, manufactured by MBRAUN, atmosphere, Ar; H2O < 1 ppm, O2 < 0.1 ppm).

[0112] [Glass tube oven] The distillation of 2-butyl-ε-caprolactone was carried out using a glass tube oven (manufactured by SIBATA, model GTO-1000).

[0113] [Size exclusion chromatography (SEC)] SEC measurements were performed under the following conditions, and the number average molecular weight (Mn, SEC) and molecular weight distribution (D) were calculated based on the molecular weight calibration curve prepared with polystyrene.

[0114] Apparatus: Shodex GPC-101 Guard column: Shodex KF-G (4.6 mm × 10 mm) Column: Shodex KF-804L (linear, 8 mm × 300 mm) × 2 Exclusion limit molecular weight: 400,000 Particle size: 7 μm Column oven: Jasco CO-2065 Plus Differential refractive index detector: Jasco RI-2031 Plus Liquid delivery pump: Jasco PU-980 Degassing device: Jasco PU-4180 Solvent: THF Measurement temperature: 40 °C Flow rate: 1.0 mL / min Standard substance: Polystyrene (molecular weights: 2,170; 3,070; 4,430; 10,200; 19,600; 55,100; 133,000; 275,000; 815,000; 1,320,000)

[0115] [Preparative size exclusion chromatography] The fractionation of the polymer was carried out using a preparative size exclusion chromatography apparatus.

[0116] Apparatus: LaboACE LC-7080 Column: JAIGEL-2.5HR (20 mm × 600 mm; exclusion limit, 2×10 5 ) Measurement temperature: Room temperature Solvent: CHCl3 Flow rate: 10 mL / min

[0117] [Fractional chromatography] The purification of the monomer was carried out using fractional chromatography.

[0118] Apparatus: Pure C-815 Flash Column: Flash Pure Silica 40 μm irregular 220 g Laser output of evaporative light scattering detector: 0.3 mW, Gas flow rate of evaporative light scattering detector: 2 - 2.5 L / min UV detector: Diode array detector Solvent: n-hexane / ethyl acetate = 69 / 31 (v / v) Flow rate: 100 mL / min

[0119] [Measurement of average particle diameter, PdI, and zeta potential of lipid nanoparticles] The average particle diameter, polydispersity index (PdI), and zeta potential (ζ-potential) of lipid nanoparticles were measured by dynamic light scattering (DLS) using a DLS apparatus (product name: "Zetasizer Nano ZS ZEN3600", manufactured by Malvern Panalytical; He-Ne laser, λ = 632 nm). The hydrodynamic diameter of lipid nanoparticles was measured in percent intensity mode, and the average of three independent measurements was taken as the average particle diameter of the lipid nanoparticles.

[0120] [Evaluation of nucleic acid recovery rate and encapsulation rate into lipid nanoparticles] For lipid nanoparticles encapsulating nucleic acid, the nucleic acid recovery rate and nucleic acid encapsulation rate (encapsulation efficiency) into lipid nanoparticles were evaluated using a nucleic acid quantification reagent (product name: "Quant-iT RiboGreen assay", manufactured by Thermo Fisher Scientific). The lipid nanoparticle solution was diluted 40-fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8), and mixed in equal amounts with each of the RiboGreen diluent containing dextran sulfate, the diluent containing dextran sulfate and Triton X-100 to prepare an analytical sample. This analytical sample was measured for fluorescence (λex = 321 nm, λem = 447 nm) using a fluorescence plate reader (Tecan Infinite M200 Pro, manufactured by Tecan). Based on the calibration curve prepared using a nucleic acid solution with a known concentration, the amount of nucleic acid after lipid nanoparticle preparation and the amount of nucleic acid not encapsulated in the lipid nanoparticles were calculated, and the recovery rate and nucleic acid encapsulation rate were calculated using the following formulas.

[0121] [Recovery rate (%)] = [Total amount of nucleic acid after preparation (μg) (amount of nucleic acid when dextran sulfate and Triton X-100 are added)] / [Amount of nucleic acid added at the beginning of the experiment (μg)] × 100

[0122] [Nucleic acid encapsulation rate (%)] = 100 - [Amount of nucleic acid outside (μg) (fluorescence when only dextran sulfate is added)] / [Total amount of nucleic acid after preparation (μg) (amount of nucleic acid when dextran sulfate and Triton X-100 are added)] × 100

[0123] [Example 1] An amino polyester library was constructed by changing the structure and substitution position of the side chain of the polymer unit, the structure of the polymerization initiator (amino alcohol), the degree of polymerization, etc.

[0124] [Synthesis of caprolactone derivative and amino alcohol] First, a caprolactone derivative and an amino alcohol were synthesized. The amino alcohol was synthesized in two steps in the same manner as the method of Hu et al. (Non-Patent Document 2).

[0125] In addition, 6-hexyl-ε-caprolactone (E6: >97.0%, manufactured by FUJIFILM Wako Pure Chemical Corporation), 6-butyl-ε-caprolactone (E4: ≧99%, manufactured by Sigma-Aldrich), N-methyldiethanolamine (AA02; >99.0%, manufactured by TCI), 2-{[2-(dimethylamino)ethyl]methylamino}ethanol (AA05; 98.0%, manufactured by Sigma-Aldrich), triethanolamine (AA04; >98.0%, manufactured by TCI), and ε-caprolactone (CL: >99.0%, manufactured by TCI) were used after distillation under reduced pressure in the presence of calcium hydride (CaH2). Mentide (B1E3: ≧98.0%, manufactured by Sigma-Aldrich) and 1,4-bis(2-hydroxyethyl)piperazine (AA14; >98.0%, manufactured by TCI) were used after purification by sublimation.

[0126] [Chemical formula]

[0127] [Chemical formula]

[0128] [Chemical formula]

[0129] (1) Synthesis of 6-methyl-ε-caprolactone (E1) by Baeyer-Villiger oxidation

[0130] [Chemical formula]

[0131] In a 2 L three-necked flask equipped with a mechanical stirrer, 2-methylcyclohexanone (25.0 g, 222 mmol) was dissolved in CH2Cl2 (1.00 L), and mCPBA (46.2 g, 267 mmol) and NaHCO3 (28.1 g, 334 mmol) were added. After stirring at room temperature for 24 hours, the by-product sodium m-chlorobenzoate was removed by filtration. The filtrate was washed with saturated aqueous NaHSO3 (100 mL × 3 times), saturated aqueous NaHCO3 (100 mL × 3 times), and distilled water (100 mL × 3 times). The organic layer was dried over anhydrous MgSO4, and then the solvent was distilled off under reduced pressure. Finally, the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2 / 1 (v / v), Rf = 0.30) and vacuum distillation (b.p. = 40 °C, 3.0 Pa) to obtain 6-methyl-ε-caprolactone (E1) as a colorless transparent liquid (yield: 3.65 g, yield: 12.7%).

[0132] 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.46 (dt, J = 14.9, 6.5 Hz, 1H, -OCH(CH3)CH2-), 2.70 - 2.58 (m, 2H, -CH2CH2CO2-), 1.95 - 1.53 (m, 6H, -CH2CH2CH2CH2CO2-), 1.36 (d, J = 6.4 Hz, 3H, -OCH(CH3)CH2-).

[0133] 13 C NMR (100 MHz, CDCl3): δ (ppm) 175.7 (-CO2-), 76.9 (-OCH(CH3)CH2-), 36.3 (-OCH(CH3)CH2-), 35.1 (-CH2CO2-), 28.4 (-CH2CH2CH2CO2-), 23.0 (-CH2CH2CO2-), 22.7 (-OCH(CH3)CH2-).

[0134] (2) Synthesis of 6-sec-butyl-ε-caprolactone (E4sec) by Baeyer-Villiger oxidation

[0135] [Chemical]

[0136] In a 2 L three-necked flask equipped with a mechanical stirring device, 2-sec-butylcyclohexanone (20.0 g, 129 mmol) was dissolved in CH2Cl2 (800 mL), and mCPBA (26.9 g, 156 mmol) and NaHCO3 (16.3 g, 194 mmol) were added. After stirring at room temperature for 24 hours, sodium m-chlorobenzoate, a by-product, was removed by filtration. The filtrate was washed with saturated aqueous NaHSO3 (80 mL × 3 times), saturated aqueous NaHCO3 (80 mL × 3 times), and distilled water (80 mL × 3 times). The organic layer was dried over anhydrous MgSO4, and then the solvent was distilled off under reduced pressure. Finally, the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 7 / 3 (v / v), Rf = 0.35) and vacuum distillation (b.p. = 66 °C, 3.0 Pa) to obtain 6-sec-butyl-ε-caprolactone (E4sec) as a pale yellow transparent liquid (yield: 6.44 g, yield: 29.2%).

[0137] 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.16 (ddd, J = 18.5, 8.6, 3.9 Hz, 1H, -OCH(CH(CH3)CH2CH3)CH2-), 2.66 - 2.62 (m, 2H, -CH2CH2CO2-), 2.01 - 1.45 (m, 8H, -CH2CH2CH2CH(CH(CH3)CH2CH3)-), 1.30 - 1.19 (m, 1H, -OCH(CH(CH3)CH2CH3)CH2-), 0.97 - 0.89 (m, 6H, -OCH(CH(CH3)CH2CH3)CH2-).

[0138] 1313C NMR (100 MHz, CDCl3): δ (ppm) 176.0 (-CO2-), 84.1, 83.3 (-OCH(CH(CH3)CH2CH3)CH2-), 40.5, 39.8 (-OCH(CH(CH3)CH2CH3)CH2-), 34.9 (-CH2CO2-), 32.0, 30.6 (-CH2CH2CH2CH2CO2-), 28.6, 28.5 (-CH2CH2CH2CO2-), 25.5, 24.8 (-OCH(CH(CH3)CH2CH3)CH2-), 23.3 (-CH2CH2CO2-), 14.6, 13.7 (-OCH(CH(CH3)CH2CH3)CH2-), 11.8 (-OCH(CH(CH3)CH2CH3)CH2-).

[0139] (3) Synthesis of 6-propyl-ε-caprolactone (E3) by Baeyer-Villiger oxidation

[0140] [Chemical Structure]

[0141] In a 1 L three-necked flask equipped with a mechanical stirrer, 2-propylcyclohexanone (10.0 g, 71.3 mmol) was dissolved in CH2Cl2 (400 mL), and mCPBA (18.5 g, 107 mmol) and NaHCO3 (11.2 g, 134 mmol) were added. After stirring at room temperature for 24 hours, the by-product sodium m-chlorobenzoate was removed by filtration. The filtrate was washed with saturated aqueous NaHSO3 (40 mL × 3 times), saturated aqueous NaHCO3 (40 mL × 3 times), and distilled water (40 mL × 3 times). The organic layer was dried over anhydrous MgSO4, and then the solvent was distilled off under reduced pressure. Finally, the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 4 / 1 (v / v), Rf = 0.30) and vacuum distillation (b.p. = 66 °C, 3.0 Pa) to obtain 6-propyl-ε-caprolactone (E3) as a pale yellow transparent liquid (yield: 2.06 g, yield rate: 18.5%).

[0142] 11H NMR (400 MHz, CDCl3): δ (ppm) 4.25 (td, J = 8.1, 4.0 Hz, 1H, -OCH(C3H7)CH2-), 2.70 - 2.57 (m, 2H, -CH2CH2CO2-), 1.96 - 1.34 (m, 10H, -CH2CH2CH2CH(CH2CH2CH3)-), 0.93 (t, J = 7.2 Hz, 3H, -OCH(CH2CH2CH3)CH2-).

[0143] 13 13C NMR (100 MHz, CDCl3): δ (ppm) 175.9 (-CO2-), 80.3 (-OCH(C3H7)CH2-), 38.5 (-CH2CH2CH2CH2CO2-), 35.0 (-CH2CH2CH2CH2CO2-), 28.4 (-OCH(CH2CH2CH3)CH2-), 23.1 (-CH2CH2CO2-), 18.7 (-OCH(CH2CH2CH3)CH2-), 13.9 (-OCH(CH2CH2CH3)CH2-).

[0144] (4)Synthesis of 4-(4-Butylcyclohexyl)-ε-caprolactone (G8) by Baeyer-Villiger Oxidation

[0145]

Chemical Structure

[0146] In a 1 L three-necked flask equipped with a mechanical stirrer, 4-(4-butylcyclohexyl)cyclohexanone (15.0 g, 63.5 mmol) was dissolved in CH2Cl2 (600 mL), and mCPBA (13.1 g, 76.1 mmol) and NaHCO3 (8.00 g, 95.3 mmol) were added. After stirring at room temperature for 24 hours, the by-product sodium m-chlorobenzoate was removed by filtration. The filtrate was washed with saturated aqueous NaHSO3 (60 mL × 3 times), saturated aqueous NaHCO3 (60 mL × 3 times), and distilled water (60 mL × 3 times). The organic layer was dried over anhydrous MgSO4, and then the solvent was distilled off under reduced pressure. Finally, the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2 / 1 (v / v), Rf = 0.30), and the water contained in trace amounts in the product was removed by azeotropic distillation with toluene to obtain 4-(4-butylcyclohexyl)cyclohexanone (G8) as a white solid (yield: 4.33 g, yield: 27.0%).

[0147] 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.32 (ddd, J = 12.7, 5.7, 1.7 Hz, 1H, -OCH2-), 4.15 (dd, J = 12.6, 10.3 Hz, 1H, -OCH2-), 2.73-2.67 (m, 1H, -CH2CO2-), 2.61-2.53 (m, 1H, -CH2CO-), 1.95-1.85 (m, 4H, -CH2CH2CO2CH2CH2-), 1.78 (d, J = 12.6 Hz, 2H), 1.64 (td, J = 6.4, 3.3 Hz, 2H), 1.55 (s, 3H), 1.52-1.38 (m, 2H), 1.28-1.11 (m, 7H), 1.08-0.98 (m, 1H), 0.91-0.81 (m, 5H).

[0148] 1313C NMR (100 MHz, CDCl3): δ (ppm) 176.4 (-CH2CO2CH2-), 68.8 (-OCH2-), 46.2 (-CH2CH(C6H9O2)CH2-), 43.3 (-CO2CH2CH2CH-), 37.8 (-CHCH2CH2CH2CH3), 37.1 (-CH2CH2CH3), 33.7 (-CH2CO-), 33.5 (-CH2CH(C6H9O2)CH2-), 32.6 (-OCH2CH2-), 29.8 (-CH2CH(C4H9)CH2-), 29.3 (-CH2CH2CH2CH3), 26.2 (-CH2CH2CO-), 23.1 (-CH2CH3), 14.3 (-CH2CH3).

[0149] (5) Synthesis of 4-butyl-ε-caprolactone (G4)

[0150] [Chemical formula]

[0151] In a 500 mL Erlenmeyer flask, pyridinium chlorochromate (51.3 g, 238 mmol) was dissolved in CH2Cl2 (300 mL), and 4-butylcyclohexanol (25.0 g, 160 mmol) was added dropwise. After stirring at room temperature for 3 hours, the chromium residue, which is a by-product, was removed by filtration using Celite 545. The filtrate was washed with saturated aqueous CuSO4 solution (100 mL × 5), and the organic layer was dried over anhydrous MgSO4. Then, the solvent was distilled off under reduced pressure. Finally, the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 4 / 1 (v / v), Rf = 0.30) to obtain 4-butylcyclohexanone as a pale yellow transparent liquid (yield: 17.1 g, yield rate: 69.3%).

[0152] 11H NMR (400 MHz, CDCl3): δ (ppm) 2.40 - 2.28 (m, 4H, -CH2COCH2-), 2.08 - 2.02 (m, 2H, -CH2CH2COCH2CH2-), 1.72 - 1.67 (m, 1H, -CH2CH(C4H9)CH2-), 1.44 - 1.24 (m, 8H, -CH2CH(CH2CH2CH2CH3)CH2-), 0.93 - 0.88 (m, 3H, -CH2CH2CH2CH3).

[0153] 13 13C NMR (100 MHz, CDCl3): δ (ppm) 212.8 (-CH2COCH2-), 41.0 (-CH2COCH2-), 36.1 (-CH2CH2CH2CH3), 35.3 (-CH2CH2CH2CH3), 32.9 (-CH2CH2COCH2CH2-), 29.6 (-CH2CH(C4H9)CH2-), 23.0 (-CH2CH2CH2CH3), 14.2 (-CH2CH2CH2CH3).

[0154] Subsequently, in a 1 L three-necked flask equipped with a mechanical stirrer, the obtained 4-butylcyclohexanone (17.1 g, 111 mmol) was dissolved in CH2Cl2 (600 mL), and mCPBA (19.1 g, 133 mmol) and NaHCO3 (14.0 g, 166 mmol) were added. After stirring at room temperature for 24 hours, the by-product sodium m-chlorobenzoate was removed by filtration. The filtrate was washed with saturated aqueous NaHSO3 (60 mL × 3 times), saturated aqueous NaHCO3 (60 mL × 3 times), and distilled water (60 mL × 3 times). The organic layer was dried over anhydrous MgSO4, and then the solvent was distilled off under reduced pressure. Finally, the residue was purified by vacuum distillation (b.p. = 88 - 90 °C, 6.0 Pa) to obtain 4-butyl-ε-caprolactone (G4) as a pale yellow transparent liquid (yield: 5.91 g, yield: 31.3%).

[0155] 11H NMR (400 MHz, CDCl3): δ (ppm) 4.30 (qd, J = 6.1, 1.8 Hz, 1H, -OCH2-), 4.17 (dd, J = 12.6, 9.9 Hz, 1H, -OCH2-), 2.71 - 2.56 (m, 2H, -CH2CO-), 2.02 - 1.89 (m, 2H, -CH2CH(C4H9)CH2-), 1.63 - 1.43 (m, 2H, -CH2CH(C4H9)CH2CH2O-), 1.37 - 1.29 (m, 7H, -CH2CH(CH2CH2CH2CH3)CH2CH2O-), 0.90 (t, J = 6.7 Hz, 3H, -CH2CH3).

[0156] 13 13C NMR (100 MHz, CDCl3): δ (ppm) 176.3 (-CH2CO2-), 68.3 (-OCH2-), 40.3 (-CH2CH(C4H9)CH2-), 36.2 (-CH2CH2CH3), 35.5 (-OCH2CH2-), 33.3 (-CH2CO-), 29.1, 29.0 (-CH2CH(CH2CH2CH2CH3)CH2CH2O-), 22.9 (-CH2CH3), 14.1 (-CH2CH3).

[0157] (6) Synthesis of 2-butyl-ε-caprolactone (A4)

[0158]

Chemical Structure

[0159] In a 1 L three-necked flask, under an argon atmosphere, a solution of diisopropylamine (14.1 mL, 100 mmol) in THF (300 mL) was cooled to -78 °C, and then n-BuLi (38.4 mL, 50.0 mmol, a 2.60 mol / L stock solution dissolved in n-hexane) was added dropwise to the solution, and the mixture was stirred for 15 minutes to prepare lithium diisopropylamide (LDA). Then, a solution of ε-caprolactone (10.3 g, 90.0 mmol) in THF (50 mL) was added dropwise over 1 hour or more. After stirring the reaction solution for 30 minutes, a solution of crotyl bromide (14.0 mL, 139 mmol) in HMPA (24.5 mL, 141 mmol) was added dropwise. After stirring the reaction solution for 30 minutes, the reaction was stopped by gradually adding saturated aqueous NH4Cl solution (10.0 mL). The solution was allowed to stand to room temperature, and the solvent was distilled off under reduced pressure. The residue was extracted with diethyl ether (100 mL) and washed with saturated brine (50 mL × 3 times). The organic layer was dried over anhydrous MgSO4, and then the solvent was distilled off under reduced pressure and purified by flash column chromatography (hexane / ethyl acetate = 4 / 1 (v / v)) to obtain 2-(2-butenyl)-ε-caprolactone as a pale yellow transparent liquid (yield: 2.22 g, yield: 14.5%).

[0160] 1 H NMR (400 MHz, CDCl3): δ (ppm) 5.70-5.34 (m, 2H, CH3CH=CH-), 4.32-4.20 (m, 2H, -CO2CH2-), 2.60-2.43 (m, 1H, -CHCO2-), 2.10-1.24 (m, 11H, -CH2CH2CH2CH2CH(CH2CH=CHCH3)CO2-).

[0161] Subsequently, in a 300 mL Schlenk flask, the obtained 2-(2-butenyl)-ε-caprolactone (2.22 g, 13.2 mmol) was added to a suspension of Pd / C (4.80 g, 2.03 mmol) in ethyl acetate (135 mL), and the reaction mixture was vigorously stirred for 30 minutes under a hydrogen atmosphere (1 atm). The resulting mixture was filtered through Celite using ethyl acetate. The obtained filtrate was distilled off under reduced pressure, and 2-butyl-ε-caprolactone was obtained by vacuum distillation using a glass tube oven (Yield: 1.22 g, Yield rate: 54.6%).

[0162] 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.30 - 4.20 (m, 2H, -OCH2-), 2.54 - 2.48 (m, 1H, -CH2CH(C4H7)CO2-), 1.99 - 1.24 (m, 12H, -CH2CH2CH2CH(CH2CH2CH2CH3)CO-), 0.90 (t, J = 6.6 Hz, 3H, -CH3).

[0163] 13 C NMR (100 MHz, CDCl3): δ (ppm) 177.8 (-CO2-), 68.4 (-CO2CH2-), 43.0 (-CHCO2-), 32.5 (CH3CH2CH2CH2-), 30.2, 29.7, 29.0, 28.5 (-CH2CH2CH(CH2CH2CH2CH3)CO2-), 22.9 (-CH2CH3), 14.2 (-CH2CH3).

[0164] (7) Synthesis of Amino Alcohol AA07

[0165]

Chemical Structure

[0166] In an argon atmosphere, N,N - diisopropylethylenediamine (5.00 g, 34.7 mmol) was dissolved in dehydrated methanol (21 mL) and stirred at room temperature for 30 minutes. To prevent polymerization, after covering the reaction system with aluminum foil, methyl acrylate (7.46 g, 86.7 mmol) was added dropwise. After stirring at room temperature for 48 hours, the solvent was distilled off under reduced pressure to obtain dimethyl 3,3’ - ((2 - (diisopropylamino)ethyl)azanediyl)diisopropionate as a pale yellow transparent liquid (yield: 10.7 g, yield rate: 97.5%).

[0167] 1 H NMR (400 MHz, CDCl3): δ (ppm) 3.67 (s, 6H, -OCH3), 3.00 - 2.94 (m, 2H, (CH3)2CH-), 2.78 (t, J = 7.2 Hz, 4H, -CH2CO2-), 2.45 (t, J = 7.2 Hz, 8H, -CH2NCH2CH2N-), 1.00 (d, J = 6.3 Hz, 12H, (CH3)2CH-).

[0168] 13 C NMR (100 MHz, CDCl3): δ (ppm) 173.2 (-CO2-), 51.7 ((CH3)2CH-), 56.4 (-CH2CH2CO2-), 50.0 (-OCH3), 49.5 ((CH3)2CHNCH2CH2-), 44.2 ((CH3)2CHNCH2-), 32.8 (-CH2CO2-), 20.9 ((CH3)2CH-).

[0169] In an argon atmosphere, 3,3'-((2-(diisopropylamino)ethyl)azanediyl)diisopropionate (10.7 g, 33.8 mmol) was dissolved in dehydrated THF (35 mL), cooled to 0 °C, and stirred for 30 minutes. LiAlH4 (7.18 g, 189 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 48 hours. Then, the reaction was stopped by adding H2O (7.4 mL) at 0 °C. Subsequently, NaOH (15 wt%, 7.4 mL) was added to the solution, and the mixture was stirred until the gray solid turned white. After adding H2O (22.1 mL) to the solution, it was washed with methanol using celite. The reaction solution was dissolved in n-butanol, and LiOH, which is a by-product, was completely removed using ultracentrifugation (12,000 rpm, 20 minutes, 20 °C). Finally, n-butanol was removed by azeotropic distillation with toluene to obtain 3,3'-((2-(diisopropylamino)ethyl)azanediyl)bis(propan-1-ol) (AA07) as a yellow liquid (yield: 5.93 g, yield: 67.4%).

[0170] 1 H NMR (400 MHz, CDCl3): δ (ppm) 7.27 (s, 2H, -OH), 3.73 (t, J = 5.6 Hz, 4H, -CH2OH), 3.05 - 2.95 (m, 2H, (CH3)2CH-), 2.64 - 2.42 (m, 8H, -NCH2CH2NCH2CH2CH2OH), 1.75 - 1.69 (m, 4H, -CH2CH2OH), 0.98 - 1.05 (d, 12H, (CH3)2CH-).

[0171] 13 C NMR (100 MHz, CDCl3): δ (ppm) 62.5 (-CH2OH), 56.0 (-CH2NCH2CH2CH2OH), 53.4 (-CH2CH2CH2OH), 49.3 ((CH3)2CH-), 43.3 (-NCH2CH2NCH2CH2CH2OH), 28.9 (-CH2CH2OH), 20.8 ((CH3)2CH-).

[0172] (8) Synthesis of amino alcohol AA17

[0173]

Chem.

[0174] Under an argon atmosphere, 1-(3-aminopropyl)imidazole (5.00 g, 39.9 mmol) was dissolved in dehydrated methanol (21 mL) and stirred at room temperature for 30 minutes. To prevent polymerization, after covering the reaction system with aluminum foil, methyl acrylate (8.60 g, 99.9 mmol) was added dropwise. After stirring the reaction solution at room temperature for 48 hours, the solvent was distilled off under reduced pressure to obtain dimethyl 3,3'-((3-(1H-imidazol-1-yl)azanediyl)dipropionate as a pale yellow transparent liquid (yield: 11.6 g, yield: 97.6%).

[0175] 1 H NMR (400 MHz, CDCl3): δ (ppm) 7.52 (s, 1H, -NCHN-), 7.06 (s, 1H, -CH2CH2NCHCHN-), 6.96 (s, 1H, -CH2CH2NCHCHN-), 3.95 (t, J = 7.0 Hz, 2H, -CH2CH2N-), 3.68 (s, 6H, -OCH3), 2.74 (t, J = 6.7 Hz, 4H, -CH2CO2-), 2.40 (dt, J = 17.5, 6.5 Hz, 6H, -CH2NCH2-), 1.95 - 1.88 (m, 2H, -NCH2CH2CH2N-).

[0176] 13 C NMR (100 MHz, CDCl3): δ (ppm) 173.0 (-CO2-), 137.5 (-NCHN-), 129.5 (-CH2CH2NCHCHN-), 119.0 (-CH2CH2NCHCHN-), 51.7 (-CH2CH2CO2-), 50.5 (-CH2CH2CH2NCHCHN-), 49.2 (-OCH3), 44.5 (-CH2CH2CH2NCHCHN-), 32.4 (-CH2CO2-), 29.0 (-NCH2CH2CH2N-).

[0177] In an argon atmosphere, dimethyl 3,3'-((3-(1H-imidazol-1-yl)azanediyl)dipropionate (11.6 g, 39.0 mmol) was dissolved in dehydrated THF (35 mL), cooled to 0 °C, and stirred for 30 minutes. LiAlH4 (8.19 g, 189 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 48 hours. Then, the reaction was stopped by adding H2O (8.3 mL) at 0 °C. Subsequently, NaOH (15 wt%, 8.0 mL) was added to the solution, and the mixture was stirred until the gray solid turned white. After adding H2O (25.8 mL) to the solution, it was washed with methanol using celite. The reaction solution was dissolved in n-butanol, and LiOH, a by-product, was completely removed using ultracentrifugation (12,000 rpm, 20 minutes, 20 °C). Finally, n-butanol was removed by azeotropic distillation with toluene to obtain 3,3'-((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(propan-1-ol) (AA17) as a yellow liquid (yield: 3.72 g, yield: 39.6%).

[0178] 1 H NMR (400 MHz, CDCl3): δ (ppm) 7.50 (s, 1H, -NCHN-), 7.28 (s, 2H, -OH), 7.05 (s, 1H, -CH2CH2NCHCHN-), 6.94 (s, 1H, -CH2CH2NCHCHN-), 3.99 (t, J = 7.1 Hz, 2H, -CH2CH2N-), 3.72 (t, J = 5.7 Hz, 4H, -CH2OH), 2.60 (t, J = 6.4 Hz, 4H, -CH2CH2CH2OH), 2.41 (t, J = 7.1 Hz, 2H, -NCH2CH2CH2NCH2-), 2.02-1.95 (m, 2H, -NCH2CH2CH2N-), 1.72-1.66 (m, 4H, -CH2CH2OH).

[0179] 1313C NMR (100 MHz, CDCl3): δ (ppm) 137.3 (-NCHN-), 129.6 (-CH2CH2NCHCHN-), 118.8 (-CH2CH2NCHCHN-), 62.3 (-CH2OH), 52.9 (-CH2CH2OH), 50.9 (-CH2CH2CH2OH), 44.9 (-CH2CH2NCHCHN-), 28.7 (-CH2CH2CH2OH), 28.5 (-NCH2CH2CH2N-).

[0180] (9) Synthesis of Amino Alcohol AA11

[0181] [Chemical formula]

[0182] Under an argon atmosphere, tris[2-(methylamino)ethyl]amine (4.50 g, 23.9 mmol) was dissolved in dehydrated methanol (21 mL) and stirred at room temperature for 30 minutes. To prevent polymerization, after covering the reaction system with aluminum foil, methyl acrylate (8.23 g, 95.6 mmol) was added dropwise. After stirring the reaction solution at room temperature for 48 hours, the solvent was distilled off under reduced pressure to obtain trimethyl 3,3’,3’’-((nitrilotris(ethane-2,1-diyl))tris(methylazanediyl))tripropionate as a pale yellow transparent liquid (yield: 10.3 g, yield rate: 96.9%).

[0183] 1 1H NMR (400 MHz, CDCl3): δ (ppm) 3.68 (s, 9H, -OCH3), 2.71 (t, J = 7.2 Hz, 6H, -CH2CO2-), 2.60 (dd, J = 8.8, 5.6 Hz, 6H, -CH2CH2CO2-), 2.47 (q, J = 7.3 Hz, 12H, -NCH2CH2N-), 2.24 (s, 9H, CH3N-).

[0184] 1313C NMR (100 MHz, CDCl3): δ (ppm) 173.1 (-CO2-), 55.6 (-NCH2CH2N(CH3)-), 53.3 (-CH2CH2CO2-), 53.0 (-NCH2CH2N(CH3)-), 51.7 (-OCH3), 42.5 (CH3N-), 32.4 (-CH2CO2-).

[0185] Under an argon atmosphere, trimethyl 3,3’,3’’-((nitrilotris(ethane-2,1-diyl))tris(methylazanediyl))tripropionate (10.3 g, 23.2 mmol) was dissolved in dehydrated THF (35 mL), cooled to 0 °C, and stirred for 30 minutes. To the reaction solution was added LiAlH4 (7.40 g, 195 mmol), and the mixture was stirred at room temperature for 48 hours. Then, the reaction was stopped by adding H2O (7.4 mL) at 0 °C. Subsequently, NaOH (15 wt%, 7.1 mL) was added to the solution, and the mixture was stirred until the gray solid turned white. After adding H2O (22.1 mL) to the solution, it was washed with methanol using celite. The reaction solution was dissolved in n-butanol, and LiOH, which is a by-product, was completely removed using ultracentrifugation (12,000 rpm, 20 minutes, 20 °C). Finally, n-butanol was removed by azeotropic distillation with toluene to obtain 3,3’,3’’-((nitrilotris(ethane-2,1-diyl))tris(methylazanediyl))tris(propan-1-ol) (AA11) as a yellow liquid (yield: 4.69 g, yield: 55.8%).

[0186] 1 1H NMR (400 MHz, CDCl3): δ (ppm) 7.28 (s, 3H, -OH), 3.76 (t, J = 5.2 Hz, 6H, -CH2OH), 2.62 - 2.47 (m, 18H, -NCH2CH2N(CH3)CH2-), 2.26 (s, 9H, -CH2N(CH3)CH2-), 1.71 - 1.66 (m, 6H, -CH2CH2OH).

[0187] 1313C NMR (100 MHz, CDCl3): δ (ppm) 63.7 (-CH2OH), 57.7 (-NCH2CH2N(CH3)CH2-), 55.6 (-CH2CH2CH2OH), 52.6 (-NCH2CH2N(CH3)CH2-), 42.7 (-CH2N(CH3)CH2-), 28.1 (-CH2CH2OH).

[0188] (10) Synthesis of Amino Alcohol AA08

[0189] [Chemical formula]

[0190] Under an argon atmosphere, N,N'-dimethyl-1,6-hexanediamine (5.00 g, 34.7 mmol) was dissolved in dehydrated methanol (21 mL) and stirred at room temperature for 30 minutes. To prevent polymerization, after covering the reaction system with aluminum foil, methyl acrylate (7.46 g, 86.7 mmol) was added dropwise. After stirring the reaction solution at room temperature for 48 hours, the solvent was distilled off under reduced pressure to obtain dimethyl 3,3'-(hexane-1,6-diylbis(methylazanediyl))dipropionate as a pale yellow transparent liquid (yield: 10.7 g, yield: 96.4%).

[0191] 1 1H NMR (400 MHz, CDCl3): δ (ppm) 3.68 (s, 6H, -OCH3), 2.69 (t, J = 7.4 Hz, 4H, -CH2CO2-), 2.48 (t, J = 7.4 Hz, 4H, -CH2CH2CO2-), 2.33 (t, J = 7.4 Hz, 4H, -NCH2CH2CH2-), 2.22 (s, 6H, CH3N-), 1.44 (d, J = 6.7 Hz, 4H, -NCH2CH2CH2-), 1.30 (t, J = 7.0 Hz, 4H, -NCH2CH2CH2-).

[0192] 1313C NMR (100 MHz, CDCl3): δ (ppm) 173.2 (-CO2-), 57.6 (-NCH2CH2CH2-), 52.8 (-CH2CH2CO2-), 51.7 (-OCH3), 42.1 (CH3N-), 32.4 (-CH2CO2-), 27.5 (-NCH2CH2CH2-), 27.3 (-NCH2CH2CH2-).

[0193] Under an argon atmosphere, dimethyl 3,3'-(hexane-1,6-diylbis(methylazanediyl))dipropionate (10.6 g, 33.4 mmol) was dissolved in dehydrated THF (35 mL), cooled to 0 °C, and stirred for 30 minutes. LiAlH4 (7.11 g, 187 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 48 hours. Then, the reaction was stopped by adding H2O (7.4 mL) at 0 °C. Subsequently, NaOH (15 wt%, 7.3 mL) was added to the solution, and the mixture was stirred until the gray solid turned white. After adding H2O (22.0 mL) to the solution, it was washed with methanol using celite. The reaction solution was dissolved in n-butanol, and LiOH, a by-product, was completely removed using ultracentrifugation (12,000 rpm, 20 minutes, 20 °C). Finally, n-butanol was removed by azeotropic distillation with toluene to obtain 3,3'-(hexane-1,6-diylbis(methylazanediyl)bis(propan-1-ol) (AA08) as a yellow liquid (yield: 4.47 g, yield: 51.4%).

[0194] 11H NMR (400 MHz, CDCl3): δ (ppm) 7.28 (s, 2H, -OH), 3.79 (t, J = 5.2 Hz, 4H, -CH2OH), 2.58 (t, J = 5.6 Hz, 4H, -CH2CH2CH2OH), 2.35 (t, J = 7.4 Hz, 4H, -CH2N(CH3)CH2CH2CH2OH), 2.23 (s, 6H, -CH2N(CH3)CH2-), 1.72 - 1.66 (m, 4H, -CH2CH2OH), 1.47 (q, J = 6.6 Hz, 4H, -NCH2CH2CH2CH2CH2CH2N-), 1.32 - 1.29 (m, 4H, -NCH2CH2CH2CH2CH2CH2N-).

[0195] 13 13C NMR (100 MHz, CDCl3): δ (ppm) 64.9 (-CH2OH), 58.8 (-CH2CH2CH2OH), 58.3 (-CH2N(CH3)CH2CH2CH2OH), 42.1 (-CH2N(CH3)CH2-), 27.7 (-CH2CH2OH), 27.3, 27.3 (-NCH2CH2CH2CH2CH2CH2N-).

[0196] (11) Synthesis of Amino Alcohol AA09

[0197]

Chemical Structure

[0198] In an argon atmosphere, N,N'-bis[3-(methylamino)propyl]methylamine (5.00 g, 28.9 mmol) was dissolved in dehydrated methanol (21 mL) and stirred at room temperature for 30 minutes. To prevent polymerization, the reaction system was covered with aluminum foil, and then methyl acrylate (6.21 g, 72.1 mmol) was added dropwise. After stirring the reaction solution at room temperature for 48 hours, the solvent was distilled off under reduced pressure to obtain dimethyl 3,3'-(((methylazanediyl)bis(propane-3,1-diyl))bis(methylazanediyl)dipropionate as a pale yellow transparent liquid (yield: 9.65 g, yield: 96.8%).

[0199] 1 H NMR (400 MHz, CDCl3): δ (ppm) 3.68 (s, 6H, -OCH3), 2.70 (t, J = 7.4 Hz, 4H, -CH2CO2-), 2.48 (t, J = 7.2 Hz, 4H, -CH2CH2CO2-), 2.38-2.31 (m, 8H, -NCH2CH2CH2N-), 2.22 (d, J = 9.9 Hz, 9H, CH3N-), 1.66-1.58 (m, 4H, -NCH2CH2CH2N-).

[0200] 13 C NMR (100 MHz, CDCl3): δ (ppm) 173.2 (-CO2-), 55.9, 55.7 (-NCH2CH2CH2N-), 52.9 (-CH2CH2CO2-), 51.7 (-OCH3), 42.4 (-CH2CH2CH2N(CH3)CH2CH2CH2-), 42.1 (-OCOCH2CH2N(CH3)CH2-), 32.5 (-CH2CO2-), 25.2 (-NCH2CH2CH2N-).

[0201] In an argon atmosphere, dimethyl 3,3'-(((methylazanediyl)bis(propane-3,1-diyl))bis(methylazanediyl)dipropionate (9.65 g, 27.9 mmol) was dissolved in dehydrated THF (35 mL), cooled to 0 °C, and stirred for 30 minutes. LiAlH4 (5.94 g, 189 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 48 hours. Then, the reaction was stopped by adding H2O (5.9 mL) at 0 °C. Subsequently, NaOH (15 wt%, 5.7 mL) was added to the solution, and the mixture was stirred until the gray solid turned white. After adding H2O (17.8 mL) to the solution, it was washed with methanol using celite. The reaction solution was dissolved in n-butanol, and LiOH, which is a by-product, was completely removed using ultracentrifugation (12,000 rpm, 20 minutes, 20 °C). Finally, n-butanol was removed by azeotropic distillation with toluene to obtain 3,3'-(((methylazanediyl)bis(propane-3,1-diyl))bis(methylazanediyl))bis(propane-1-ol) (AA09) as a yellow liquid (yield: 5.23 g, yield: 64.8%).

[0202] 1 H NMR (400 MHz, CDCl3): δ (ppm) 7.28 (s, 2H, -OH), 3.78 (t, J = 5.2 Hz, 4H, -CH2OH), 2.58 (t, J = 5.6 Hz, 4H, -CH2CH2CH2OH), 2.36 (dt, J = 19.3, 7.4 Hz, 8H, -NCH2CH2CH2N-), 2.25 (s, 6H, -CH2N(CH3)CH2CH2CH2OH), 2.21 (s, 3H, -NCH2CH2CH2N(CH3)CH2CH2CH2N-), 1.72 - 1.62 (m, 8H, -CH2CH2N(CH3)CH2CH2CH2OH)

[0203] 1313C NMR (100 MHz, CDCl3): δ (ppm) 64.6 (-CH2OH), 58.5 (-CH2CH2CH2OH), 56.4, 55.8 (-NCH2CH2CH2N-), 42.4 (-NCH2CH2CH2N(CH3)CH2CH2CH2N-), 42.1 (-CH2N(CH3)CH2CH2CH2OH), 27.8 (-CH2OH), 25.3 (-NCH2CH2CH2N-).

[0204] (12) Synthesis of Amino Alcohol AA06 Ethylenediamine anhydride (manufactured by TCI, >98.0%) (2.0 g, 33.3 mmol, 1 equivalent) was dissolved in dry methanol (21 mL), placed in a two-necked flask, and degassed at room temperature for 30 minutes by argon bubbling. Then, to prevent polymerization of methyl acrylate, the two-necked flask was covered with an aluminum film. Subsequently, methyl acrylate (15.0 mL, 166.4 mmol, 5 equivalents) was added dropwise to the solution in the two-necked flask. The resulting reaction mixture was stirred at room temperature for 2 days under an inert atmosphere. Thereafter, the solvent was removed from the reaction mixture under reduced pressure to obtain an ester. Subsequently, the generated ester (12.0 g, 29.7 mmol, 1 equivalent) was suspended in anhydrous THF (30 mL), and the reaction flask was cooled to 0 °C. The resulting suspension was stirred at 0 °C for 30 minutes under an argon atmosphere, and then LiAlH4 (6.3 g, 166.1 mmol, 5.6 equivalents) was added very slowly to the flask, and the resulting mixture was stirred at room temperature for 48 hours. Then, the reaction was quenched by adding water (6.3 mL) very slowly to the flask at 0 °C. Further, an NaOH solution (15 wt%, 6.14 mL) was added to the mixture in the flask, and the mixture was stirred until all the gray solids turned white. After adding water (18.9 mL) to the obtained solid, it was filtered through celite and washed with methanol. To completely remove LiOH, the obtained product was solubilized in butanol, and LiOH was removed by centrifugation (15000 rpm, 15 minutes at 20 °C), and then butanol was removed by co-evaporation with toluene to obtain amino alcohol AA06 as a yellow liquid (1.7 g, 5.8 mmol, yield: 17.4%).

[0205] (13) Synthesis of amino alcohol AA10 Using 3,3'-diaminodipropylamine (manufactured by TCI, >98.0%), amino alcohol AA10 was synthesized in the same manner as the synthesis of amino alcohol AA06.

[0206] 1 H-NMR (400 MHz, METHANOL-D4): δ 5.06 (s, 5H, -OH), 3.65 (td, J = 6.2, 3.0 Hz, 10H, -CH2OH), 2.61 (t, J = 7.3 Hz, 10H, -NCH2-), 2.51 (dt, J = 10.4, 4.2 Hz, 8H, -NCH2-), 1.68 - 1.77 (m, 14H, -CH2-).

[0207] (14) Synthesis of amino alcohol AA12 Using tris(2-aminoethyl)amine (manufactured by TCI, >98.0%), amino alcohol AA07 was synthesized in the same manner as the synthesis of amino alcohol AA06. The NMR spectrum of the synthesized AA07 was the same as that reported by Kowalski et al. (Non-Patent Document 1).

[0208] 1 H-NMR (500 MHz, CDCl3) δ 4.55 (bs, 8H), 3.68 (t, J = 5.7 Hz, 12H), 2.59 (m, 24H), 1.69 (m, 12H).

[0209] (15) Synthesis of amino alcohol AA16 Using homopiperazine (manufactured by TCI, >98.0%), amino alcohol AA16 was synthesized in the same manner as the synthesis of amino alcohol AA06.

[0210] 1H-NMR (400 MHz, METHANOL-D4): δ 5.11 (s, 2H, -OH), 3.61 (t, J = 6.2 Hz, 4H, -CH2OH), 2.72-2.75 (m,8H, -NCH2-), 2.59 (t, J = 7.5 Hz, 4H, -NCH2-), 1.82 (t, J = 5.9 Hz, 2H, -CH2-), 1.70 (dt, J = 14.6, 6.3 Hz, 4H, -CH2-).

[0211] <Synthesis of Amino Polyester> Using an amino alcohol as a polymerization initiator, a ring-opening polymerization reaction of a caprolactone derivative was carried out to synthesize an amino polyester.

[0212]

Chemical Structure

[0213] The polymerization conditions were appropriately optimized according to the monomers and polymerization initiators selected. Also, the polymerization was carried out by adjusting the molar ratio of the monomer and the polymerization initiator so that the degree of polymerization of the amino polyester was around 10 or around 20. Each amino polyester was characterized by NMR and size exclusion chromatography. The degree of polymerization of the amino polyester was calculated from NMR. The amino polyester was denoted as X-Y-Z based on the type of initiator (X), the type of monomer (Y), and the target degree of polymerization (Z).

[0214] The polymerization reaction using amino alcohol AA02 as the initiator and caprolactone derivative (G4) as the monomer was carried out as follows. In a glove box, TBD (1.70 mg, 12.0 μmol), a caprolactone derivative (G4) (200 mg, 1.17 mmol), and amino alcohol AA02 (4.90 mg, 41.1 μmol) were added to a test tube. After stirring at 80 °C for 0.2 hours, the reaction was stopped by adding an excess amount of chloroform. Then, the resulting reaction product was purified by preparative size exclusion chromatography to obtain poly(4-butyl-ε-caprolactone) (AA02-G4-20) as a colorless viscous substance.

[0215] Using amino alcohols AA04, AA05, AA06, AA07, AA08, AA10, AA12, AA14, AA16, and AA17 as initiators, and various caprolactone derivatives, polymerization was carried out in the same manner as the polymerization reaction using amino alcohol AA02 and caprolactone derivative (G4).

[0216] The polymerization reaction using amino alcohol AA11 as an initiator and caprolactone derivative (E4) as a monomer was carried out as follows. In a glove box, TBD (1.49 mg, 10.7 μmol), caprolactone derivative (E4) (200 mg, 1.17 mmol), amino alcohol AA11 (19.4 mg, 53.4 μmol), and CH2Cl2 (25 mL) were added to a test tube. After stirring at room temperature for 0.16 hours, the reaction was stopped by adding benzoic acid. Then, purification by preparative size exclusion chromatography gave poly(6-butyl-ε-caprolactone) (AA11-E4-20) as a colorless viscous substance.

[0217] When using amino alcohol AA09 as an initiator, polymerization was carried out in the same manner as the polymerization reaction using amino alcohol AA11 and caprolactone derivative (E4).

[0218] The properties of each synthesized aminopolyester are shown in Tables 1 to 26 together with the reaction conditions of the polymerization reaction. In the tables, "[lactone]0 / [initiator]0 / [catalyst]" is the molar ratio of the monomer (caprolactone derivative), initiator (amino alcohol), and catalyst (TBD) used in the polymerization reaction, "Temperature (°C)" is the temperature of the polymerization reaction, "Reaction time (h)" is the reaction time (hours) of the polymerization reaction, "Yield (%)" is the yield (%) of the polymerization reaction, and "Conv. (%)" is 1 the monomer conversion rate (%) calculated from the 1H NMR spectrum. "DP" is the degree of polymerization determined by the 1 1H NMR spectrum performed in CDCl3, and "M n,th " is the theoretical number average molecular weight calculated from the molar ratio of the monomer and the initiator and the monomer conversion rate ([M n,th =[molecular weight of amino alcohol]+[molecular weight of lactone]×[conv.(%)]×[number of moles of lactone] / [number of moles of amino alcohol]), and "M n,NMR " is 1 the number average molecular weight calculated based on the measurement results of 1H NMR, "M n,SEC " is the number average molecular weight calculated based on the measurement results of SEC, and "D" is the dispersity.

[0219]

Chemical formula

[0220]

Table 1

[0221]

Table 2

[0222]

Chemical formula

[0223]

Table 3

[0224]

Table 4

[0225]

Chem.

[0226]

Table 5

[0227]

Table 6

[0228]

Chem.

[0229]

Table 7

[0230]

Table 8

[0231]

Chem.

[0232]

Table 9

[0233]

Table 10

[0234] [Chemical formula]

[0235] [Table 11]

[0236] [Table 12]

[0237] [Chemical formula]

[0238] [Table 13]

[0239] [Table 14]

[0240] [Chemical formula]

[0241] [Table 15]

[0242] [Table 16]

[0243] [Chemical formula]

[0244] [Table 17]

[0245]

Table 18

[0246]

Chem.

[0247]

Table 19

[0248]

Table 20

[0249]

Chem.

[0250]

Table 21

[0251]

Table 22

[0252]

Chem.

[0253]

Table 23

[0254]

Table 24

[0255] [Chemical]

[0256] [Table 25]

[0257] [Table 26]

[0258] [Example 2] Using the aminopolyester synthesized in Example 1, lipid nanoparticles encapsulating mRNA were prepared.

[0259] As the constituent lipids, a lipid nanoparticle with a molar ratio of aminopolyester, cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2k) of 50 / 25 / 23.5 / 1.5 was encapsulated with firefly luciferase (Fluc) mRNA as the nucleic acid. For 10 μg of mRNA, the total lipid amount was 500 nmol.

[0260] Specifically, aminopolyester (APE) was dissolved in t-butyl alcohol to prepare a 5 mM stock solution (APE solution). The prepared APE solution (50.0 μL, 250 nmol), 5 mM cholesterol ethanol solution (24.0 μL, 118 nmol), 5 mM DOPE ethanol solution (25.0 μL, 125 nmol), 0.5 mM DMG-PEG 2k ethanol solution (1.50 μL, 7.50 nmol), and ethanol (140 μL) were mixed. While stirring the mixed lipid solution using a vortex mixer, a mixed solution of Fluc mRNA (10 μL) and acetate buffer (30 μL) was added dropwise. Subsequently, the obtained mixed solution of lipid and mRNA was added dropwise to acetate buffer (750 μL) under vortex mixer stirring. After standing for 10 minutes, PBS (2000 μL) was added and mixed to prepare luciferase-expressing lipid nanoparticles (Fluc-expressing lipid nanoparticles).

[0261] Also, for the polymer using ε-caprolactone as a monomer, it did not completely dissolve under the same conditions, so ethanol with the same volume as t-butyl alcohol was added to prepare a 2.5 mM stock solution. Thereafter, without changing the amount of substance and volume of each component, nanoparticles were prepared in the same procedure.

[0262] Also, as a control, lipid nanoparticles in which Fluc mRNA was similarly encapsulated in the commercially available transfection reagent lipid nanoparticles “In vivoJet PEI” (manufactured by Polyplus-transfection) were also prepared.

[0263] The characterization results of each lipid nanoparticle are shown in Tables 27 to 39. As an overall trend, the average particle diameter of the lipid nanoparticles was about 100 to 200 nm, and most of the lipid nanoparticles had a polydispersity index (PdI) of 0.15 or less. It was found that the lipid nanoparticles using caprolactone derivative (G8) as a monomer had a generally wide particle size distribution (PdI > 0.3).

[0264]

Table 27

[0265]

Table 28

[0266]

Table 29

[0267]

Table 30

[0268]

Table 31

[0269]

Table 32

[0270]

Table 33

[0271]

Table 34

[0272]

Table 35

[0273]

Table 36

[0274]

Table 37

[0275]

Table 38

[0276]

Table 39

[0277] [Example 3] The lipid nanoparticles for Fluc expression prepared in Example 2 were administered to mice, and delivery to major organs was examined. Specifically, ICR and C57BL / 6 mice (female, 4 - 6 weeks old) were administered lipid nanoparticles for Fluc expression by tail vein injection so that the dose of Fluc mRNA per kg body weight was 0.15 mg. 4 hours and 50 minutes after administration, each mouse was anesthetized with isoflurane, and 3 mg of d - luciferin (manufactured by GoldBio, 150 μL of a 20 mg / mL PBS solution) was administered by intraperitoneal injection. 10 minutes after administration under anesthesia, each mouse was euthanized, and the major organs, namely the lung, liver, spleen, kidney, and heart, were excised and placed on a black sheet. Then, the luminescence of Fluc was measured using an in vivo bioluminescence imaging system (IVIS Lumina LT Series III, manufactured by Perkin Elmer), and the average radiance (P / s / cm 2 / sr) of the luminescence of each organ was quantified and compared. The images obtained by the bioluminescence imaging system were processed with Living Image software v.4.3 (64 - bit, manufactured by Caliper Life Sciences). The average of two independent measured values was used as the luminescence intensity of each organ. For those with particularly excellent organ selectivity, the presence or absence of a significant difference due to the type of aminopolyester was confirmed by an independent t - test. The measurement results of the luminescence intensity of each tissue in the mice administered with each lipid nanoparticle for Fluc expression are shown in Tables 40 - 52.

[0278]

Table 40

[0279]

Table 41

[0280]

Table 42

[0281]

Table 43

[0282]

Table 44

[0283]

Table 45

[0284]

Table 46

[0285]

Table 47

[0286]

Table 48

[0287]

Table 49

[0288]

Table 50

[0289]

Table 51

[0290]

Table 52

[0291] As shown in Tables 40 to 52, most of these lipid nanoparticles for Fluc expression showed the highest gene expression in the spleen. More specifically, it is as follows. For the lipid nanoparticles for Fluc expression of the AA02-Y-Z series, spleen-specific gene expression was confirmed regardless of the monomer structure and degree of polymerization. Among them, AA02-G4-20 showed the highest gene expression in the spleen. For the lipid nanoparticles for Fluc expression of the AA04-Y-Z series, spleen-specific gene expression was confirmed except for AA04-A4-10. Among them, AA04-E6-20 showed the highest gene expression in the spleen. For the lipid nanoparticles for Fluc expression of the AA05-Y-Z series, spleen-specific gene expression was confirmed regardless of the monomer structure and degree of polymerization. Among them, AA05-A4-10 showed particularly high gene expression in the spleen. For the lipid nanoparticles for Fluc expression of the AA06-Y-Z series, high gene expression was confirmed in all spleens. In particular, AA06-A4-10, AA06-E6-20, AA06-G8-10, and AA06-G8-20 showed spleen-specific gene expression. AA06-G4-10 showed high gene expression not only in the spleen but also in the lung. Also, it was found that the degree of polymerization does not affect organ specificity and expression efficiency so much. For the lipid nanoparticles for Fluc expression of the AA07-Y-Z series, spleen-specific gene expression was confirmed regardless of the monomer structure and degree of polymerization. Among them, AA07-G4-10 and AA07-G4-20 showed particularly high gene expression in the spleen. For the lipid nanoparticles for Fluc expression in the AA08-Y-Z series, high gene expression was confirmed in the spleen regardless of the monomer structure and degree of polymerization. AA08-G4-20 showed high gene expression not only in the spleen but also in the lung. For the lipid nanoparticles for Fluc expression in the AA09-Y-Z series, comparable gene expression was confirmed in the spleen and lung, and differences in expression efficiency between the spleen and lung were observed depending on the monomer structure. For the lipid nanoparticles for Fluc expression in the AA10-Y-Z series, high gene expression was confirmed in the spleen regardless of the monomer structure and degree of polymerization. The AA10-A-Z series and AA10-E-Z series showed spleen-specific gene expression, while the AA10-G-Z series showed the highest gene expression in the lung in addition to the spleen. For the lipid nanoparticles for Fluc expression in the AA11-Y-Z series, gene expression specific to both the spleen and lung was confirmed, but no significant difference in expression efficiency was observed between the two organs. For the lipid nanoparticles for Fluc expression in the AA12-Y-Z series and AA14-Y-Z series, high gene expression was confirmed in the spleen regardless of the monomer structure and degree of polymerization. Among them, AA12-G4-20 in the AA12-Y-Z series and AA14-G4-10 in the AA14-Y-Z series showed the highest gene expression in the spleen. For the lipid nanoparticles for Fluc expression in the AA16-Y-Z series, high gene expression was confirmed in the spleen regardless of the monomer structure and degree of polymerization. AA16-A4-20 showed high gene expression in the lung as well. For the lipid nanoparticles for Fluc expression in the AA17-Y-Z series, high gene expression was confirmed in the spleen regardless of the monomer structure and degree of polymerization. Among them, AA17-A4-10 showed the highest gene expression in the spleen.

[0292] Among the lipid nanoparticles with high gene expression observed in the lung, AA06-G4-10, AA08-G4-20, AA09-A4-10, AA09-E4-20, AA09-G4-20, AA10-G4-10, AA10-G4-20, AA11-A4-20, and AA11-E4-20 all had gene expression efficiency superior to or comparable to that of the commercially available transfection reagent In vivoJet PEI, which is a carrier for lung-specific genes. From these results, it was confirmed that these lipid nanoparticles are suitable as gene carriers targeting particularly lung tissue.

[0293] The structures of both the initiator and the monomer affected organ specificity and expression efficiency, and the degree of polymerization did not seem to have much effect. In particular, it was confirmed that amino polyesters having an initiator structure with a large number of nitrogens tended to have higher expression efficiency.

[0294] As shown by these results, the lipid nanoparticles composed of the amino polyesters according to the present invention can efficiently encapsulate nucleic acids for gene expression, are efficiently taken up by spleen tissue and lung tissue, and in addition, are relatively safe. Therefore, the lipid nanoparticles are useful as carriers for delivering basic pharmaceutical ingredients for the treatment of spleen diseases and lung diseases to spleen tissue and lung tissue, and can particularly serve as active ingredients of pharmaceutical compositions for spleen diseases and lung diseases with few effective drugs, such as spleen cancer, splenomegaly, acute respiratory distress syndrome, lung cancer, and pulmonary hypertension.

Claims

1. The following general formula (P-1) 【Chemical Formula 1】 [In the formula, Z １ is an alkylene group having 1 to 6 carbon atoms; among the alkylene groups of said Z １ one hydrogen atom bonded to a carbon atom not bonded to either an oxygen atom or a nitrogen atom may be substituted with -O-[D １ n2-H; D １ is the following general formula (P) 【Chemical Formula 2】 (In the formula, the carbon atom of the carbonyl group is bonded to said Z １ via an oxygen atom; R １１ to R １３ one of these is an alkyl group having 1 to 12 carbon atoms and the rest are hydrogen atoms; R １４ is a hydrogen atom or a methyl group; Z ２ is a 1,4-cyclohexylene group; when R １１ is an alkyl group having 1 to 12 carbon atoms, n11 is 0 or 1, and when R １１ is a hydrogen atom, n11 is 0; when R １２ is an alkyl group having 1 to 12 carbon atoms, n12 is 0 or 1, and when R １２ is a hydrogen atom, n12 is 0; when R １３ is an alkyl group having 1 to 12 carbon atoms, n13 is 0 or 1, and when R １３ is a hydrogen atom, n13 is 0: the black circle represents a bond to another group) is a divalent group represented by; n1 is an integer of 1 or more; n2 is an integer of 0 or more: the black circle represents a bond to another group] An aminopolyester having a structure represented by.

2. The number of D １ present in one molecule is 5 to 200, the aminopolyester according to claim 1.

3. The following general formula (A-1) or (A-2) 【Chemical Formula 3】 [In the formula, R ０Ｈ is a group represented by the following general formula (H-1) or (H-2) [Chemical Formula 4] (wherein a1 is an integer of 1 to 6; a2 and a3 are 1 or more, and the sum of the two is an integer of 2 to 6; the black circle represents a bond with another group) and is a group represented by; R ａ is an alkyl group having 1 to 6 carbon atoms which may be substituted with a dialkylamino group, a 1-pyrrolidyl group, or a 1-imidazolyl group; R １ is a group represented by the following general formula (H-3) or (H-4) [Chemical Formula 5] (wherein R ０Ｈ and R ａ are the same as described above; c1 is an integer of 1 to 6; c2 is an integer of 1 to 6; the black circle represents a bond with another group) and is a group represented by; p is an integer of 0 or more and 3 or less, q is an integer of 0 or more and 3 or less, r is an integer of 0 or more and 2 or less, but p + q + r = 3; R ２ is R ０Ｈ or R １ ; R ３ is R ０Ｈ or R ａ or R １ ; A is a 5- to 7-membered ring having two nitrogen atoms]] Any of the amino alcohols represented by the formula, and the following general formula (La), (Lg), or (Le) [Chemical Formula 6] [In the formula, R １１ to R １３ are each independently an alkyl group having 1 to 12 carbon atoms; Z ２ is a 1,4-cyclohexylene group; when R １１ is an alkyl group having 1 to 12 carbon atoms, n11 is 0 or 1, and when R １１ is a hydrogen atom, n11 is 0; when R １２ is an alkyl group having 1 to 12 carbon atoms, n12 is 0 or 1, and when R １２ is a hydrogen atom, n12 is 0; when R １３ When it is an alkyl group having 1 to 12 carbon atoms, n13 is 0 or 1, and R １３ is a hydrogen atom, n13 is 0] The aminopolyester according to claim 1, which is obtained by ring-opening polymerization of a caprolactone derivative represented by

4. The amino alcohol is represented by the following general formulas (A-1-1) to (A-1-9) and (A-2-1) to (A-2-4) 【Chemical Formula 7】 [In the formula, R ０Ｈ , R ａ , and R １ are the same as those in the general formula (A-1)] The aminopolyester according to claim 3, which is an amino alcohol represented by any of

5. The amino alcohol is represented by the following formulas (AA01) to (AA17) 【Chemical Formula 8】 【Chemical Formula 9】 The aminopolyester according to claim 3, which is an amino alcohol represented by any of

6. The caprolactone derivative is represented by the following formula (E1), (E3), (E4), (E6), (E4sec), (G4), (G8), (A4), or (B1E3) 【Chemical Formula 10】 The aminopolyester according to claim 3, which is a caprolactone derivative represented by any of

7. The amino alcohol is represented by the following formulas (AA01) to (AA17) 【Chemical Formula 11】 【Chemical Formula 12】 The amino alcohol is an amino alcohol represented by any of the following, and the caprolactone derivative is represented by the following formula (E1), (E3), (E4), (E6), (E4sec), (G4), (G8), (A4), or (B1E3) 【Chemical Formula 13】 The aminopolyester according to claim 3, which is a caprolactone derivative represented by any one of them.

8. The following general formulas (P-1-1) to (P-1-9) and (P-2-1) to (P-2-4) 【Chemical Formula 14】 [In the formula, R ＯＰ is the above R ＯＨ or at least one hydroxy group in the above R ＯＨ is a group substituted with -O-[D １ n1-H; R １Ｐ is the above R １ or at least one hydroxy group in the above R １ is a group substituted with -O-[D １ n1-H; D １ and n1 are the same as those in the general formula (P-1); R ａ is the same as that in the general formula (A-1); in one molecule, it has at least 5 D １ The aminopolyester according to claim 3, which is represented by any one of them.

9. The following general formula (A-1) or (A-2) 【Chemical Formula 15】 [In the formula, R ０Ｈ is a group represented by the following general formula (H-1) or (H-2) 【Chemical Formula 16】 (In the formula, a1 is an integer from 1 to 6; a2 and a3 are 1 or more, and the sum of the two is an integer from 2 to 6; the black circle represents a bond with another group) and is a group represented by; R ａ is an alkyl group having 1 to 6 carbon atoms, which may be substituted with a dialkylamino group, a 1-pyrrolidyl group, or a 1-imidazolyl group; R １ is the following general formula (H-3) or (H-4) 【Chemical Formula 17】 (In the formula, R ０Ｈ and R ａ is the same as described above; c1 is an integer from 1 to 6; c2 is an integer from 1 to 6; the black circle represents a bond with another group) is a group represented by; p is an integer from 0 to 3, q is an integer from 0 to 3, r is an integer from 0 to 2, provided that p + q + r = 3; R ２ is R ０Ｈ or R １ ; R ３ is R ０Ｈ , R ａ , or R １ ; A is a 5- to 7-membered ring having two nitrogen atoms] with any of the amino alcohols represented by, and the following general formula (La), (Lg), or (Le) [Chemical Formula 18] [In the formula, R １１ to R １３ are each independently an alkyl group having 1 to 12 carbon atoms; Z ２ is a 1,4-cyclohexylene group; when R １１ is an alkyl group having 1 to 12 carbon atoms, n11 is 0 or 1, and when R １１ is a hydrogen atom, n11 is 0; when R １２ is an alkyl group having 1 to 12 carbon atoms, n12 is 0 or 1, and when R １２ is a hydrogen atom, n12 is 0; when R １３ is an alkyl group having 1 to 12 carbon atoms, n13 is 0 or 1, and when R １３ is a hydrogen atom, n13 is 0] to carry out ring-opening polymerization with a caprolactone derivative represented by, to produce an aminopolyester, a method for producing an aminopolyester.

10. Lipid nanoparticles containing the aminopolyester according to any one of Claims 1 to 8.

11. Further, the lipid nanoparticles according to Claim 10, containing a polyalkylene glycol-modified lipid.

12. The lipid nanoparticles according to Claim 10, containing a nucleic acid.

13. The lipid nanoparticle according to claim 12, wherein the nucleic acid is a gene to be expressed in parenchymal cells of spleen tissue or lung tissue.

14. A pharmaceutical composition comprising, as an active ingredient, a lipid nanoparticle containing the amino polyester according to any one of claims 1 to 8.

15. The pharmaceutical composition according to claim 14, which is used for the treatment of spleen diseases or lung diseases.

16. A method for expressing a foreign gene, comprising administering, to a test animal (excluding humans), a lipid nanoparticle containing the amino polyester according to any one of claims 1 to 8 and encapsulating a foreign gene for the purpose of expression in cells of spleen tissue or lung tissue, and expressing the foreign gene inside the spleen tissue or lung tissue of the test animal.

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