Polyion complex micelles

Abstract

A polyion complex micelle comprising a block copolymer having a hydrophilic block portion, a cationic hydrophobic block portion, and a crosslinking block portion located between the hydrophilic block and the cationic hydrophobic block, and an anionic molecular drug encapsulated in the block copolymer, wherein the crosslinking block has a hydrazone bond, the block copolymer comprises a first block copolymer chain and a second block copolymer chain, the first block copolymer chain and the second block copolymer chain are crosslinked to each other by the crosslinking block portion, the hydrophilic block portion comprises a first hydrophilic block of the first block copolymer chain and a second hydrophilic block of the second block copolymer chain, and the cationic hydrophobic block portion comprises a first cationic hydrophobic block of the first block copolymer chain.

Description

[Technical Field]

[0001] This invention relates to polyion complex micelles. This application claims priority under U.S. Provisional Patent Application No. 63 / 130,861, filed on 28 December 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Conventionally, polyion complex micelles (sometimes referred to as "PIC micelles") have been used for nucleic acids (e.g., pDNA, mRNA, siRNA, ASO) and large negatively charged molecules that have ionic interactions with cationic polymers (e.g., PEG-polylysine, PEG-poly(N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide). For example, Non-Patent Documents 1 and 2 describe polyion complex micelles comprising a poly(ethylene glycol)-poly(lysine) diblock copolymer and an anionic drug encapsulated in the diblock copolymer. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Wang C,Chen Q,Wang Z,Zhang X.An enzyme-responsive polymeric superamphiphile.Angew.Chemie-Int.Ed.49(46), 8612-8615(2010). [Non-Patent Document 2] HSMin, HJKim, M. Naito, S. Ogura, K. Toh, K. Hayashi, BSKim, S. Fukushima, Y. Anraku, K. Miyata, K. Kataoka. Systemic brain delivery of antisense oligonucleotides across the blood-brain barrier with a glucose-installed polymeric nanocarrier.Angew.Chem.Int.Ed.59(21),8173-8180(2020) [Overview of the project] [Problems that the invention aims to solve]

[0004] In conventional polyion complex micelles, such as those described in Non-Patent Document 1, the formation of polyion complex micelles with a hydrophobic inner core and a PEG outer layer is triggered by simple ionic interactions between the drug and the polymer. However, conventional polyion complex micelles readily disintegrate under physiological conditions because the ionic interactions between the polymer and the drug are disrupted. Furthermore, free polymers cause non-selective cytotoxicity when released. Therefore, encapsulation has been difficult because negatively charged small molecules, being water-soluble, easily leak out of the delivery system, leading to premature release.

[0005] The present invention has been made in consideration of the above circumstances, and its objective is to provide a polyion complex micelle that enables the stable encapsulation of negatively charged molecules. [Means for solving the problem]

[0006] To solve the above problems, the present invention employs the following configuration. (1) A block copolymer having a hydrophilic block portion, a cationic hydrophobic block portion, and a crosslinked block portion located between the hydrophilic block portion and the cationic hydrophobic block portion, Anionic molecular drugs encapsulated in block copolymers and A polyion complex micelle containing, The cross-linked block portion has a hydrazone bond, The block copolymer comprises a first block copolymer chain and a second block copolymer chain, and the first block copolymer chain and the second block copolymer chain are crosslinked with each other at the crosslinked block portion. The hydrophilic block portion includes a first hydrophilic block of the first block copolymer chain and a second hydrophilic block of the second block copolymer chain. A polyion complex micelle comprising a cationic hydrophobic block portion containing a first cationic hydrophobic block of a first block copolymer chain. (2) The polyion complex micelle according to (1) above, wherein the cationic hydrophobic block portion further comprises a second cationic hydrophobic block of a second block copolymer chain. (3) The block copolymer is of formula (I)

[0007] [ka] (In the formula, A represents a repeating unit constituting the first hydrophilic block or the second hydrophilic block; B represents a repeating unit constituting the first cationic hydrophobic block or the second cationic hydrophobic block; m represents 1 or 2; L) 1 R represents a divalent linking group. 1 R represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group. 2 L represents a hydrogen atom or a methyl group. 2 (where n represents a single bond or a divalent linking group, and n represents 1 or 2) A polyion complex micelle as described in (2) above. (4) The block copolymer is of formula (II)

[0008] [ka] (In the formula, A represents a repeating unit constituting the first hydrophilic block or the second hydrophilic block; B represents a repeating unit constituting the first cationic hydrophobic block; m represents 1 or 2; L) 1 R represents a divalent linking group. 1 R represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group. 2 L represents a hydrogen atom or a methyl group. 2 (where n represents a single bond or a divalent linking group, and n represents 1 or 2) A polyion complex micelle as described in (1) above. (5) A polyion complex micelle according to any one of (1) to (4) above, wherein the first cationic hydrophobic block is composed of a repeating structure derived from polylysine. (6) The polyion complex micelle according to (2) or (3) above, wherein the second cationic hydrophobic block is composed of a repeating structure derived from polylysine. (7) A polyion complex micelle according to any one of (1) to (6) above, having a particle size of 20 to 100 nm and a polydispersity index of 0.05 to 0.3. (8) A polyion complex micelle according to any one of (1) to (7) above, wherein the net negative charge of the anionic molecular drug is -25 to -1 at physiological pH. (9) A polyion complex micelle according to any one of (1) to (8) above, wherein the anionic molecular drug is a nucleic acid drug. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide polyion complex micelles capable of stably encapsulating negatively charged molecules. In particular, the present invention can provide polyion complex micelles useful for encapsulating negatively charged small molecules. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1 is a schematic diagram showing one embodiment of the polyion complex micelle of the present invention. [Figure 2] Figure 2 is a schematic diagram showing one embodiment of the polyion complex micelle of the present invention. [Figure 3] Figure 3 shows the results of a cell uptake experiment using RPMI2650. [Figure 4] Figure 4 shows the results of a cell uptake experiment using RPMI2650. [Figure 5] Figure 5 shows the timeline of RPMI2650 culture for the Transwell permeability experiment. [Figure 6] Figure 6 shows the results of the Transwell permeability experiment. [Figure 7] Figure 7 is a schematic diagram showing non-polyion complex micelles prepared for cell uptake experiments using brain cells. [Figure 8] Figure 8 shows the results of a cell uptake experiment using KT-5 (astrocytic cells). [Figure 9] Figure 9 shows the results of a cell uptake experiment using BV-2 (microglia). [Figure 10] Figure 10 shows the results of a cell uptake experiment using GT1-7-5 (neuronal cell). [Figure 11] Figure 11 shows the results of a cell uptake experiment using primary rat brain endothelial cells. [Modes for carrying out the invention]

[0011] <Polyion complex micelles> The polyion complex micelle according to this embodiment comprises a block copolymer having a hydrophilic block portion, a cationic hydrophobic block portion, and a crosslinked block portion located between the hydrophilic block and the cationic hydrophobic block, and an anionic molecular drug encapsulated in the block copolymer. The crosslinked block portion has a hydrazone bond. The block copolymer comprises a first block copolymer chain and a second block copolymer chain. The first block copolymer chain and the second block copolymer chain are crosslinked with each other at the crosslinked block portion. The hydrophilic block portion comprises a first hydrophilic block of the first block copolymer chain and a second hydrophilic block of the second block copolymer chain. The cationic hydrophobic block portion comprises a first cationic hydrophobic block of the first block copolymer chain.

[0012] First embodiment: Figure 1 is a schematic diagram showing one embodiment of the polyion complex micelle of the present invention. As shown in Figure 1, the polyion complex micelle 1 is formed by the self-assembly of a block copolymer 2 and an anionic molecular drug 3. Specifically, a core B1 on which the anionic molecular drug 3 is supported is formed by ionic interactions between a cationic hydrophobic block portion B and the anionic molecular drug 3. A crosslinked block portion C surrounds the core B1 and stabilizes the core B1. The hydrophilic block portion A forms a shell, thereby forming the polyion complex micelle 1.

[0013] Because the cross-linking block C surrounds core B1, it can prevent the leakage of anionic molecular drug 3 unless there is a physiological trigger such as a decrease in the pH of intracellular endosomes.

[0014] Block copolymer 2 is composed of a first block copolymer chain 4 and a second block copolymer chain 5. The first block copolymer chain 4 includes a first hydrophilic block 4A, a first crosslinked block 4C, and a first cationic hydrophobic block 4B in that order. The second block copolymer chain 5 includes a second hydrophilic block 5A, a second crosslinked block 5C, and a second cationic hydrophobic block 5B in that order. Block copolymer 2 is formed by crosslinking between the first crosslinked block 4C of the first block copolymer chain 4 and the second crosslinked block 5C of the second block copolymer chain 5. Hydrophilic block portion A includes the first hydrophilic block 4A and the second hydrophilic block 5A. Hydrophobic block portion B includes the first hydrophobic block 4B and the second hydrophobic block 5B.

[0015] The particle size of the polyion complex micelles according to this embodiment is preferably 20 nm to 100 nm, and more preferably 35 nm to 50 nm. Furthermore, the polyion complex micelles preferably have a polydispersity index of 0.05 to 0.3, and more preferably 0.05 to 0.1.

[0016] (Block copolymer) In block copolymers, the "hydrophilicity" and "hydrophobicity" of hydrophilic and hydrophobic blocks are relative. The "hydrophilicity" and "hydrophobicity" of hydrophilic and hydrophobic blocks are sometimes defined by the logP value. The logP value is the logarithm of the octanol / water partition coefficient (Pow) and is a useful parameter for characterizing the hydrophilicity / hydrophobicity of a wide range of compounds. In other words, a logP value greater than 0, increasing towards the positive side, indicates higher hydrophobicity, while an increase towards the negative side indicates higher hydrophilicity.

[0017] Each of the first hydrophilic block and the first hydrophobic block may have one type of repeating unit or two or more types of repeating units. Each of the second hydrophilic block and the second hydrophobic block may have one type of repeating unit or two or more types of repeating units. Hereinafter, the first hydrophilic block and the second hydrophilic block may be collectively referred to as "hydrophilic blocks," and the first hydrophobic block and the second hydrophobic block may be collectively referred to as "hydrophobic blocks."

[0018] The number and molecular weight of the repeating units of the hydrophilic block can be appropriately controlled according to the molecular weight of the anionic molecular drug. The number of repeating units of the hydrophilic block may be, for example, 1 or more, 5 or more, 10 or more, 20 or more, or 45 or more. Furthermore, the number of repeating units of the hydrophilic block may be, for example, 1000 or less, 700 or less, or 450 or less. The molecular weight of the hydrophilic block may be, for example, 1,000 Da or more, 2,000 Da or more, or 5,000 Da or more. The molecular weight of the hydrophilic block may be, for example, 40,000 Da or less, 30,000 Da or less, or 20,000 Da or less.

[0019] The number and molecular weight of the repeating units of the hydrophobic block can be appropriately controlled depending on the molecular weight of the anionic molecular drug. The number of repeating units in the hydrophobic block may be, for example, 5 or more, 10 or more, or 20 or more. The number of repeating units in the hydrophobic block may be, for example, 1000 or less, 800 or less, 600 or less, 500 or less, 300 or less, 200 or less, 100 or less, or 60 or less. The molecular weight of the hydrophobic block may be, for example, 1,000 Da or more, 2,000 Da or more, 3,000 Da or more, or 5,000 Da or more. The molecular weight of the hydrophobic block may be, for example, 50,000 Da or less, 30,000 Da or less, 16,000 Da or less, or 10,000 Da or less.

[0020] Specific examples of hydrophilic blocks include blocks having at least one repeating unit selected from the group consisting of repeating units derived from polyethylene glycol, poly(ethylethylene phosphate), polyvinyl alcohol, polyvinylpyrrolidone, poly(oxazoline), and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA). Among these examples, blocks having repeating units derived from polyethylene glycol are preferred as hydrophilic blocks.

[0021] Examples of hydrophobic blocks include blocks having at least one repeating unit selected from the group consisting of repeating units derived from amino acids and their derivatives, preferably repeating units derived from polyamino acids and their derivatives. Examples of polyamino acids include polylysine, polyornithine, poly(2,6-diaminoheptanoic acid), poly(2,8-diaminooctanoic acid), poly(2,9-diaminononanoic acid), polyarginine, and polyhistidine. Examples of amino acid derivatives include poly[N-(2-aminoethyl)aspartamide](PAsp-(EDA)), poly{N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide}(PAsp(DET)), poly(N-{N'-[N”-(2-aminoethyl)-2-aminoethyl]-2-aminoethyl}aspartamide)(PAsp(TET)), and poly[N-(N'-{N”-[N'''-(2-aminoethyl)-2-aminoethyl]-2-aminoethyl}-2-aminoethyl)aspartamide](PAsp(TEP)). Among these examples, the hydrophobic block is preferably composed of a repeating structure derived from polylysine.

[0022] The first hydrophilic block of the first block copolymer chain and the second hydrophilic block of the second block copolymer chain may be the same or different. The number of repeating units in the first hydrophilic block of the first block copolymer chain and the number of repeating units in the second hydrophilic block of the second block copolymer chain may be the same or different. The first hydrophobic block of the first block copolymer chain and the second hydrophobic block of the second block copolymer chain may be the same or different. The number of repeating units in the first hydrophobic block of the first block copolymer chain and the number of repeating units in the second hydrophobic block of the second block copolymer chain may be the same or different.

[0023] The crosslinked block portion is not particularly limited as long as it has a hydrazone bond. The crosslinked block portion may consist of a first crosslinked block and a second crosslinked block that are crosslinked with each other. The linking group that crosslinks the first crosslinked block and the second crosslinked block has a hydrazone bond. Hereinafter, the first crosslinked block and the second crosslinked block may be collectively referred to as the "crosslinked block". Typically, the crosslinked block portion has repeating units in which repeating units of a first crosslinked block and repeating units of a second crosslinked block are linked by hydrazone bonds. Examples of amino acids and their derivatives include aspartic acid, glutamic acid, lysine, ornithine, benzyl aspartic acid, benzyl glutamic acid, and their derivatives. The crosslinked block may be composed of polyamino acids or their derivatives, such as polyaspartic acid, polyglutamic acid, polylysine, polyornithine, poly(benzyl aspartic acid), and poly(benzyl glutamic acid).

[0024] Hereinafter, a repeating unit in which a repeating unit of the first crosslinking block and a repeating unit of the second crosslinking block are linked by a hydrazone bond may be referred to as "repeating unit (c1)". A repeating unit of the first crosslinking block crosslinked to a repeating unit of the second crosslinking block may be referred to as "repeating unit (c1-1)". A repeating unit of the second crosslinking block crosslinked to a repeating unit of the first crosslinking block may be referred to as "repeating unit (c1-2)". In one embodiment, the repeating unit (c1) is preferably represented by the formula (c1). The crosslinking block portion may have one type of repeating unit (c1) or may have two or more types of repeating units (c1).

[0025]

Chemical formula

[0026] L 1 、R 1 、R 2 、L 2 、m and n are the same as those defined for L 1 、R 1 、R 2 、L 2 、m and n in the following formula (I).

[0027] The first crosslinked block may have repeating units (c1-1) and uncrosslinked repeating units (c2-1) of the second crosslinked block. The second crosslinked block may have repeating units (c1-2) and uncrosslinked repeating units (c2-2) of the first crosslinked block. Repeating units (c2-1) and (c2-2) may be derived from amino acids or their derivatives. Examples of amino acids and their derivatives are the same as those described above. The first crosslinked block may have one type of repeating unit (c2-1) or two or more types of repeating units (c2-1). The second crosslinked block may have one type of repeating unit (c2-2) or two or more types of repeating units (c2-2). The ratio of repeating units (c1-1) to all repeating units constituting the first crosslinked block may be 30 mol% or more, 40 mol% or more, 50 mol% or more, or 60 mol% or more. The ratio of repeating units (c1-2) to all repeating units constituting the second cross-linking block may be 30 mol% or more, 40 mol% or more, 50 mol% or more, or 60 mol% or more.

[0028] In this embodiment, the block copolymer is preferably represented by formula (I).

[0029] [ka] In the above equation, A represents a repeating unit constituting the first hydrophilic block or the second hydrophilic block; B represents a repeating unit constituting the first cationic hydrophobic block or the second cationic hydrophobic block; m represents 1 or 2; L 1 R represents a divalent linking group. 1 R represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group. 2 L represents a hydrogen atom or a methyl group. 2 represents a single bond or a divalent linking group, and n represents 1 or 2.

[0030] In formula (I), A represents a repeating unit that constitutes a hydrophilic block, and the same repeating unit as described above can be used for the hydrophilic block. In formula (I), B represents a repeating unit that constitutes a cationic hydrophobic block, and the same repeating units as described above can be used for the hydrophobic block. m represents 1 or 2, preferably 1. n represents 1 or 2, preferably 1.

[0031] In formula (I), L 1 represents a divalent linking group. The divalent linking group is not particularly limited, but preferred examples include divalent hydrocarbon groups which may have substituents, and divalent linking groups which contain heteroatoms.

[0032] L 1 If is a divalent linking group which may have substituents, the hydrocarbon group may be either an aliphatic hydrocarbon group or an aromatic hydrocarbon group.

[0033] L 1 Examples of aliphatic hydrocarbon groups include linear or branched aliphatic hydrocarbon groups, and aliphatic hydrocarbon groups that contain a ring in their structure. The linear or branched aliphatic hydrocarbon group preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, and most preferably 1 to 3 carbon atoms. A linear alkylene group is preferred as the linear aliphatic hydrocarbon group. Specific examples include the methylene group [-CH2-], ethylene group [-(CH2)2-], trimethylene group [-(CH2)3-], tetramethylene group [-(CH2)4-], and pentamethylene group [-(CH2)5-]. Preferred branched aliphatic hydrocarbon groups are branched alkylene groups, and specific examples include alkylmethylene groups such as -CH(CH3)-, -CH(CH2CH3)-, -C(CH3)2-, -C(CH3)(CH2CH3)-, -C(CH3)(CH2CH2CH3)-, and -C(CH2CH3)2-; alkylethylene groups such as -CH(CH3)CH2-, -CH(CH3)CH(CH3)-, -C(CH3)2CH2-, -CH(CH2CH3)CH2-, and -C(CH2CH3)2-CH2-; alkyltrimethylene groups such as -CH(CH3)CH2CH2- and -CH2CH(CH3)CH2-; and various alkylalkylene groups, including alkyltetramethylene groups such as -CH(CH3)CH2CH2CH2- and -CH2CH(CH3)CH2CH2-. A linear alkyl group having 1 to 5 carbon atoms is preferred as the alkyl group within the alkylalkylene group. Linear or branched aliphatic hydrocarbon groups may or may not have substituents. Examples of substituents include fluorine atoms, fluorinated alkyl groups having 1 to 5 carbon atoms, and carbonyl groups.

[0034] L 1 Examples of hydrocarbon groups containing a ring in their structure include, for example, a cyclic aliphatic hydrocarbon group that contains a heteroatom in its ring structure and may have substituents (groups from which two hydrogen atoms have been removed from an aliphatic hydrocarbon ring), a group in which a cyclic aliphatic hydrocarbon group is bonded to the end of the above-mentioned linear aliphatic hydrocarbon group, and a group in which a cyclic aliphatic group is interposed within the above-mentioned linear or branched aliphatic hydrocarbon group. The same groups as described above can be used as linear or branched aliphatic hydrocarbon groups. The cyclic aliphatic hydrocarbon group preferably has 3 to 20 carbon atoms, more preferably 3 to 12 carbon atoms. The cyclic aliphatic hydrocarbon group may or may not have substituents. Examples of substituents include alkyl groups, alkoxy groups, halogen atoms, alkyl halides, hydroxyl groups, and carbonyl groups.

[0035] L 1The aromatic hydrocarbon group is a hydrocarbon group having at least one aromatic ring. The aromatic ring is not particularly limited as long as it is a cyclic conjugated compound having (4n+2)π electrons, and may be monocyclic or polycyclic. The aromatic ring preferably has 5 to 30 carbon atoms, more preferably 5 to 20 carbon atoms, even more preferably 6 to 15 carbon atoms, and most preferably 6 to 12 carbon atoms. Here, the number of carbon atoms in one or more substituents is not included in the number of carbon atoms of the aromatic hydrocarbon group. Examples of aromatic rings include aromatic hydrocarbon rings such as benzene, naphthalene, anthracene, and phenanthrene; and aromatic heterocycles in which some of the carbon atoms constituting the above aromatic hydrocarbon rings are substituted with heteroatoms. Examples of heteroatoms in aromatic heterocycles include oxygen atoms, sulfur atoms, and nitrogen atoms. Specific examples of aromatic heterocycles include pyridine rings and thiophene rings. Specific examples of aromatic hydrocarbon groups include groups (or heteroarylene groups) obtained by removing two hydrogen atoms from the above-mentioned aromatic hydrocarbon ring or aromatic heterocycle; groups obtained by removing two hydrogen atoms from aromatic compounds having two or more aromatic rings (such as biphenyl and fluorene); and groups in which one hydrogen atom of the above-mentioned aromatic hydrocarbon ring or aromatic heterocycle is substituted with an alkylene group (such as the above-mentioned arylalkyl groups such as benzyl, phenethyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-naphthylethyl, or 2-naphthylethyl groups, or groups obtained by removing one hydrogen atom from the aryl group in a heteroarylalkyl group). The alkylene group bonded to the above-mentioned aryl group or heteroaryl group preferably has 1 to 4 carbon atoms, more preferably 1 or 2 carbon atoms, and most preferably 1 carbon atom. L 1 With respect to the aromatic hydrocarbon group, the hydrogen atoms within the aromatic hydrocarbon group may be substituted with substituents. For example, the hydrogen atoms bonded to the aromatic ring within the aromatic hydrocarbon group may be substituted with substituents. Examples of substituents include alkyl groups, alkoxy groups, halogen atoms, alkyl halides, and hydroxyl groups.

[0036] L 1 When represents a divalent linking group containing a heteroatom, preferred examples of linking groups include -O-, -C(=O)-O-, -C(=O)-, -OC(=O)-O-, -C(=O)-NH-, -NH-, -NH-C(=NH)- (which may be substituted with substituents such as alkyl or acyl groups), -S-, -S(=O)2-, -S(=O)2-O-, and the general formula: -Y 21 -OY 22 -, -Y 21 -O-, -Y 21 -C(=O)-O-, -C(=O)-OY 21 -,-[Y 21 -C(=O)-O] m” -Y 22 -, -Y 21 -OC(=O)-Y 22 - or -Y 21 -S(=O)2-OY 22 -[where, Y 21 and Y 22 Examples of groups represented by [where each independently represents a divalent hydrocarbon group which may have substituents, O represents an oxygen atom, and m' represents an integer from 0 to 3] include groups represented by [where each independently represents a divalent hydrocarbon group which may have substituents, O represents an oxygen atom, and m' represents an integer from 0 to 3]. When the divalent linking group containing a heteroatom is -C(=O)-NH-, -C(=O)-NH-C(=O)-, -NH-, or -NH-C(=NH)-, H may be substituted with substituents such as alkyl groups or acyl groups. The substituent (alkyl group, acyl group, etc.) preferably has 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and most preferably 1 to 5 carbon atoms. General formula-Y 21 -OY 22 -, -Y 21 -O-, -Y 21 -C(=O)-O-, -C(=O)-OY 21 -,-[Y 21 -C(=O)-O] m” -Y 22 -, -Y 21 -OC(=O)-Y 22 - or -Y 21 -S(=O)2-OY 22 -In Y 21 and Y 22Each of these independently represents a divalent hydrocarbon group that may have substituents. Examples of divalent hydrocarbon groups include the same groups described above as "divalent hydrocarbon groups that may have substituents" in the explanation of divalent linking groups. Y 21 The preferred group is a linear aliphatic hydrocarbon group, more preferably a linear alkylene group, and even more preferably a linear alkylene group having 1 to 5 carbon atoms, with methylene or ethylene groups being particularly desirable. Y 22 The group is preferably a linear or branched aliphatic hydrocarbon group, more preferably a methylene group, an ethylene group, or an alkylmethylene group. The alkyl group within the alkylmethylene group is preferably a linear alkyl group having 1 to 5 carbon atoms, more preferably a linear alkyl group having 1 to 3 carbon atoms, and most preferably a methyl group. Formula - [Y 21 -C(=O)-O] m” -Y 22 In the base represented by -, m'' represents an integer between 0 and 3, preferably between 0 and 2, more preferably 0 or 1, and most preferably 1. That is, formula -[Y 21 -C(=O)-O] m” -Y 22 The base represented by - is given by formula -Y 21 -C(=O)-OY 22 It is particularly desirable that the group be represented by -(CH2) a’ -C(=O)-O-(CH2) b’ A base represented by - is preferred. In the formula, a' is an integer from 1 to 10, preferably an integer from 1 to 8, more preferably an integer from 1 to 5, even more preferably 1 or 2, and most preferably 1. b' is an integer from 1 to 10, preferably an integer from 1 to 8, more preferably an integer from 1 to 5, even more preferably 1 or 2, and most preferably 1.

[0037] In formula (I), L 1 This is preferably a divalent linear or branched hydrocarbon group or a divalent aromatic hydrocarbon group, more preferably a group from which one hydrogen atom has been removed from the benzyl group.

[0038] In formula (I), R 1 represents a hydrogen atom, an aliphatic hydrocarbon group or an aromatic hydrocarbon group. Examples of the aliphatic hydrocarbon group as R 1 include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group. R 1 The aliphatic hydrocarbon group as R may have a substituent. Examples of the substituent include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a tert-pentyl group, a cyclohexyl group, and a trihalomethyl group. Examples of the aromatic hydrocarbon group as R 1 include a phenyl group, a benzyl group, a pyridyl group, a naphthyl group, a hydroxyphenyl group, a methoxyphenyl group, an ethoxyphenyl group, a xylyl group, a methylphenyl group, a nitrophenyl group, a chlorophenyl group, a fluorophenyl group, an iodophenyl group, and a bromophenyl group. Among these, R 1 is preferably a hydrogen atom or an aliphatic hydrocarbon group, more preferably a hydrogen atom or a methyl group.

[0039] In formula (I), examples of the divalent linking group of L 2 include the same groups as those described above for L 1 . Further, the divalent linking group of L 2 may be a group represented by the formula -L R -NH-N=C(R 11 )-L 21 -NH-. In the formula, R 11 and L 21 are the same as those defined above for R 1 and L 1 in formula (I), respectively. L R represents the divalent residue of a linker. Examples of the linker include a dihydrazide linker, a disulfide linker, an acetal linker, and a ketal linker. Among the above examples, L 2 is a single bond or the formula -L R -NH-N=C(R11 )-L 21 A group represented by -NH- is preferred, and a single bond is more preferred.

[0040] (Anionic molecular drugs) As used herein, “anionic molecular drug” refers to a drug molecule having a net negative charge. Anionic molecular drugs may be small molecule drugs, medium molecule drugs, macromolecule drugs, or nucleic acid drugs. Examples of nucleic acid drugs include, but are not limited to, antisense nucleic acids, small interfering nucleic acids (e.g., siRNA), miRNA, mRNA, and plasmid DNA. Anionic molecular drugs preferably have a molecular weight of 20,000 Da or less. The molecular weight of anionic molecular drugs may be 15,000 Da or less, 10,000 Da or less, 8,000 Da or less, 5,000 Da or less, 3,000 Da or less, 2,000 Da or less, or 1,000 Da or less. More specifically, small molecule drugs may have a molecular weight of 1,000 Da or less. Nucleic acid drugs may have a molecular weight of 20,000 Da or less. Anionic molecular drugs preferably have a net negative charge of -25 to -1 at physiological pH. Physiological pH may be pH 6.5 to 8, preferably pH 7 to 7.5. In one embodiment, physiological pH may be approximately pH 7.4. Examples of anionic molecular drugs include cytarabine triphosphate, gemcitabine triphosphate, fludarabine triphosphate, cladribine triphosphate, capecitabine triphosphate, troxacitabine triphosphate, clofarabine triphosphate, combretastatin A1 diphosphate, adenosine triphosphate, cyclic guanosine monophosphate-adenosine monophosphate, cyclic diguanosine monophosphate, palmitoyl coenzyme A, and malonyl coenzyme A. Among these examples, cytarabine triphosphate and gemcitabine triphosphate are preferred. Specific examples of nucleic acid drugs, though limited, include luciferase ASO, homivirsen, mipomersen, defibrotide, eteplirsen, pegaptinib, and nusinersen. Among these examples, luciferase ASO is preferred.

[0041] Second embodiment: Figure 2 is a schematic diagram showing another embodiment of the polyion complex micelle of the present invention. As shown in Figure 2, the polyion complex micelle 1' is formed by the self-assembly of a block copolymer 2' and an anionic molecular drug 3. Specifically, ionic interactions between the cationic hydrophobic block portion B and the anionic molecular drug 3 form a core B2 on which the anionic molecular drug 3 is supported. The crosslinked block portion C surrounds the core B2 and stabilizes it. The hydrophilic block portion A forms a shell, thereby forming the polyion complex micelle 1'.

[0042] Because the cross-linking block portion C surrounds core B2, it can prevent the leakage of anionic molecular drug 3 unless there is a physiological trigger such as a decrease in the pH of intracellular endosomes.

[0043] Block copolymer 2' is composed of a first block copolymer chain 4 and a second block copolymer chain 5'. The first block copolymer chain 4 includes a first hydrophilic block 4A, a first crosslinked block 4C, and a first cationic hydrophobic block 4B in that order. The second block copolymer chain 5' includes a second hydrophilic block 5A and a second crosslinked block 5C. Block copolymer 2' is formed by crosslinking between the crosslinked block 4C of the first block copolymer chain 4 and the crosslinked block 5C of the second block copolymer chain 5'. Hydrophilic block portion A includes the first hydrophilic block 4A and the first hydrophilic block 5A. Hydrophobic block portion B includes the first hydrophobic block 4B.

[0044] The particle size of the polyion complex micelles according to this embodiment is preferably 20 nm to 100 nm, and more preferably 35 nm to 50 nm. Furthermore, the polyion complex micelles preferably have a polydispersity index of 0.05 to 0.3, and more preferably 0.05 to 0.15.

[0045] (Block copolymer) The hydrophilic block portion, the first hydrophilic block, and the second hydrophilic block are the same as those described above. The crosslinked block portion, the first crosslinked block, and the second crosslinked block are the same as those described above. In the present embodiment, the second block copolymer chain does not include a hydrophobic block. The hydrophobic block portion may be composed of the first hydrophobic block of the first block copolymer chain. The first hydrophobic block is the same as the first hydrophobic block of the first embodiment.

[0046] The first hydrophilic block of the first block copolymer chain and the second hydrophilic block of the second block copolymer chain may be the same or different. The number of repeating units of the first hydrophilic block of the first block copolymer chain and the number of repeating units of the second hydrophilic block of the second block copolymer chain may be the same or different.

[0047] In the present embodiment, the block copolymer is preferably represented by the formula (II).

[0048] [Chemical formula] In the above formula, A represents a repeating unit constituting the first hydrophilic block or the second hydrophilic block; B represents a repeating unit constituting the first cationic hydrophobic block: m represents 1 or 2: L 1 represents a divalent linking group: R 1 represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group: R 2 represents a hydrogen atom or a methyl group: L 2 represents a single bond or a divalent linking group: n represents 1 or 2.

[0049] A, B, m, L 1 R 1 R 2 L 2 and n are respectively A, B, m, L of the formula (I) 1 R 1, R 2 , L 2 This is the same as the definition for n.

[0050] The terminal groups of the second crosslinking block are not particularly limited. Examples of terminal groups of the second crosslinking block include, but are not limited to, a hydrogen atom, an acyl group having 1 to 5 carbon atoms (e.g., an acetyl group), an amino group, an alkyl group having 1 to 5 carbon atoms (e.g., a methyl group), and an alkoxy group having 1 to 5 carbon atoms (e.g., a methoxy group).

[0051] (Anionic molecular drugs) The anionic molecular drug is the same as the anionic molecular drug in the first embodiment described above.

[0052] <Method for preparing polyion complex micelles> First embodiment: The polyion complex micelle according to the first embodiment can be prepared by reacting a compound (Ia-1) represented by formula (Ia-1) with a compound (Ia-2) represented by formula (Ia-2) to obtain a block copolymer represented by formula (I), and then self-assembling this block copolymer together with an anionic molecular drug.

[0053] [ka] In the above equation, A, B, and m are the same as those defined for A, B, and m in equation (I); Ra 11 represents a hydrogen atom; La 1 represents an alkylene group, an arylene group, or an aralkylene group, however, La 1 It may have a hydrazide group or a hydrazine group and an inert substituent; Ra 12 represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group; Ra 21 represents a hydrogen atom or a methyl group; Ra 22 and Ra 23 This represents a hydrogen atom.

[0054] In formula (Ia-1), Ra 11 This represents a hydrogen atom. In formula (Ia-1), Ra 12 R in equation (I) 1 This is the same as the definition given for [the subject]. La 1 As an alkylene group, arylene group, or aralkylene group, the L of formula (I) 1 The divalent linking group may be the same alkylene group, arylene group, or aralkylene group as described above.

[0055] In formula (Ia-2), Ra 21 represents a hydrogen atom or a methyl group, preferably a hydrogen atom.

[0056] An example of a reaction scheme for producing polyion complex micelles according to this embodiment is shown below.

[0057] [ka]

[0058] Alternatively, L in equation (I) 2 is formula -L R -NH-N=C(R 1 )-L 1 If the group is represented by -NH-, the block copolymer represented by formula (Ib) may be crosslinked with a linker to obtain the block copolymer represented by formula (I).

[0059] [ka] In the above equation, A, B, and m are the same as those defined for A, B, and m in equation (I); Lb 1 , Rb 11 and Rb 12 These are La in equation (Ia-1), respectively. 1 Ra 11 and Ra 12 This is the same as the definition given for [the subject].

[0060] An example of an alternative reaction scheme for producing polyion complex micelles according to this embodiment is shown below.

[0061] [ka]

[0062] Second embodiment: The polyion complex micelle according to the second embodiment can be prepared by reacting a compound (IIa-1) represented by formula (IIa-1) with a compound (IIa-2) represented by formula (IIa-2) to obtain a block copolymer represented by formula (II), and then self-assembling this block copolymer together with an anionic molecular drug.

[0063] [ka] In the above equation, A, B, and m are the same as those defined for A, B, and m in equation (I); Ra 11 represents a hydrogen atom; La 1 represents an alkylene group, an arylene group, or an aralkylene group, however, La 1 It may have a hydrazide group or a hydrazine group and an inert substituent; Ra 12 represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group; Ra 21 represents a hydrogen atom or a methyl group; Ra 22 and Ra 23 This represents a hydrogen atom.

[0064] Ra in equation (IIa-1) 11 Ra 12 and La 1 This is Ra in equation (Ia-1). 11 Ra 12 and La 1 This is the same as the definition given for [the subject]. Ra in equation (IIa-2) 21 Ra 22 and Ra 23 This is Ra in equation (Ia-2).21 Ra 22 and Ra 23 This is the same as the definition given for [the subject].

[0065] An example of a reaction scheme for producing polyion complex micelles according to this embodiment is shown below.

[0066] [ka]

[0067] Alternatively, L in equation (II) 2 is formula -L R -NH-N=C(R 1 )-L 1 When the group is represented by -NH-, the block copolymer represented by formula (IIb-1) and the block copolymer represented by formula (IIb-2) can be crosslinked using a linker to obtain the block copolymer represented by formula (II).

[0068] [ka] In the above equation, A, B, and m are the same as those defined for A, B, and m in equation (I); Rb 11 and Rb 21 represents a hydrogen atom; Lb 1 and Lb 2 represents an alkylene group, an arylene group, or an aralkylene group, however, Lb 1 and Lb 2 It may have a hydrazide group or a hydrazine group and an inert substituent; Rb 12 and Rb 22 represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group.

[0069] Rb in equation (IIb-1) 11 , Rb 12 and Lb 1 These are Ra in equation (Ia-1), respectively. 11 Ra 12 and La 1This is the same as the definition given for [the subject]. Rb in equation (IIb-2) 21 , Rb 22 and Lb 2 These are Ra in equation (Ia-1), respectively. 11 Ra 12 and La 1 This is the same as the definition given for [the subject].

[0070] An example of an alternative reaction scheme for producing polyion complex micelles according to this embodiment is shown below.

[0071] [ka]

[0072] Polyion complex micelles may contain one type of block copolymer or two or more types of block copolymers. Polyion complex micelles may contain one type of anionic molecular drug or two or more types of anionic molecular drugs. The block copolymer contained in the polyion complex micelle may be bound to a functional molecule. Examples of functional molecules include, for example, targeting molecules for delivering the polyion complex micelle to a target site. Examples of targeting molecules include specific binding molecules that can specifically bind to a particular molecule, such as peptides, antibodies or fragments thereof, and ligand molecules. The functional molecule may be bound to either or both the end of the first hydrophilic block of the first block copolymer chain and the end of the second hydrophilic block of the second block copolymer chain. The functional molecule can be bound to the block copolymer by conventional methods such as click chemistry.

[0073] The polyion complex micelle according to the present invention described above includes a block copolymer having a hydrophilic block portion, a cationic hydrophobic block portion, and a crosslinked block portion located between the hydrophilic block portion and the cationic hydrophobic block portion. As described above, the crosslinked block portion surrounds the core on which the anionic molecular drug is supported, and the hydrophilic block portion forms a shell, thereby stabilizing the core. As a result, it becomes possible to stably encapsulate the anionic molecular drug. In particular, the polyion complex micelle according to this embodiment can be applied to anionic molecular drugs that cannot be stably encapsulated by conventional methods. Furthermore, the polyion complex micelles according to this embodiment have the following advantages: Monodisperse particles with a size of approximately 40-50 nm are formed, and most crosslinked micelles maintain narrow polydispersity in physiological saline compared to conventional non-crosslinked micelles. In addition, the drug release rate in physiological saline is slower than that of conventional non-crosslinked micelles. Moreover, various properties can be imparted by modifying the polymer structure. Furthermore, the polyion complex micelles according to this embodiment have excellent cell membrane permeability, allowing for efficient uptake into cells. [Examples]

[0074] The present invention will be described in detail based on the following examples. However, the embodiments of the present invention are not limited to those described.

[0075] [Synthesis Example 1: Synthesis of Triblock Copolymer, PEG-PBLA-PLys (TFA)] The triblock copolymer PEG-PBLA-PLys (TFA) was synthesized by N-carboxyanhydride (NCA) ring-opening polymerization (ROP) as follows. The initiator for the first ROP step was α-methoxy-ω-amino-poly(ethylene glycol) (Mw 12,000; PEG-NH2), which produced PEG-poly(β-benzyl L-aspartic acid) diblock copolymer (PEG-PBLA). PEG-NH2 was dried overnight under vacuum and dissolved in DMF. BLA-NCA (22 equivalents) was also dissolved in DMF and then added to the PEG-NH2 solution under an Ar atmosphere, and the mixture was reacted at 35°C for 72 hours. The polymer was separated from the reaction mixture by precipitation in a mixture of n-hexane and ethyl acetate (6:4), followed by filtration and vacuum drying.

[0076] Next, the obtained PEG-PBLA was used as the initiator for the second ROP of Lys(TFA)-NCA to obtain PEG-PBLA-PLys(TFA). The PEG-PBLA was dried overnight under vacuum and dissolved in DMSO. Lys(TFA)-NCA (40 equivalents) was also dissolved in DMSO and then added to the PEG-PBLA solution under an Ar atmosphere, and the reaction was carried out at 35°C for 72 hours. The triblock copolymer was separated from the reaction mixture by precipitation in a mixture of n-hexane and ethyl acetate (6:4), followed by filtration and finally vacuum drying.

[0077] [ka]

[0078] [Synthesis Example 2: Aminolysis and Deprotection of PEG-PBLA-PLys(TFA)] PEG-PBLA-PLys(TFA) (50 mg) was dissolved in DMF to which the aromatic aminoacetal linker, 1-[4-(dimethoxymethyl)phenyl]methaneamine (30 equivalents), was added. The reaction mixture was stirred at 40°C for 72 hours. Subsequently, the PLys(TFA) chain was deprotected by adding 3 mL of methanol and 100 μL of 5N NaOH. The reaction was allowed to proceed overnight. Next, the mixture was dialyzed against dilute acid and water for 48 hours using a 7500 Da molecular weight cutoff (MWCO) dialysis bag with the dialysate changed five times. Upon dialyzing against acid, the acetals were converted to aldehyde functional groups. This solution was freeze-dried under vacuum to obtain a modified triblock copolymer ((PEG-PAsp(ArAld)-PLys)).

[0079] [ka]

[0080] [Synthesis Example 3: Hydrazine degradation and deprotection of PEG-PBLA-PLys(TFA)] The hydrazine degradation of PEG-PBLA-PLys(TFA) was performed as follows: PEG-PBLA-PLys(TFA) (50 mg) was dissolved in DMF to which an excess amount of hydrazine monohydrate (50 μL) was added. The reaction mixture was stirred at 40°C for 4 hours. Subsequently, the PLys(TFA) chain was deprotected by adding 3 mL of methanol and 100 μL of 5N NaOH. The reaction was allowed to proceed overnight. Next, the mixture was dialyzed against dilute acid and water for 48 hours using a 7500 Da molecular weight cutoff (MWCO) dialysis bag in which the dialysate had been changed 5 times. This solution was freeze-dried under vacuum to obtain a modified triblock copolymer (PEG-PAsp(Hyd)-PLys).

[0081] [ka]

[0082] [Example 1: Preparation of polyion complex micelles (1)] Polymer solutions of the triblock copolymer PEG-PAsp(ArAld)-PLys(1) and the triblock copolymer (PEG-PAsp(Hyd)-PLys) were dispersed at a concentration of 2 mg / mL in 10 mM phosphate buffer (PB) pH 5. The resulting solutions were simply mixed with 1 mM gemcitabine triphosphate to achieve a 1:1 cation-to-anion ratio, diluted to the desired concentration with 10 mM PB pH 7.4, and then vortexed. The micelles were crosslinked at 4°C for 48 hours and then passed through a 0.22 μM syringe filter. As shown in Figure 1, crosslinked micelles (polyion complex micelles (1)) were formed by the automatic self-assembly of polymers containing anionic drug cargoes, and hydrazone bond formation occurred between the polymers as shown in the reaction scheme below.

[0083] [ka]

[0084] [Example 2: Preparation of polyion complex micelles (2)] Polyion complex micelles (2) were obtained by following the same procedure as in Example 1, except that a triblock copolymer PEG-PAsp(ArAld)-PLys(2), in which PAsp(ArAld) has a chain length of 12 to 22 repeating units, was used instead of PEG-PAsp(ArAld)-PLys(1).

[0085] [Example 3: Preparation of polyion complex micelles (3)] Polyion complex micelles (3) were obtained by following the same procedure as in Example 1, except that the triblock copolymer PEG-PAsp(aromatherapy ketone)-PLys (PEG-PAsp(ArKet)-PLys(1)) was used instead of the triblock copolymer PEG-PAsp(ArAld)-PLys.

[0086] [Example 4: Preparation of polyion complex micelles (4)] Polyion complex micelles (4) were obtained by following the same procedure as in Example 3, except that a triblock copolymer PEG-PAsp(ArKet)-PLys(2), in which PAsp(ArKet) has a chain length of 12 to 22 repeating units, was used instead of PEG-PAsp(ArKet)-PLys(1).

[0087] [Example 5: Preparation of polyion complex micelles (5)] Polyion complex micelles (5) were obtained by following the same procedure as in Example 1, except that cytarabine triphosphate was used instead of gemcitabine triphosphate.

[0088] [Comparative Example 1: Preparation of Comparative Polyion Complex Micelle (1)] A polymer solution of the diblock copolymer PEG-PLys was dispersed at a concentration of 2 mg / mL in 10 mM phosphate buffer (PB) pH 5. This was simply mixed with 1 mM gemcitabine triphosphate to achieve a 1:1 cation-to-anion ratio, diluted to the desired concentration with 10 mM PB pH 7.4, and then vortexed. Micelles were formed by the auto-self-assembly of the polymer containing the anionic drug cargo. Next, the solution was passed through a 0.22 μM syringe filter to obtain comparative polyion complex micelles (1).

[0089] [Comparative Example 2: Preparation of Comparative Polyion Complex Micelle (2)] Comparative polyion complex micelles (2) were obtained by following the same procedure as in Comparative Example 1, except that cytarabine triphosphate was used instead of gemcitabine triphosphate.

[0090] [Evaluation of micelle diameter and polyvariance index (1)] For polyion complex micelles (1), polyion complex micelles (5), comparative polyion complex micelles (1), and comparative polyion complex micelles (2), micelle diameter and polydispersity index (PDI) were obtained using dynamic light scattering (DLS) technology. The measurement conditions were as follows. Temperature: 25°C, Measurement angle: 173° backscatter, Sample holder: Quartz cuvette, Automated attenuator detection, No filter. The results are shown in Table 1.

[0091] [Table 1]

[0092] [Evaluation of drug release] Drug release was evaluated for polyion complex micelles (1) and comparative polyion complex micelles (1) as follows. The micelle solution was pipetteed into an Amicon Ultra-0.5 mL centrifuge filter (MWCO10000) and rotated (14000 g, 15 min, 4°C). The filtrate was then collected, weighed, and transferred to a UV-transmitting 96-well plate. Its UV absorption at 259 nm was measured using a microplate reader. The drug encapsulation amount was calculated by obtaining the ratio of the absorbance of the filtrate to the absorbance of the original (±)-C75-CoA solution added to form the micelles. The results are shown in Table 2.

[0093] [Table 2]

[0094] [Example 6: Preparation of polyion complex micelles (6)] Polyion complex micelles (6) were obtained by following the same procedure as in Example 1, except that luciferase ASO was used instead of gemcitabine triphosphate.

[0095] [Comparative Example 3: Preparation of Comparative Polyion Complex Micelle (3)] Comparative polyion complex micelles (3) were obtained by following the same procedure as in Comparative Example 1, except that luciferase ASO was used instead of gemcitabine triphosphate. [Evaluation of micelle diameter and polyvariance index (2)] Regarding the polyion complex micelles (6) and the comparative polyion complex micelles (3), the micelle diameter and the polydispersity index (PDI) were evaluated in the same manner as the above-mentioned "Evaluation of Micelle Diameter and Polydispersity Index (1)". The results are shown in Table 3.

[0096]

Table 3

[0097] [Evaluation of Cell Uptake] <Preparation of Crosslinked Polyion Complex Micelles> Solutions of PEG-PAsp(ArAld)-PLys 12-22 (chain length of PAsp(ArAld): 12 - 22 repeating units) and PEG-PAsp(Hyd)-PLys 12-22 (chain length of PAsp(Hyd): 12 - 22 repeating units) polymers were each dispersed in 10 mM phosphate buffer (PB) pH 5 at a concentration of 10 mg / mL. These solutions were simply mixed with 7.88 mM Fluor-CoA (Paraiso WKD et al., Biomater. Sci. 9(21), 7076 - 7091 (2021)) to achieve a 1:1 cation-to-anion ratio, diluted to the desired concentration with 10 mM PB pH 7.4, and then vortexed. After crosslinking the micelles at 4 °C for 24 hours, they were passed through a 0.22 μM syringe filter. The size of the obtained crosslinked polyion complex micelles was 44 ± 0.4 nm and the polydispersity index was 0.083.

[0098] <Preparation of DET Micelles> A polymer solution of PEG-PAsp(DET)12-69 was dispersed in 10 mM phosphate buffer (PB) pH 5 at a concentration of 10 mg / mL. This solution was simply mixed with 7.88 mM Fluor-CoA to achieve a 1:1 cation-to-anion ratio, diluted to the desired concentration with 10 mM PB pH 7.4, and then vortexed. Next, the solution was passed through a 0.22 μM syringe filter.

[0099] <Evaluation Method> The cellular uptake of polyion complex micelles was evaluated using the RPMI2650 permeability model (Reichl S, Becker K. J. Pharm. Pharmacol. 64(11), 1621-1630 (2012)). Permeability coefficients for polyion complex micelles and Fluor-CoA were measured, as reported by Gonzalez-Carter D et al. (J. Neuroendocrinol. 28(6) (2016)). RPMI2650 (human nasal epithelial carcinoma, mucin-expressing cells) were used as the cells.

[0100] <Result> The results are shown in Figures 3 and 4. As shown in Figure 3, cross-linked polyion complex micelles showed greater cellular uptake than DET micelles due to their higher stability.

[0101] [Evaluation of Transwell permeability] <Preparation of cross-linked polyion complex micelles> The procedure was the same as described in [Evaluation of Cellular Uptake], except that 4kDa FITC-dextran was used instead of Fluor-CoA.

[0102] <Evaluation Method> RPMI2650 culture using Transwell was performed according to the timeline shown in Figure 5. Transwell permeability was measured by the method described in Reichl S et al. (J. Pharm. Pharmacol. 64(11), 1621-1630 (2012)).

[0103] <Result> The results are shown in Figure 6. FITC-dextran 4kDa showed minimal paracellular transport due to its low permeability. FITC-dextran 70kDa could not pass through the membrane at all. In contrast, cross-linked polyion complex micelles showed good transwell permeability.

[0104] [Evaluation of cellular uptake into brain cells] <Preparation of cross-linked polyion complex micelles> The preparation of PEG-PAsp(Hyd)12-38 (PAsp(Hyd) chain length: 12-38 repeat units) was carried out according to the method described by S, Cabral H et al. (J. Control. Release 188, 67-77 (2014)).

[0105] [ka]

[0106] Polymer solutions of PEG-PAsp(ArAld)-PLys 12-22 and PEG-PAsp(Hyd) 12-38 were dispersed at a concentration of 10 mg / mL in 10 mM phosphate buffer (PB) pH 5. These solutions were simply mixed with 7.88 mM Fluor-CoA to achieve a 1:1 cation-to-anion ratio, diluted to the desired concentration with 10 mM PB pH 7.4, and then vortexed. The micelles were crosslinked at 4°C for 24 hours and then passed through a 0.22 μM syringe filter. The resulting crosslinked polyion complex micelles had a size of 43 ± 0.43 nm and a polydispersity index of 0.13. Figure 2 shows a schematic diagram of the crosslinked polyion complex micelles.

[0107] <Preparation of non-crosslinked polyion complex micelles> A polymer solution of PEG-PAsp(ArAld)-PLys 12-22 was dispersed at a concentration of 10 mg / mL in 10 mM phosphate buffer (PB) pH 5. This solution was simply mixed with 7.88 mM Fluor-CoA to achieve a 1:1 cation-to-anion ratio, diluted to the desired concentration with 10 mM PB pH 7.4, and then vortexed. The solution was then passed through a 0.22 μM syringe filter. Figure 7 shows a schematic diagram of the non-crosslinked polyion complex micelles. The obtained non-crosslinked polyion complex micelles had a size of 43 ± 0.97 nm and a polydispersity index of 0.13.

[0108] <Evaluation Method> The same procedure as described in [Evaluation of Cell Uptake] was used, except that KT-5 (astrocytocytes), BV-2 (microglia), GT1-7 (neurons), or primary rat brain endothelial cells were used instead of RPMI2650.

[0109] <Result> The results are shown in Figures 8-11. In all cell types, cross-linked polyion complex micelles were taken up more efficiently than non-cross-linked polyion complex micelles.

[0110] [Preparation of peptide-bonded micelles] The same procedure as described in [Evaluation of Cellular Uptake into Brain Cells] was used, except that azide-terminated PEG-PAsp(Hyd)12-38 was used instead of PEG-PAsp(Hyd)12-38 (with a methoxy terminus). Click conjugation of the DBCO-binding peptide was performed using the freeze-thaw method, as described by Takemoto H et al. (Bioconjug. Chem. 23(8), 1503-1506 (2012)). Briefly, the DBCO peptide and azide-terminated PEG-PAsp(Hyd) were mixed at equimolar concentrations and frozen at -30°C for 8 hours. The resulting peptide-binding polymer was thawed at 4°C for 2 hours, then dialyzed against ammonium bicarbonate buffer and lyophilized to obtain the resulting polymer.

[0111] While preferred embodiments of the present invention have been described and illustrated above, it should be understood that these are illustrative and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the invention. Therefore, the present invention should not be considered limited by the foregoing description, but only by the appended claims.

Claims

1. A block copolymer having a hydrophilic block portion, a cationic hydrophobic block portion, and a crosslinked block portion located between the hydrophilic block portion and the cationic hydrophobic block portion, and an anionic molecular drug encapsulated in the block copolymer. A polyion complex micelle containing, The aforementioned block copolymer is of formula (II): 【Chemistry 1】 (In the formula, A represents a repeating unit constituting a hydrophilic block; B represents a repeating unit constituting a cationic hydrophobic block; m represents 1 or 2; L1 represents a divalent linking group; R1 represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group; R2 represents a hydrogen atom or a methyl group; L2 represents a single bond or a divalent linking group; n represents 1 or 2) A polyion complex micelle, represented by the term.

2. A block copolymer having a hydrophilic block portion, a cationic hydrophobic block portion, and a crosslinked block portion located between the hydrophilic block portion and the cationic hydrophobic block portion, and an anionic molecular drug encapsulated in the block copolymer. A polyion complex micelle containing, The block copolymer is of formula (I): 【Chemistry 2】 (In the formula, A represents a repeating unit constituting a hydrophilic block; B represents a repeating unit constituting a cationic hydrophobic block; m represents 1 or 2; L １ R represents a divalent aliphatic hydrocarbon group or an aromatic hydrocarbon group; １ R represents a hydrogen atom, an aliphatic hydrocarbon group, or an aromatic hydrocarbon group; ２ L represents a hydrogen atom or a methyl group; ２ (where n represents a single bond; n represents 1 or 2) A polyion complex micelle, represented by the term.

3. The polyion complex micelle according to claim 1 or 2, wherein the cationic hydrophobic block is composed of a repeating structure derived from polylysine.

4. A polyion complex micelle according to any one of claims 1 to 3, wherein the particle size is 20 to 100 nm and the polydispersity index is 0.05 to 0.

3.

5. The polyion complex micelle according to any one of claims 1 to 4, wherein the net negative charge of the anionic molecular drug is -25 to -1 at a physiological pH.

6. The polyion complex micelle according to any one of claims 1 to 5, wherein the anionic molecular drug is a nucleic acid drug.

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