Polyrotaxane, composite containing same, pharmaceutical composition containing same, and method for producing said composite

Abstract

Provided are: a polyrotaxane which is selective for target receptors and is usable as an accumulating agent for the target receptors (for example, applicable as a delivery carrier); a composite containing the polyrotaxane; a pharmaceutical composition containing the polyrotaxane; and a method for producing the composite. This polyrotaxane A comprises a plurality of macrocyclic molecules a, an axial molecule a piercing through the rings of the macrocyclic molecules a, and caps a bonded to terminals of the axial molecule a. At least some of the macrocyclic molecules a each have one or more ligand groups. One or more of the ligand groups possessed by the macrocyclic molecules a are recognized by target receptors that each recognize one or more of the ligand groups. The number of the ligand groups possessed by the macrocyclic molecules a is less than the number of the ligand groups recognized by the target receptors, or the number of the ligand groups possessed by the macrocyclic molecules a is the same as or greater than the number of the ligand groups recognized by the target receptors.

Description

Polyrotaxane, complex containing same, pharmaceutical composition containing same, and method for producing said complex

[0001] The present invention relates to a polyrotaxane that can be used as an agent that accumulates at a target receptor and is suitable for delivering a biological material, which is an active ingredient, to a target receptor, a complex containing the polyrotaxane, a pharmaceutical composition containing the complex, and a method for producing the complex.

[0002] In recent years, there has been a strong demand for the development of technologies for safely and efficiently introducing proteins, nucleic acid polymers, etc., into cells, for example, protein / nucleic acid complexes such as the complex of Cas9 protein and guide RNA (Cas9 RNP) used to induce genome editing, and nucleic acid molecules such as siRNA.

[0003] The present inventors have previously developed a polyrotaxane (PRX)-based intracellular delivery carrier (hereinafter also referred to simply as "protean polymer (5G)") that recognizes and transforms the shape, charge distribution, etc. of genome editing molecules, nucleic acids, proteins, etc., to interact with the above with high efficiency. It is noteworthy that this carrier exhibits higher intracellular delivery efficiency and safety than Lipofectamine CRISPRMAX, the most commonly used carrier among commercially available Cas9RNP delivery reagents (Patent Document 1).

[0004] International Publication No. 2022 / 163729A1

[0005] However, in theory, such carriers do not have tissue selectivity. As described above, the development of a carrier capable of delivering biological materials (e.g., genome editing molecules) that are active ingredients selectively to target tissues is an urgent issue. The present invention has been made in consideration of the problems of the prior art described above, and aims to provide a polyrotaxane that is target receptor selective and can be used as an agent that accumulates at a target receptor (e.g., can be applied to a delivery carrier), a complex containing the polyrotaxane, a pharmaceutical composition containing the polyrotaxane, and a method for producing the complex.

[0006] As a result of intensive research into the above-mentioned problems, the present inventors have discovered that both ligand-modified polyrotaxanes (PRXs) having a ligand-containing group containing one ligand and ligand-modified PRXs having ligand-containing groups containing multiple ligands exhibit significantly more specific interactions with target receptors than conventional polymers due to highly efficient multivalent interactions between the ligand and receptor, which are achieved by eliminating spatial mismatches due to the dynamic properties of PRXs. They further discovered that by further incorporating the ligand-modified PRX into the complex of the protean polymer (5G) and genome editing molecule, it is possible to deliver the genome editing molecule to target tissues (liver, cancer). The present invention has been completed based on the above findings. Specifically, the present invention is as follows.

[0007] <1> A polyrotaxane A having a plurality of macrocyclic molecules a, an axis molecule a that penetrates the rings of the macrocyclic molecules a, and caps a that are bonded to the ends of the axis molecules a, wherein at least some of the macrocyclic molecules a have one or more ligand groups, the one or more ligand groups possessed by the macrocyclic molecules a are recognized by a target receptor that recognizes the one or more ligand groups, and the number of the ligand groups possessed by the macrocyclic molecules a is either less than the number of the ligand groups recognized by the target receptor, or the number of the ligand groups possessed by the macrocyclic molecules a is equal to or greater than the number of the ligand groups recognized by the target receptor. <2> The polyrotaxane A according to <1>, wherein the number of the ligand groups possessed by the macrocyclic molecules a is less than the number of the ligand groups recognized by the target receptor. <3> The polyrotaxane A according to <1>, wherein the number of the ligand groups possessed by the macrocyclic molecule a is equal to or greater than the number of the ligand groups recognized by the target receptor. <4> The polyrotaxane A according to <1>, wherein the macrocyclic molecule a has two or more of the ligand groups, and the two or more ligand groups are covalently bonded to the macrocyclic molecule a via a linking group, and the linking group is a branched hydrocarbon chain having 5 to 80 carbon atoms, which may contain a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group, and the ligand group is bonded to each of two or more ends of the branched hydrocarbon chain. <5> The polyrotaxane A according to <1>, wherein the cap a has a drug group directly or via a linker. <6> The polyrotaxane A according to <5>, wherein the drug group is an antibody or an antibody fragment. <7> The polyrotaxane A according to <1>, which is to be contained in a composition containing the following polyrotaxane B and used. [Polyrotaxane B: a polyrotaxane having a plurality of macrocyclic molecules b, an axial molecule b that passes through the rings of the macrocyclic molecules, and a cap b that is bound to an end of the axial molecule, and having the ability to form a polyion complex with a biological material, wherein the cap b has a host-guest interaction and / or an inclusion interaction with the cap a.<8> The polyrotaxane A according to <7>, wherein at least a portion of the macrocyclic molecules b in the polyrotaxane B has an amine-containing group b, and the amine-containing group b has a monovalent proton at neutral pH and a divalent proton at acidic pH. <9> The polyrotaxane A according to <8>, wherein at least a portion of the macrocyclic molecules b further has an amino group via an intracellularly degradable bond in addition to the amine-containing group b. <10> The polyrotaxane A according to <7>, wherein the composition further contains a biological material, and the biological material is capable of forming a polyion complex with the polyrotaxane B. <11> The polyrotaxane A according to <10>, wherein the biological material is a nucleic acid molecule or a complex of Cas9 protein and guide RNA (Cas9 RNP).

[0008] <12> A complex comprising the polyrotaxane A and polyrotaxane B according to <1> above, and a biological material, wherein the polyrotaxane B and the biological material form a polyion complex, wherein the polyrotaxane B has a plurality of macrocyclic molecules b, an axis molecule b passing through rings of the macrocyclic molecules, and a cap b bonded to an end of the axis molecule, and the polyrotaxane A and the polyrotaxane B are bonded via a host-guest interaction and / or an inclusion interaction between the cap a and the cap b. <13> A pharmaceutical composition comprising the polyrotaxane A according to <5> above. <14> A pharmaceutical composition comprising the following polyrotaxane A, the following polyrotaxane B, and a biological material, wherein the biological material is capable of forming a polyion complex with the polyrotaxane B. [Polyrotaxane A: the polyrotaxane having a plurality of macrocyclic molecules a, an axis molecule a penetrating the rings of the macrocyclic molecules a, and caps a bonded to the ends of the axis molecules a, wherein at least a portion of the macrocyclic molecules a have one or more ligand groups, wherein the one or more ligand groups possessed by the macrocyclic molecules a are recognized by a target receptor that recognizes the one or more ligand groups, and wherein the number of the ligand groups possessed by the macrocyclic molecules a is less than the number of the ligand groups recognized by the target receptor, or the number of the ligand groups possessed by the macrocyclic molecules a is equal to or greater than the number of the ligand groups recognized by the target receptor.] [Polyrotaxane B: the polyrotaxane having a plurality of macrocyclic molecules b, an axis molecule b penetrating the rings of the macrocyclic molecules, and caps b bonded to the ends of the axis molecules, and capable of forming a polyion complex with the biological material, wherein the caps b have host-guest interaction and / or inclusion interaction with the caps a. <15> The pharmaceutical composition according to <13> or <14>, which is for introducing the biological material into a cell and delivering it to a target. <16> A method for producing the complex according to <12> above, the method comprising mixing polyrotaxane A and polyrotaxane B, and mixing a biological material.

[0009] According to the present invention, it is possible to provide a polyrotaxane that is target receptor-selective (preferably target receptor-specific) and can be used as an agent that accumulates at a target receptor (for example, can be applied to a delivery carrier), a complex containing the polyrotaxane, a pharmaceutical composition containing the polyrotaxane, and a method for producing the complex. According to the present invention, it is possible to provide a polyrotaxane that has target receptor directivity suitable as a component for delivering a biological material as an active ingredient described below to the target receptor. According to the present invention, it is possible to provide a polyrotaxane that can be used for the target receptor-selective delivery of a biological material as an active ingredient, a complex containing the polyrotaxane, a pharmaceutical composition containing the polyrotaxane, and a method for producing the complex.

[0010] 1 is a schematic diagram of the recognition (interaction) of a ligand group possessed by a macrocyclic molecule a in polyrotaxane A with a target receptor. As an example, it is a schematic diagram of a case where the number of ligand groups recognized by a target receptor is three, while the number of ligand groups possessed by a macrocyclic molecule a is one. It is a schematic diagram of a ternary complex consisting of polyrotaxane A, polyrotaxane B, and a biological material. It is a diagram showing the results of a test on the cellular uptake efficiency of monoGalNAc-PRX, triGalNAc-PRX, monoGalNAc-DEX, and triGalNAc-DEX. It is a diagram showing the results of a test on ASGPR competitive inhibition by a competitive inhibitor (free GalNAc) of the cellular uptake of monoGalNAc-PRX, triGalNAc-PRX, monoGalNAc-DEX, and triGalNAc-DEX.

[0033] Figure 1 shows test results for the cellular uptake efficiency of FA-PRX and FA-DEX. Figure 1 shows test results for the uptake of a ternary complex of GalNAcsystem and Cas9RNP into target cells (HepG2 cells). Figure 1 shows test results for the uptake of a ternary complex of FAsystem and Cas9RNP into target cells (HeLa cells). Figure 2 shows test results for the uptake of GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP into target cells under competitive inhibition conditions. Figure 3 shows test results for in vivo genome editing effect in target tissue (liver) after a single administration of GalNAcsystem / Cas9RNP into the tail vein of a mouse. Figure 4 shows test results for in vivo genome editing effect in target tissue (cancer) after a single administration of FAsystem / Cas9RNP into the tail vein of a mouse.

[0033] Figure 1 shows the results of a complex formation test between polyrotaxane A (Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX) and polyrotaxane B (5G 35k CD-cap). Figure 2 shows the results of a ternary complex (polyion complex) formation test using polyrotaxane A (Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX) and polyrotaxane B (5G 35k CD-cap) with Cas9RNP. Figure 3 shows the results of a pharmacokinetics test of a ternary complex (polyion complex) using the GalNAc system and Cas9RNP.

[0033] Figure 1 shows the results of a pharmacokinetic test of a ternary complex (polyion complex) using the FAsystem and Cas9RNP. Figure 2 shows an outline of a test to confirm the reactivity of DBCO-GalNAc-PRX (20k). Figure 3 shows the results of a test to confirm the reactivity of DBCO-GalNAc-PRX (20k). Figure 4 shows an outline and test results of an in vitro test of the antigen delivery ability of monoGalNAc-PRX-antibody (LYTAC). Figure 5 shows an outline and test results of an in vivo test of the antigen delivery ability of monoGalNAc-PRX-antibody (LYTAC) to target cells. Figure 6 shows a summary of the test results of the ability of monoGalNAc-PRX-antibody (LYTAC) to deliver target substances to target cells.

[0011] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0012] The definitions of the terms used in this specification are as follows. In this specification, a "polyrotaxane" is a so-called supramolecule that consists of multiple macrocyclic molecules and an axial molecule that penetrates the rings of the macrocyclic molecules and has caps on both ends. A polypseudorotaxane has multiple macrocyclic molecules and an axial molecule that penetrates the rings of the macrocyclic molecules, but does not have a cap structure.

[0013] As used herein, a "macrocyclic molecule" refers to a cyclic molecule having an internal opening (cavity) large enough for the axis molecule to penetrate and having a chemically modifiable group (e.g., a hydroxyl group). Examples of macrocyclic molecules include cyclic polyethers, cyclic polyesters, cyclic polyetheramines, cyclic polyamines, crown ethers, cucurbit[n]urils, calixarenes, cyclic amides, transition metal complexes, cyclodextrins, cyclodextrin derivatives, and any combination thereof. "Cyclodextrin" refers to a cyclic oligosaccharide compound, and includes, for example, α-cyclodextrin (hexasaccharide), β-cyclodextrin (heptasaccharide), or γ-cyclodextrin (octasaccharide). The macrocyclic molecule may be substituted with a substituent other than the ligand group or "amine-containing group b" described below, such as a methyl group, a hydroxyethyl group, a hydroxypropyl group, an acetyl group, a carboxymethyl group, a succinyl group, a glucosyl group, a carboxyethyl group, a sulfobutyl group, an amino group, a halogen atom, or any combination thereof.

[0014] As used herein, an "axle molecule" is typically a polymer, and examples thereof include a polymer of a single monomer (homopolymer) and a copolymer of two or more types of monomers. A "polymer" refers to a macromolecule formed by the repeated bonding of many of one or more monomers. A monomer is typically a molecule having one carbon-carbon double bond or a molecule having at least two functional groups per molecule. A copolymer may be a random copolymer, an alternating copolymer, and / or a block copolymer. When the axial molecule is a homopolymer, examples of the polymer include polyalkylene oxide, polyethylene glycol (PEG), polypropylene glycol, polyvinyl ether, polymethyl vinyl ether, polyethyleneimine, poly(trimethylene oxide), poly(ε-caprolactone), polylactic acid, polyamino acid, poly(ε-lysine), polyamide, poly(iminooligomethylene), ionene, poly(vinyldiene chloride), polypropylene, oligopropylene, polyethylene, oligoethylene, poly(alkylenebenzimidazole), polyurethane, poly(viologen), poly(N-dimethyldecamethyleneammonium), poly(dimethylsiloxane), polyaniline, polycarbonate, poly(methyl methacrylate), poly(N-acylethyleneimine), poly(4-vinylpyridine)-dodecylbenzenesulfonic acid complex, fullerene-polyethylene glycol conjugate, and hydrophobized polysaccharide. When the axial molecule is a copolymer, examples thereof include a copolymer of polyethylene glycol and polypropylene glycol, a copolymer of poly(ε-caprolactone) and polylactic acid, a copolymer of polylactic acid and polyethylene glycol, a polyethylene glycol-polysaccharide graft copolymer, and a polypropylene glycol-polysaccharide graft copolymer. The axial molecule may also be a branched multi-arm polymer, such as a star-shaped polyethylene glycol, a hyperbranched polyether, a hyperbranched oligoethylene glycol, or a hyperbranched oligopropylene glycol. These polymers may be modified or substituted with a substituent, as necessary.The degree of polymerization, molecular weight, etc. of the axial molecule may be any length that allows a required amount of macrocyclic molecules to pass through depending on the target nucleic acid / protein to be introduced into cells. For example, polymers with number-average molecular weights (Mn) of about 200 to 1,000,000 Da, about 400 to 50,000 Da, about 500 to 40,000 Da, or about 1,000 to 36,000 Da can be used.

[0015] In this specification, the term "cap" (CAP) refers to a bulky substituent bonded to an end of a polyrotaxane. More specifically, from the viewpoint of preventing the macrocyclic molecule from detaching from the axis molecule, the cap is preferably bonded to at least one end of the axis molecule in the polyrotaxane, and more preferably bonded to both ends of the axis molecule. Examples of the cap include a group having a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin), a group having a polycyclic alicyclic ring (adamantyl, norbornyl, isonorbornyl, etc.), dinitrophenyl groups, trityl groups, fluoresceins, silsesquioxanes, pyrenes, substituted benzenes (optionally substituted with one or more substituents, which may be alkyl, alkyloxy, hydroxy, halogen, cyano, sulfonyl, carboxyl, amino, and phenyl), steroids, amino acids, oligopeptides, oligosaccharides, sugar derivatives, a group having one or more benzene rings (benzyloxycarbonyl (Z) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyl ester (OBz) group), or a group having one or more tertiary butyl groups (tertiary butylcarbonyl (Boc) group, amino acid tert-butyl ester (OBu) group). As the cap, a group having a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin), a group having a polycyclic alicyclic ring (adamantyl group, norbornyl group, isonorbornyl group, etc.), a dinitrophenyl group, a trityl group, a fluorescein, a silsesquioxane, or a pyrene is preferred, and a group having a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin), a group having a polycyclic alicyclic ring (adamantyl group, norbornyl group, isonorbornyl group, etc.) is more preferred. By bonding to the end of the polypseudorotaxane, the cap can prevent the macrocyclic molecule threaded (threaded) by the axis molecule from detaching from the axis molecule. Therefore, it is preferable that the cap have sufficient steric bulkiness to block the macrocyclic molecule from escaping from the axis molecule.

[0016] The cap is preferably linked to the stalk molecule via an intracellularly degradable bond. Here, "an intracellularly degradable bond" refers to a bond that is easily degraded under conditions in the intracellular environment that differ from those outside the cell, such as physicochemical conditions such as pH or biological conditions such as intracellular enzymes. Preferably, the intracellularly degradable bond is a bond that is not easily degraded outside the cell. Here, the terms "degradable" and "not easily degradable" are not absolute and do not mean completely degradable or not at all degradable, respectively.

[0017] "Degradable" and "hard to degrade" mean that a substance is relatively easy to degrade and hard to degrade, respectively, when compared between the extracellular and intracellular environments. For example, the condition in the intracellular environment that differs from the extracellular environment may be a GSH concentration. In this case, the bond that is degradable within the cell may be a bond that is hard to degrade at a GSH concentration (extracellular concentration) of up to 0.2 mM, but is degradable at a GSH concentration (intracellular concentration) of 2 to 10 mM. Examples of "bonds that are degradable within the cell" include carbamate (-NH(C=O)O-), ketal (-OC(CH ３ ) ２ O-), amide (-NHCO-), disulfide (-S-S-), acetal (-C(OH)O-), orthoester, vinyl ether (-CH ２ ═CH—O—), hydrazide, and ester (—COO—) bonds.

[0018] More preferably, the cap is bonded to the stalk molecule via a carbamate bond. Bonding of the cap to the stalk molecule via a bond that can be degraded in cells is described, for example, in Journal of Controlled Release 76 (2001) 11-25; Biomacromolecules 2003, 4, 1426-1432; Langmuir 2011, 27 (2), 612-617; Angew. Chem. Int. Ed. 2013, 52, 7300-7305; J. Mater. Chem. B, 2013, 1, 3535-3544; Biomaterials, 34, 2480-2491 (2013); Scientific Reports, 3, 2252 (2013), etc.

[0019] In polyrotaxanes and polypseudorotaxanes, the axial molecules penetrate the macrocyclic molecules by penetrating through the openings of the macrocyclic molecules. Polyrotaxanes and polypseudorotaxanes have multiple macrocyclic molecules sewn onto the axial molecules in this way. The number of macrocyclic molecules in one polyrotaxane molecule can be set depending on the purpose, but can be, for example, an average of about 5 to about 200, or about 10 to about 100. The macrocyclic molecules are not covalently bonded to the axial molecule, and therefore can move in both the rotational and axial directions relative to the axial molecule.

[0020] <Polyrotaxane A> A first aspect of the present invention is a polyrotaxane A having a plurality of macrocyclic molecules a, an axis molecule a penetrating the ring of the macrocyclic molecule a, and a cap a bonded to the end of the axis molecule a, wherein at least some of the macrocyclic molecules a have one or more ligand groups, and the one or more ligand groups are recognized by a target receptor that recognizes the one or more ligand groups, and the number of ligand groups possessed by the macrocyclic molecule a is either less than the number of ligand groups recognized by the target receptor, or the number of ligand groups possessed by the macrocyclic molecule a is equal to or greater than the number of ligand groups recognized by the target receptor. In the present invention, the ligand group refers to a group in which a ligand that specifically binds to the target receptor is covalently bonded to the macrocyclic molecule a directly or via an arbitrary linking group. When the target receptor includes a plurality of subunits, it is preferable that one ligand group specifically binds to one of the subunits.

[0021] FIG. 1 is a schematic diagram showing the recognition (interaction) between a ligand group carried by a macrocyclic molecule a in polyrotaxane A and a target receptor. In polyrotaxane A according to the first embodiment, the macrocyclic molecule a is not covalently bonded to the axial molecule a and can move rotationally and axially relative to the axial molecule a, which also has flexibility. Due to this mobility or dynamic property of polyrotaxane A, as shown in FIG. 1, one or more of the ligand groups carried by the macrocyclic molecule a can move in accordance with the distribution of target receptors in the system (preferably target receptors present on the cell membrane at the cell surface), thereby eliminating spatial mismatches with the target receptors. As a result, the recognition (interaction) between the target receptor and the ligand group can be enhanced. Furthermore, when the target receptor is a target receptor present on the cell membrane on the cell surface, the enhanced recognition (interaction) can improve the efficiency of cellular uptake by endocytosis (e.g., receptor-dependent endocytosis including clathrin-independent carriers / glycosylphosphatedylinositol-anchored-protein-enriched endosomal compartment (CLIC / GEEC) endocytosis, clathrin endocytosis, caveolae endocytosis, etc.).

[0022] Furthermore, when the target receptor recognizes two or more of the ligand groups (when the target receptor interacts in a multivalent manner), the spatial mismatch (recognition loss, interaction loss) may increase due to the steric configuration, steric hindrance, etc. between the two or more ligand recognition sites. Even when such spatial mismatch may increase, the polyrotaxane A according to the first aspect can resolve the spatial mismatch with the target receptor due to its mobility or dynamic properties. Specific examples of "when the target receptor recognizes (interacts with) two or more of the ligand groups" include when one target receptor has two or more (e.g., 2, 3, 4, 5, 6, etc.) ligand recognition sites, and when a target receptor multimer (e.g., dimer, trimer, tetramer, pentamer, hexamer, etc.) containing two or more target receptor subunits is formed.

[0023] Furthermore, even when the macrocyclic molecule a contains two or more of the ligand groups (when the macrocyclic molecule a interacts with the target receptor in a polyvalent manner of divalent or greater), the spatial mismatch may occur due to the steric configuration or steric hindrance on the macrocyclic molecule a. However, whether the target receptor recognizes one of the ligand groups (when the target receptor interacts in a monovalent manner) or whether the target receptor recognizes two or more of the ligand groups (when the target receptor interacts in a polyvalent manner of divalent or greater), the mobility or dynamic properties of polyrotaxane A can eliminate the spatial mismatch with the target receptor.

[0024] When the macrocyclic molecule a has one ligand group, it can be referred to as a monovalent (single-valent) ligand group. When the macrocyclic molecule a has two or more ligand groups, it can be referred to as a polyvalent (e.g., divalent or higher) ligand group. When the macrocyclic molecule a has one ligand group (i.e., a monovalent ligand group), the ligand group may be bonded to the macrocyclic molecule a directly or via an optional linking group, but is preferably bonded (substituted) to the macrocyclic molecule a via an optional linking group. Examples of the linking group include divalent linear hydrocarbon chains having 1 to 20 carbon atoms (preferably 2 to 15 carbon atoms, more preferably 3 to 10 carbon atoms) that may or may not contain a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group, and may be the above-mentioned "intracellularly degradable bond."

[0025] When the macrocyclic molecule a has two or more ligand groups (i.e., when it is a polyvalent ligand group), the two or more ligand groups may be bonded to the macrocyclic molecule a directly or via any linking group, but are preferably bonded (substituted) to the macrocyclic molecule a via any linking group. The linking group may be a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group, and the ligand group is bonded to each of two or more ends of a branched hydrocarbon chain having 5 to 80 carbon atoms (preferably 10 to 70 carbon atoms, more preferably 20 to 60 carbon atoms, and even more preferably 30 to 50 carbon atoms). From the viewpoint of bonding a larger number of ligand groups, it is preferable that a ligand group is bonded to each of three or more ends of the branched hydrocarbon chain, more preferably four or more ends of the branched hydrocarbon chain, even more preferably five or more ends of the branched hydrocarbon chain, and particularly preferably six or more ends of the branched hydrocarbon chain. Examples of the upper limit include "a ligand group is bonded to each of eight or fewer ends of the branched hydrocarbon chain" and "a ligand group is bonded to each of seven or fewer ends of the branched hydrocarbon chain."

[0026] In the first aspect, in particular, from the viewpoints of suppressing the spatial mismatch (recognition loss, interaction loss) due to steric configuration, steric hindrance, etc. on the macrocyclic molecule a, and reducing the complexity and production costs of the production of polyrotaxane A, it is preferable that the number of ligand groups possessed by the macrocyclic molecule a is smaller than the number of ligand groups recognized by the target receptor. Figure 2 is a schematic diagram illustrating an example in which the number of ligand groups recognized by the target receptor is three, while the number of ligand groups possessed by the macrocyclic molecule a is one. For example, when the number of ligand groups recognized by the target receptor is three, while the number of ligand groups possessed by the macrocyclic molecule a is one, the mobility or dynamic properties of polyrotaxane A cause polyrotaxane A to deform in response to the triangle-like three-point recognition structure of the target receptor, thereby resolving the spatial mismatch, and the three monovalent ligand groups come together to function as if they were a trivalent ligand group, thereby interacting strongly with the target receptor.

[0027] The "case where the number of ligand groups in the macrocyclic molecule a is smaller than the number of ligand groups recognized by the target receptor" is not particularly limited as long as the above-mentioned problem can be solved, and examples thereof include the case where the number of ligand groups in the macrocyclic molecule a is smaller than the number of ligand groups recognized by the target receptor by one or more. From the viewpoints of reducing the complexity and production costs of polyrotaxane A production, the interaction with the ligand, etc., the number of ligand groups in the macrocyclic molecule a is preferably smaller than the number of ligand groups recognized by the target receptor by two or more, more preferably smaller than the number of ligand groups recognized by the target receptor by three or more, even more preferably smaller than the number of ligand groups recognized by the target receptor by four or more, and particularly preferably smaller than the number of ligand groups recognized by the target receptor by five or more. The maximum difference between the number of ligand groups in the macrocyclic molecule a and the number of ligand groups recognized by the target receptor is not particularly limited, and examples thereof include 10 or less, 8 or less, 7 or less, etc. More specifically, examples of "the case where the number of ligand groups possessed by the macrocyclic molecule a is smaller than the number of ligand groups recognized by the target receptor" include: when the number of ligand groups recognized by the target receptor is 4, while the number of ligand groups possessed by the macrocyclic molecule a is 1, 2, or 3; when the number of ligand groups recognized by the target receptor is 3, while the number of ligand groups possessed by the macrocyclic molecule a is 1 or 2; and when the number of ligand groups recognized by the target receptor is 2, while the number of ligand groups possessed by the macrocyclic molecule a is 1.

[0028] On the other hand, even if the number of ligand groups possessed by the macrocyclic molecule a is equal to or greater than the number of ligand groups recognized by the target receptor, polyrotaxane A can eliminate spatial mismatch with the target receptor and enhance the recognition (interaction) between the target receptor and the ligand groups. In this case, from the viewpoint of reducing the complexity and production costs of the production of polyrotaxane A, it is preferable that the number of ligand groups possessed by the macrocyclic molecule a is the same as the number of ligand groups recognized by the target receptor. For example, when the number of ligand groups possessed by the macrocyclic molecule a is one, the number of ligand groups recognized by the target receptor is one; when the number of ligand groups possessed by the macrocyclic molecule a is two, the number of ligand groups recognized by the target receptor is two; and when the number of ligand groups possessed by the macrocyclic molecule a is three, the number of ligand groups recognized by the target receptor is three.

[0029] As described above, polyrotaxane A according to the first aspect can selectively (preferably specifically) interact with a target receptor, and is therefore suitable as an accumulating agent for a target receptor. The present invention also relates to an accumulating agent for a target receptor, including polyrotaxane A according to the first aspect. The present invention also relates to the use of polyrotaxane A according to the first aspect in an accumulating agent for a target receptor. Here, "accumulation" means being selectively (preferably specifically) directed to or localized at a target receptor, and it is preferable that it is directed to or localized selectively (preferably specifically) at a target receptor, rather than through a non-specific interaction. For example, when polyrotaxane A according to the first aspect has an optional label, as described below, it can be used as an accumulating agent for any treatment (e.g., theranostic treatment) or diagnostic purposes.

[0030] In the first aspect, the target receptor is not particularly limited as long as it can solve the above problem, but is preferably a target receptor present on the cell membrane of a cell in a biological tissue from the viewpoint of intracellular uptake by endocytosis (e.g., receptor-mediated endocytosis including CLIC / GEEC endocytosis, clathrin endocytosis, caveolae endocytosis, etc.) etc. Furthermore, the target receptor is also preferably a target receptor that recognizes two or more of the ligand groups (multivalent recognition), from the viewpoint of the ligand group moving in accordance with the distribution of the target receptors on the cell surface and being able to perform multivalent interactions with multiple target receptors while resolving spatial mismatches. The biological tissue is not particularly limited as long as it can solve the above-mentioned problem, and examples include biological tissues in or derived from internal organs (e.g., pancreas, kidneys, lungs, liver, heart, stomach, intestines, etc.), reproductive organs (e.g., ovaries, testes), fertilized eggs, embryos, fetuses, bone marrow (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, etc., and cancer tissue. From the viewpoint of suitability as a target for treatment, diagnosis, etc., biological tissues in or derived from internal organs or cancer tissue are preferred, liver tissue, lung tissue, pancreatic tissue, kidney tissue, or cancer tissue is more preferred, and liver tissue, lung tissue, or cancer tissue is even more preferred.

[0031] The target receptor is not particularly limited as long as it can recognize a ligand, but from the viewpoint of intracellular uptake by endocytosis (e.g., receptor-dependent endocytosis including CLIC / GEEC endocytosis, clathrin endocytosis, caveolae endocytosis, etc.), a cell surface receptor (e.g., a transmembrane protein) is preferred, and a cell surface receptor (e.g., a transmembrane protein) responsible for receptor-dependent endocytosis is more preferred. Examples of the cell surface receptor (e.g., a transmembrane protein) include lectins, vitamin receptors or coenzyme receptors, antibodies or antibody fragments (i.e., antibodies or antibody fragments as receptors for antigens or epitopes), antigen or epitope receptor proteins, antibody or antibody fragment receptor proteins, etc., and from the viewpoint of intracellular uptake by endocytosis, a lectin, or a vitamin receptor or coenzyme receptor is preferred, and a lectin or vitamin receptor is more preferred. Specific examples of lectins include C-type lectins, P-type lectins, galectins, calnexins, calreticulins, annexins, I-type lectins (e.g., siglecs), lactose receptors, etc., and from the viewpoint of solving the problems of the present invention, C-type lectins including asialoglycoprotein receptor (ASGPR), selectins (E-selectin, L-selectin, P-selectin, etc.), collectins, etc. are preferred, with asialoglycoprotein receptor (ASGPR) being more preferred.

[0032] ASGPR is known as a receptor highly expressed on the surface of hepatocytes. ASGPR is a C-type lectin that recognizes galactose and / or GalNAc. Approximately 500,000 copies of ASGPR are expressed per hepatocyte, of which approximately 5-10% are presented as receptors on the cell surface. Furthermore, upon binding to substrates such as galactose, ASGPR is rapidly internalized, recycled, and presented again on the cell membrane within approximately 15 minutes. (Springer A.D., Dowdy S.F., GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics. Nucleic Acid Ther.,28,109-118(2018).、Debacker A.J.,Voutila J.,Catley M.,Blakey D.,Habib N.,Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug. Mol. Ther. , 28, 1759-1771 (2020).) Thus, ASGPR has a high receptor density and is rapidly internalized and re-presented, making it suitable for liver-targeted drug delivery.

[0033] ASGPR is known to recognize GalNAc approximately 50 times more strongly than galactose. Furthermore, ASGPR is known to exist as a hetero-oligomer on the cell membrane, consisting of constituent proteins called ASGR1 and ASGR2. The binding affinity increases as the ligand valency increases from 1 to 3, reaching a plateau at 4 valency, and it is therefore believed to be a trimer consisting primarily of three subunits (trimer model). Furthermore, it has been reported that the sugar recognition site in the ASGPR trimer has a triangle-like three-point recognition structure (intermolecular distances of 2.5 nm, 2.2 nm, and 1.5 nm). (Debanne M.T., Chindemi P.A., Regoeczi E., Binding of asialotransferrins by purified rat liver plasma membranes. J. Biol. Chem.,256,4929-4933(1981).、Baenziger J.U.,Fiete D.,Galactose and N-acetylgalactosamine-specific endocytosis of glycopeptides by isolated rat hepatocytes. Cell, 22, 611-620 (1980). , Stockert R. J. , The asialoglycoprotein receptor:relationships between structure, function, and expression. Physiol. Rev. , 75, 591-609 (1995). , Lee Y. C. , Lee R. T. , Carbohydrate-Protein Interactions: Basis of Glycobiology. Acc. of Chem. Res. , 28, 321-327 (1995). )

[0034] In the polyrotaxane A according to the first aspect, even when the macrocyclic molecule a has only one ligand group, the polyrotaxane A deforms so that three monovalent ligand groups assemble into a trivalent ligand group in response to the triangle-like three-point recognition structure of ASGPR, thereby enabling strong interaction with one ASGPR (Figure 2). Furthermore, the ligand groups move in accordance with the distribution of ASGPR on the cell surface, allowing multivalent interactions with multiple ASGPRs while resolving spatial mismatches.

[0035] It should be noted that the above trimer model of ASGPR is merely a model of the oligomer that exists primarily, and it is known that ASGPR actually exists as a trimer or more, up to a hexamer. (Stockert R.J., The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. Rev., 75, 591-609 (1995). Biochemistry, 29, 10009-10018 (1990).

[0036] The lactose receptor on liver cells is known to recognize lactose (L. Stryer, Carbohydrates. Biochemistry (3rd.) p. 345, WH Freeman and Company, New York (1988)).

[0037] Specific examples of the vitamin receptor or coenzyme receptor include folate receptors (α, β), avidin, and the like. Folate receptor-α (FR-α) is a protein that is responsible for the uptake of folate into cells and is known to be highly expressed in various epithelial cancer cells, including melanoma, ovarian cancer, kidney cancer, breast cancer, colorectal cancer, and lung cancer (Okamatsu A., Motoyama K., Onodera R., Higashi T., Koshigoe T., Shimada Y., Hattori K., Takeuchi T., Arima H., Design and evaluation of folate-appended α-, β-, and γ-cyclodextrins having a caproic acid as a tumor). Selective antitumor drug carrier in vitro and in vivo. Biomacromolecules, 14, 4420-4428 (2013). FR-α is known to be involved in FR-α-mediated endocytosis (e.g., CLIC / GEEC endocytosis). Folate receptor-β (FR-β) is known to be expressed in acute myeloid leukemia and monocyte-derived cells. Avidin contains four identical subunits (homotetramer), and each subunit is known to be able to bind biotin (vitamin B7, vitamin H) with high affinity and specificity. According to the polyrotaxane A of the first aspect, even when the number of the ligand group possessed by the macrocyclic molecule a is one, the polyrotaxane A deforms so that the four ligand groups assemble in response to the four-point recognition structure of avidin to form what appears to be a tetravalent ligand group, and can thereby strongly interact with the avidin tetramer.

[0038] In the first aspect, for example, when the macrocyclic molecule a has a hydroxyl group such as cyclodextrin, it is preferable that the hydroxyl group is substituted with a ligand group (hereinafter also collectively referred to in this paragraph as "macrocyclic molecule substituent") directly or via an arbitrary linking group (e.g., an intracellularly degradable bond). The hydroxyl group of the cyclodextrin may be a hydroxyl group in the glucose constituting the cyclodextrin. In this case, the substituent of the macrocyclic molecule a may be bonded to the oxygen atom constituting the hydroxyl group via -O-CO-NH-, -O-COO-, -O-OC-, -O-, -O-C(OH)-, or -O-C(═S)NH- (in all cases, the leftmost -O- represents an oxygen atom derived from a hydroxyl group). In the first aspect, at least a portion of the macrocyclic molecule a may or may not be bonded to a ligand group via an intracellularly degradable bond, but it is preferable that the macrocyclic molecule a is bonded to a ligand group via an intracellularly degradable bond. Here, examples of the "intracellularly degradable bond" include the same specific and preferred examples as those described above for the "cap" for the "intracellularly degradable bond." In the first aspect, the ligand in the ligand group is not particularly limited as long as it can solve the above-mentioned problem, and examples include monosaccharides or oligosaccharides, vitamins or coenzymes, antigens or epitopes, antibodies or antibody fragments, or oligopeptides or polypeptides, with monosaccharides or oligosaccharides, or vitamins or coenzymes being preferred.

[0039] The monosaccharide is not particularly limited as long as it is recognized by the target receptor, and examples thereof include N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, fucose, sialic acid, etc. The oligosaccharide is not particularly limited as long as it is recognized by the target receptor, and examples thereof include disaccharides or more, and may or may not be trisaccharides or more. The upper limit is preferably 15 sugars or less, more preferably 10 sugars or less, and even more preferably 7 sugars or less. Specific examples of oligosaccharides include oligosaccharides containing at least one constituent monosaccharide selected from the group consisting of N-acetylgalactosamine (GalNAc), N-acetylglucosamine, galactose, glucose, fucose, and sialic acid. More specific examples of oligosaccharides include disaccharides such as lactose, sucrose, trehalose, and maltose; trisaccharides such as L-fucose-α(1→2)-D-galactose-β(1→3)-N-acetyl-D-glucosamine, L-fucose-α(1→2)-D-galactose-β(1→4)-N-acetyl-D-glucosamine, raffinose, panose, maltotriose, melezitose, and gentianose; and tetrasaccharides such as stachyose.

[0040] The vitamin or coenzyme is not particularly limited as long as it is recognized by the target receptor, and examples thereof include vitamin B (e.g., folic acid (FA), biotin, etc.), vitamin A, vitamin D, vitamin H, vitamin K, coenzyme A, etc., and from the viewpoint of solving the problems of the present invention, vitamin B (e.g., folic acid (FA), biotin, etc.) is preferred, and folic acid (FA) is more preferred.

[0041] The antigen or epitope is not particularly limited as long as it is recognized by the target receptor, and examples thereof include any cancer antigen or epitope, any tumor antigen (e.g., tumor-specific antigen, tumor-associated antigen, etc.) or epitope, any pathogen antigen or epitope, etc. The antibody or antibody fragment is not particularly limited as long as it is recognized by the target receptor, and may be a monoclonal antibody, a polyclonal antibody, or a fragment of such an antibody. From the perspective of solving the problems of the present invention, examples include an antibody or antibody fragment against any cancer antigen or epitope, an antibody or antibody fragment against any tumor antigen (e.g., tumor-specific antigen, tumor-associated antigen, etc.) or epitope, and an antibody or antibody fragment against any pathogen antigen or epitope, etc. The oligopeptide or polypeptide is not particularly limited as long as it is recognized by the target receptor, and examples thereof include any oligopeptide or polypeptide.

[0042] In the first aspect, specific examples and preferred examples of the macrocyclic molecule a include those similar to the specific examples and preferred examples described above for the "macrocyclic molecule." Specific examples and preferred examples of the axis molecule a include those similar to the specific examples and preferred examples described above for the "axis molecule." Specific examples and preferred examples of the cap a include those similar to the specific examples and preferred examples described above for the "cap," with groups having a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin) and groups having a polycyclic alicyclic ring (adamantyl group, norbornyl group, isonorbornyl group, etc.) being more preferred. Furthermore, as described above for the "cap," the cap a is preferably bonded to the axis molecule a via a bond that is degradable within cells, and more preferably bonded to the axis molecule a via a carbamate bond.

[0043] The polyrotaxane A according to the first aspect may or may not have an arbitrary label (e.g., a fluorescent label, an ultraviolet label, a radioactive label, etc.). From the viewpoint of accumulation in a target receptor and arbitrary therapeutic or diagnostic applications, it is preferable that it has an arbitrary label, more preferably an arbitrary labeling group, and it is even more preferable that the cap a or the above-mentioned "substituent of the macrocyclic molecule" has an arbitrary labeling group, and it is particularly preferable that the cap a has an arbitrary labeling group. Examples of the labeling group include a fluorescent labeling group (e.g., a group containing fluorescein), an ultraviolet labeling group (e.g., a group containing a benzophenone skeleton, a group containing a benzotriazole skeleton), and a radioactive labeling group (e.g., １８ and groups containing radioactive isotopes such as F (fluoro).

[0044] Polyrotaxane A according to the first aspect may or may not have any drug group (drug group) directly or via a linker, and preferably has any drug group (drug group) directly or via a linker. When the drug group (drug group) is a drug group that captures any substance extracellularly and recruits it into lysosomes or endosomes (e.g., the LYTAC antibody described below), a drug group that is resistant to degradation in lysosomes or endosomes, or a drug group that is active in lysosomes or endosomes, polyrotaxane A having any drug group directly or via a linker is suitable for delivering such drug groups. On the other hand, from the viewpoint of protecting a biological material from degradation in lysosomes or endosomes and promoting escape from lysosomes or endosomes by forming a polyion complex, delivery of a biological material that is not resistant to degradation in lysosomes or endosomes to the cytoplasm or cell nucleus is suitable using a polyion complex with polyrotaxane B described below. The polyrotaxane A having the above-mentioned "drug group resistant to degradation in lysosomes or endosomes" is also preferably delivered to the cytoplasm or cell nucleus.

[0045] Here, the terms "resistant" and "non-resistant" are not absolute and do not mean that a substance is not degraded at all or completely degraded, respectively. "Resistant" and "non-resistant" mean that a substance is relatively less susceptible to degradation and relatively more susceptible to degradation, respectively, when compared with outside of a lysosome or outside of an endosome and inside of a lysosome or inside of an endosome. A "drug group resistant to degradation in a lysosome or endosome" refers to a drug group that is resistant to degradation due to conditions in the lysosome or endosome environment that are different from those outside of a lysosome or outside of an endosome, such as physicochemical conditions such as pH (e.g., acidic conditions) or biological conditions such as lysosomal enzymes (e.g., hydrolases). On the other hand, a "biological material that is non-resistant to degradation in a lysosome or endosome" refers to a biological material that is not resistant to degradation due to conditions in the lysosome or endosome environment that are different from those outside of a lysosome or outside of an endosome, such as physicochemical conditions such as pH or biological conditions such as lysosomal enzymes. Furthermore, the term "active drug group" refers to a drug group (drug group) that has not been deactivated, and includes at least an active drug group. Here, "deactivation" refers to the loss of the drug's ability to perform its original activity.

[0046] The drug group that polyrotaxane A may have includes, as an active ingredient, a group containing any drug or pharmaceutical agent (e.g., a biological material, an anticancer agent, an antibiotic, a steroid, etc.) to be introduced into a cell and / or delivered to the target receptor. As described above, preferred are drug groups that capture any substance extracellularly and recruit it into a lysosome or endosome, drug groups that are resistant to degradation in a lysosome or endosome, or drug groups that are active in a lysosome or endosome. More specific examples of groups containing anticancer agents resistant to degradation in a lysosome or endosome, or groups containing anticancer agents active in a lysosome or endosome, include groups containing doxorubicin. More specific examples of groups containing antibiotics resistant to degradation in a lysosome or endosome, or groups containing antibiotics active in a lysosome or endosome, include groups containing aminoglycoside antibiotics (e.g., gentamicin, amikacin). More specifically, examples of groups containing a steroid drug resistant to degradation in a lysosome or an endosome, or groups containing a steroid drug active in a lysosome or an endosome, include groups containing a fat-soluble steroid drug (e.g., corticosteroids). Other examples of groups containing a drug resistant to degradation in a lysosome or an endosome, or groups containing a drug active in a lysosome or an endosome, include groups containing a nanoparticle drug (e.g., a nanoparticle drug having a particle size of 200 nm or less, preferably a nanoparticle drug having a particle size of 100 nm or less, more preferably a nanoparticle drug having a particle size of 50 nm or less, and even more preferably a nanoparticle drug having a particle size of 20 nm or less), and groups containing silica nanoparticles are preferred.In the polyrotaxane A related to the first aspect, from the viewpoint of not interfering with the movement (slide) of the macrocyclic molecule a in the axial direction of the axis molecule a, it is more preferable that the cap a or the above-mentioned "substituent of the macrocyclic molecule a" has any drug group directly or via a linker, and from the viewpoint of not interfering with the recognition of the ligand and the target receptor in the macrocyclic molecule a, it is even more preferable that the cap a has any drug group directly or via a linker.

[0047] When the polyrotaxane A according to the first aspect has any drug group directly or via a linker, from the viewpoint of delivery of the drug group to the cytoplasm or cell nucleus, it is also preferable that at least a part of the macrocyclic molecules a have an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)). This can promote the release of polyrotaxane A taken up into an endosome in a cell by disrupting the endosome into the cytoplasm. Specific and preferred examples of the amine-containing group include those described below as specific and preferred examples of the amine-containing group b in polyrotaxane B.

[0048] The above-mentioned "biological material" (hereinafter also referred to as "first biological material") refers to a natural or artificial substance consisting of amino acids or nucleic acids. More specific examples of the above-mentioned "first biological material" include antibodies or antibody fragments, nucleic acid molecules, proteins, or peptides, or fusions or complexes thereof. The above-mentioned antibodies or antibody fragments may be monoclonal antibodies, polyclonal antibodies, or fragments of these antibodies, and examples thereof include antibodies or antibody fragments against any cancer antigen or epitope, antibodies or antibody fragments against any tumor antigen (e.g., tumor-specific antigen, tumor-associated antigen, etc.) or epitope, and antibodies or antibody fragments against any pathogen antigen or epitope.

[0049] Examples of nucleic acid molecules include artificial or natural nucleic acid molecules, and more specifically, examples include siRNA (including derivatives such as shRNA), decoys, CpG oligos, miRNA, antisense DNA, antisense RNA, aptamers, mRNA, and nucleic acid vaccines (mRNA vaccines, DNA vaccines, etc.). From the viewpoint of resistance to degradation in lysosomes or endosomes, the nucleic acid molecule is preferably an artificial nucleic acid molecule stabilized with modified nucleic acids based on Enhanced Stability Chemistry. Here, modified nucleic acids refer to nucleic acids other than natural nucleic acids, and include nucleic acids containing nucleotide residues other than natural nucleotide residues.

[0050] Specifically, the modified nucleic acid is preferably a modified nucleic acid containing at least one nucleotide having at least one structure selected from the group consisting of a phosphorothioate structure, a cross-linked structure, and an alkoxy structure, and an artificial nucleic acid molecule containing such a modified nucleic acid is preferred. For example, when the phosphodiester bond linking the nucleotides has a phosphorothioate structure, nuclease resistance can be acquired, and the improved hydrophobicity can also improve uptake into cells or nuclei. Furthermore, when the sugar moiety of the nucleotide has a bridged structure such as 2',4'-BNA (2',4'-Bridged Nucleic Acid; also known as LNA (Locked Nucleic Acid)) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid), or an alkoxy structure such as 2'-O-methylation or 2'-O-methoxyethylation (2'-MOE), nuclease resistance can be acquired and the mRNA binding ability can be improved.

[0051] Examples of proteins / peptides include functional proteins / peptides such as nucleases (e.g., Cas proteins such as Cas9 nuclease, Cas9 nickase, Cas12a, and Cas13a), deaminases (cytidine deaminase, adenosine deaminase, and the like), and reverse transcriptases, as well as labeled proteins / peptides such as luciferase and fluorescently / radioactively labeled proteins. For example, the protein / peptide may be a nucleic acid-binding protein / peptide having a DNA-binding domain (e.g., zinc finger, helix-turn-helix, helix-loop-helix, winged helix, or leucine zipper) or an RNA-binding domain (e.g., zinc finger, KH, S1, PAZ, PUF, PIWI, and RRM (RNA recognition motif) domain). Examples of fusions include zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Examples of complexes include protein-DNA complexes and protein-RNA complexes, such as a complex of a Cas protein and a guide RNA (typically, a complex of Cas9 and an sgRNA (Cas9 RNP)).

[0052] From the viewpoint that the polyrotaxane A can function as a lysosome targeting chimera (LYTAC) antibody, the first biological material is preferably an antibody or antibody fragment. Here, a lysosome targeting chimera (LYTAC) antibody refers to a chimeric antibody that can degrade a captured antigen by delivering the antigen to a lysosome or endosome within a cell (e.g., a lysosome or endosome in a hepatocyte). (G. Ahn et al., Nat. Chem. Biol., 17, 937-946 (2021). Y. Zhou et al., ACS Cent. Sci., 499-506 (2021).)

[0053] Examples of the linker include a click reaction linker between a linear linker having an azacyclooctyne group (e.g., a DBCO (dibenzocyclooctyl) group) or a cyclooctyne group (e.g., a BCN (bicyclononyne) group) and an azide group. Examples of the linear linker include a divalent linear hydrocarbon chain having 1 to 20 carbon atoms (preferably 2 to 15 carbon atoms, more preferably 3 to 10 carbon atoms) that may or may not contain a keto group, an ether bond, an amide bond, a sulfide bond, a urethane bond, and / or a divalent amino group.

[0054] The polyrotaxane A of the first aspect is preferably a polyrotaxane to be contained in a composition containing the following polyrotaxane B for use, or to be used in combination with the following polyrotaxane B. [Polyrotaxane B: a polyrotaxane having a plurality of macrocyclic molecules b, an axis molecule b that passes through the rings of the macrocyclic molecules, and a cap b that is bonded to an end of the axis molecule b, and having the ability to form a polyion complex with a biological material (hereinafter also referred to as a "second biological material"), wherein the cap b has a host-guest interaction and / or an inclusion interaction with the cap a. ] In this manner, the second biological material described below, which is an active ingredient (e.g., a component having pharmacological activity in the body of an animal, including a human, or a component that, upon contact with other substances such as microbial contaminants, undergoes a physical or chemical change in the other substances or the active ingredient itself; The physical or chemical change referred to here includes binding, transfer, rearrangement, addition, detachment, decomposition, cleavage, oxidation, reduction, labeling, color development, luminescence, etc.), polyrotaxane B capable of forming a polyion complex, and polyrotaxane A having target receptor-directivity are linked (bonded) in the composition via host-guest interaction and / or inclusion interaction to form a complex. This allows the formation of a multifunctional polyrotaxane complex having polyion complex-forming ability and target receptor-directivity, thereby achieving multifunctionalization of the polyrotaxane. This ultimately enables delivery of the active ingredient to the target receptor. Note that polyrotaxane B may correspond to the "protean polymer (5G)" described above.

[0055] With regard to "a polyrotaxane to be contained in a composition containing polyrotaxane B or to be used in combination with polyrotaxane B," as described above, polyrotaxane A and polyrotaxane B have different functions, and therefore, it is also preferable that polyrotaxane A and polyrotaxane B are used as a combination drug (compound drug) or a kit product described below. In this case, polyrotaxane A and polyrotaxane B can be used in combination simultaneously or at an interval. Furthermore, the administration routes of polyrotaxane A and polyrotaxane B may be the same or different.

[0056] The cap b of the axis molecule b in polyrotaxane B has a host-guest interaction and / or an inclusion interaction with the cap a of the axis molecule a in polyrotaxane A, thereby linking (bonding) polyrotaxane A having target receptor directivity to polyrotaxane B having the ability to form a polyion complex with a biological material. It is known that cyclodextrin (e.g., α-, β-, or γ-cyclodextrin) acts as a host and undergoes host-guest interaction and / or inclusion interaction with a guest group having a polycyclic alicyclic ring (e.g., an adamantyl group, a norbornyl group, an isonorbornyl group, etc.) (e.g., Cromwell W.C., Bystrom K., Eftink M.R., Cyclodextrin-adamantanecarboxylate inclusion complexes: studies of the variation in cavity size. J. Phys. Chem., 89, 326-332 (1985)). Specific examples of cap a and cap b are preferably a group having a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin) or a group having a polycyclic alicyclic ring (e.g., an adamantyl group, a norbornyl group, an isonorbornyl group, etc.), from the viewpoint of the above-mentioned interaction, and cap a and cap b are preferably different from each other. That is, when cap a is a group having a polycyclic alicyclic ring (e.g., an adamantyl group, a norbornyl group, an isonorbornyl group, etc.), cap b is preferably a group having a cyclodextrin (e.g., an α-, β-, or γ-cyclodextrin), and when cap a is a group having a cyclodextrin (e.g., an α-, β-, or γ-cyclodextrin), cap b is preferably a group having a polycyclic alicyclic ring (e.g., an adamantyl group, a norbornyl group, an isonorbornyl group, etc.). From the viewpoint of avoiding steric hindrance during host-guest interaction and / or inclusion interaction, it is more preferable that cap a is a group having a polycyclic alicyclic ring (adamantyl group, norbornyl group, isonorbornyl group, etc.) and cap b is a group having a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin). The group having a polycyclic alicyclic ring is preferably an adamantyl group, and the cyclodextrin involved in the cap is preferably β-cyclodextrin.

[0057] In the composition, the molar ratio of polyrotaxane A to polyrotaxane B is not particularly limited. However, as will be described later with reference to the schematic diagram of FIG. 3 , when a ternary complex is formed, a molar ratio of polyrotaxane A greater than that of polyrotaxane B can form a ternary complex that is excellent in stability in an in vivo environment (e.g., in the presence of serum), and the molar ratio may be, for example, 1:0.25 to 1:0.75.

[0058] In the first aspect, the composition preferably further comprises a biological material capable of forming a polyion complex with the polyrotaxane B. Here, the "second biological material" capable of forming a polyion complex with the polyrotaxane B refers to a natural or artificial substance composed of amino acids or nucleic acids that is intended to be introduced into cells and / or delivered to the target receptor as an active ingredient. There are no particular limitations on the second biological material, as long as it has the ability to form a polyion complex with the polyrotaxane B (is capable of forming a polyion complex). In the present invention, the term "polyion complex" refers to a complex formed or formed by electrostatic interaction between polymers having multiple charges on their surfaces in an aqueous solution, buffer solution, or the like. From the viewpoint of protecting the biological material from degradation in lysosomes or endosomes and promoting escape from lysosomes or endosomes through the formation of a polyion complex, delivery of the biological material via a polyion complex with polyrotaxane B is suitable for delivering biological materials that are not resistant to degradation in lysosomes or endosomes to the cytoplasm or cell nucleus.

[0059] The molar ratio of the second biological material to the polyrotaxane B (and / or polyrotaxane A) in the composition is not particularly limited, but may be, for example, 1:0.25 to 0.25:1, preferably 1:0.5 to 0.5:1. The polyion complex can be formed by mixing the biological material with the polyrotaxane B. Mixing in a container can be performed by stirring at room temperature in an aqueous solution, buffer solution, or culture medium. The mixing time can be appropriately set depending on the polyrotaxane B and biological material, their concentrations, etc., but is typically about 5 minutes to overnight, 5 minutes to 6 hours, 5 to 180 minutes, 5 to 120 minutes, 5 to 60 minutes, or 5 to 30 minutes.

[0060] As described above, polyrotaxane A, polyrotaxane B, and the biological material have different functions, and therefore, polyrotaxane A, polyrotaxane B, and the biological material are preferably used as a combination drug (compound drug) or a kit product described below. In this case, polyrotaxane A, polyrotaxane B, and the biological material can be used together simultaneously or at intervals. Furthermore, the administration routes of polyrotaxane A, polyrotaxane B, and the biological material may be the same or different.

[0061] More specifically, the "second biological material" may be, for example, a nucleic acid molecule, a vector, a protein, a peptide, a fusion thereof, or a complex thereof. Examples of nucleic acid molecules include guide RNA (including derivatives such as single-guide RNAs (sgRNAs) and prime-editing guide RNAs (pegRNAs) (Chow, R.D. et al. Nat Biomed Eng (2020))), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), siRNA (including derivatives such as shRNA), decoy, CpG oligo, miRNA, antisense DNA, antisense RNA, aptamer, mRNA, and nucleic acid vaccines (mRNA vaccines, DNA vaccines, etc.).

[0062] Here, in order to induce genome editing by CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated proteins 9), it is necessary to introduce the Cas9 protein and guide RNA (nucleic acid) into the cell. As the introduction method, a method of introducing a plasmid DNA encoding the Cas9 protein and guide RNA, a method of introducing an mRNA and guide RNA encoding Cas9, and a method of introducing a complex of the Cas9 protein and guide RNA (Cas9RNP) are mainly known. Of these, the method of directly introducing preassembled Cas9RNP is known to have excellent genome editing efficiency and is also safe and convenient (S. Kim et al., Genome Res., 24: 112-1019 (2014)., M. Wang et al., Proc. Natl. Acad. Sci., 113: 2868-2873 (2015)).

[0063] Examples of vectors include viral vectors and plasmid vectors. Examples of proteins / peptides include functional proteins / peptides such as nucleases (e.g., Cas proteins such as Cas9 nuclease, Cas9 nickase, Cas12a, and Cas13a), deaminases (cytidine deaminase, adenosine deaminase, and the like), and reverse transcriptases, as well as labeled proteins / peptides such as luciferase and fluorescently / radioactively labeled proteins. For example, the protein / peptide may be a nucleic acid-binding protein / peptide having a DNA-binding domain (e.g., zinc finger, helix-turn-helix, helix-loop-helix, winged helix, or leucine zipper) or an RNA-binding domain (e.g., zinc finger, KH, S1, PAZ, PUF, PIWI, and RRM (RNA recognition motif) domain). Examples of fusions include zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Examples of complexes include protein-DNA complexes and protein-RNA complexes, such as a complex of a Cas protein and a guide RNA (typically, a complex of Cas9 and an sgRNA (Cas9 RNP)).

[0064] In polyrotaxane B, at least a portion of the macrocyclic molecules b preferably have an amine-containing group b that has a monovalent proton at neutral pH and a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)). The amine-containing group b has a monovalent proton at neutral pH, making it safe for biological components, while it has a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)), which is the intraendosomal environment, and thus can disrupt the endosomal membrane. This can promote the release of the complex with polyrotaxane A taken up into an intracellular endosome by disrupting the endosome into the cytoplasm.

[0065] The amine-containing group b is preferably a group having a secondary amino group and a primary amino group, and -L１ -NH-L ２ -NH ２ (Here, L １ and L ２ may be the same or different, and is a straight-chain or branched C1-6 alkylene group) is more preferred. Examples of the "straight-chain or branched C1-6 alkylene group" include -CH ２ -, -(CH ２ ) ２ -, -(CH ２ ) ３ -, -(CH ２ ) ４ -, -(CH ２ ) ５ -, -(CH ２ ) ６ -, -CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ ) -, -CH(CH ２ CH ３ ) CH ２ -, -CH ２ CH (CH ２ CH ３ ) -, -CH(CH ３ ) CH ２ CH ２ -, -CH ２ CH (CH ３ ) CH ２ -, -CH ２ CH ２ CH (CH ３ )-, etc. More specifically, the amine-containing group b includes a diethylenetriamine group (hereinafter, also simply referred to as "DET").

[0066] In polyrotaxane B, at least some of the macrocyclic molecules b preferably further have, in addition to the amine-containing group b, a group having an amino group (hereinafter simply referred to as an "intracellularly degradable amino group") bonded via an intracellularly degradable bond. That is, the amine-containing group b and the "intracellularly degradable amino group" are preferably different groups. In this case, the amine-containing group b is preferably bonded to the macrocyclic molecule via a bond that is not degraded or is difficult to degrade intracellularly. This allows the amino groups of the amine-containing group b to maintain the bond between polyrotaxane B and the biological material (polyion complex formation), even if some of the bonds of the intracellularly degradable amino groups are degraded and the amino groups dissociate from the macrocyclic molecule before release into the cell. Furthermore, after release into the cytoplasm, most of the bonds of the intracellularly degradable amino groups are cleaved, thereby weakening the bond between the biological material and polyrotaxane B (polyion complex formation) and promoting dissociation of the biological material from polyrotaxane B.

[0067] A group having an amino group via a bond that can be degraded in a cell (an intracellularly degradable amino group) is -L ３ -XL ４ -NH ２ (Here, L ３ and L ４ may be the same or different, and are a straight-chain or branched C1-6 alkylene group or a single bond, and X is an intracellularly degradable bond). Here, examples of the "intracellularly degradable bond" include the same specific and preferred examples as those of the "intracellularly degradable bond" described above for the "cap". In the "intracellularly degradable amino group", a group having an amino group (in the above formula, -L ４ -NH ２ As the group corresponding to the above, —CH ２ -NH ２ , -(CH ２ ) ２ -NH ２ , -(CH ２ ) ３ -NH ２ , -(CH ２ ) ４ -NH２ , -(CH ２ ) ５ -NH ２ , -(CH ２ ) ６ -NH ２ , and -CH(CH ３ ) CH ２ -NH ２ Examples of the "group having an amino group via a bond that can be decomposed in a cell" (intracellularly decomposable amino group) include -(CH ２ ) ２ -S-S-(CH ２ ) ２ -NH ２ (cystamine) is preferred.

[0068] For example, when the macrocyclic molecule b has a hydroxyl group such as cyclodextrin, the hydroxyl group is preferably substituted with the amine-containing group b and / or a group having an amino group bonded via an intracellularly degradable bond (hereinafter collectively referred to as "macrocyclic molecule substituent" in this paragraph). The hydroxyl group of the cyclodextrin may be a hydroxyl group in the glucose constituting the cyclodextrin. In this case, the substituent of the macrocyclic molecule may be bonded to the oxygen atom constituting the hydroxyl group via -O-CO-NH-, -O-COO-, -O-OC-, -O-, -O-C(OH)-, or -O-C(═S)NH- (in each case, the leftmost -O- represents an oxygen atom derived from a hydroxyl group). For example, when the macrocyclic molecule b is α-cyclodextrin, the oxygen atom constituting the hydroxyl group of the α-cyclodextrin may be bonded to -L via -O-CO-NH-. １ -NH-L ２ -NH ２ When a substituent represented by the following formula is bonded, it can be represented by the following structural formula:

[0069]

[0070] In the present invention, the "imprinting rate" refers to the ratio of the total number of amines (primary amines, secondary amines, or tertiary amines) present at the terminals (including near the terminals) of the groups (amine-containing groups b and / or intracellularly degradable amino groups) bound to the macrocyclic molecules b of polyrotaxane B to the maximum number of cationic monomers that can be bound to the above-mentioned biological material capable of forming a polyion complex. For example, when the biological material is Cas9RNP, it is known that the imprinting rate can be set to 100% when the number of amines is 927 (Toru Taharabaru et al., Polyrotaxane-based multi-step transformable materials for the delivery of Cas9 ribonucleoprotein. Applied Materials Today 27 (2022) 101488; Guojun Chen et al., A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein. complex for in vivo genome editing. Nature Nanotechnology volume 14, pages 974-980 (2019)).

[0071] For example, when the group bonded to the macrocyclic molecule b is a diethylenetriamine group (DET), only the terminal amino group of the diethylenetriamine group (DET) is counted as the "amine present at the end of the group bonded to the macrocyclic molecule," and internal secondary amines are not counted. In other words, one DET group provides one amine to the macrocyclic molecule b on the polyrotaxane B. In the present invention, the imprinting rate can be, for example, 20% or more, 50% or more, 80% or more, or 90% or more. The upper limit of the imprinting rate is not particularly limited, but examples include 200% or less, 150% or less, and 130% or less.

[0072] In polyrotaxane B, specific examples and preferred examples of the macrocyclic molecule b include the same specific examples and preferred examples as those described above for the "macrocyclic molecule." Specific examples and preferred examples of the axis molecule b include the same specific examples and preferred examples as those described above for the "axis molecule."

[0073] Furthermore, the polyrotaxane B is also preferably a polyrotaxane having a plurality of macrocyclic molecules b, an axial molecule b that passes through the rings of the macrocyclic molecules b, and a cap b that is bonded to an end of the axial molecule, wherein an amine-containing group b is bonded to at least some of the macrocyclic molecules b, and a group having an amino group is bonded to at least some of the macrocyclic molecules b via an intracellularly degradable bond.

[0074] (Method for Producing Polyrotaxane A) Polyrotaxane A can be produced by a method well known to those skilled in the art, in accordance with Production Examples 1 to 10 described in paragraphs 0046 to 0067 of Patent Document 1 (WO 2022 / 163729A1).

[0075] Furthermore, when the macrocyclic molecule in a polyrotaxane having a plurality of macrocyclic molecules, an axial molecule a penetrating the rings of the macrocyclic molecules, and a cap a bonded to the end of the axial molecule a has at least one hydroxyl group, the polyrotaxane A is preferably produced by a production method including the following (A), (B), or (C): [(A) when the ligand group has a carboxyl group directly or via a linking group, forming a urethane bond by condensing one end of a linear linker having amino groups at both ends with at least one of the hydroxyl groups, and introducing a linear linker having an amino group at the other end into the macrocyclic molecule, and forming an amide bond between the carboxyl group and the amino group at the end, thereby constructing at least some of the macrocyclic molecules having one ligand group via the linear linker including the amide bond and the urethane bond. ] [(b) when the ligand group has an amino group via a linear linker, constructing at least a portion of the macrocyclic molecule having one ligand group via the linear linker containing the urethane bond by condensing at least one of the hydroxyl group and the amino group to form a urethane bond.] [(c) when the ligand group has an amino group via a linear linker, preparing a branched linker having a branch having a primary amino group and at least two protected carboxy groups, condensing the primary amino group of the branched linker with at least one hydroxyl group of the macrocyclic molecule to form a urethane bond, thereby introducing the branched linker having the carboxy groups protected at at least two ends into the macrocyclic molecule, deprotecting the protected carboxy groups at the at least two ends, forming an amide bond between the amino group that the ligand has via the linear linker and the carboxy groups at the at least two ends to construct at least a portion of the macrocyclic molecule having two or more ligand groups via the branched linker containing the urethane bond and the at least two amide bonds. ]

[0076] The urethane bond formed by the condensation may be formed by any condensing agent (e.g., N,N-carbonyldiimidazole). The amide bond may be formed by any condensing agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide). The linking group may be a divalent linear hydrocarbon chain having 1 to 20 carbon atoms (preferably 2 to 15 carbon atoms, more preferably 3 to 10 carbon atoms) which may or may not contain a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group. The linear linker may be a divalent linear hydrocarbon chain having 1 to 20 carbon atoms (preferably 2 to 15 carbon atoms, more preferably 3 to 10 carbon atoms) which may or may not contain a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group.

[0077] Examples of the "protected carboxy group" include a carboxy group protected with any protecting group (e.g., a tert-butoxycarbonyl group, etc.). From the viewpoint of binding a larger number of ligands, the "at least two protected carboxy groups" are preferably "at least three protected carboxy groups," more preferably "at least four protected carboxy groups," and even more preferably "at least five protected carboxy groups." Examples of the upper limit include "eight or fewer protected carboxy groups," "seven or fewer protected carboxy groups," etc. Examples of the "branched linker having a branch having a primary amino group and at least two protected carboxy groups" include a branched hydrocarbon chain having 4 to 25 carbon atoms (preferably 6 to 20 carbon atoms, more preferably 7 to 15 carbon atoms), which may or may not contain a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group. A specific example of the above-mentioned "branched linker having a branch having a primary amino group and at least two protected carboxy groups" is Tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine.

[0078] (Method for Producing Polyrotaxane B) Polyrotaxane B can be produced by a method well known to those skilled in the art, in accordance with Production Examples 1 to 10 described in paragraphs 0046 to 0067 of Patent Document 1.

[0079] <<Complex (Ternicomplex)>> A second aspect of the present invention is a complex comprising the polyrotaxane A according to the first aspect, polyrotaxane B, and a second biological material, wherein the polyrotaxane B and the biological material form a polyion complex, wherein the polyrotaxane B has a plurality of macrocyclic molecules b, an axis molecule b penetrating the rings of the macrocyclic molecules, and a cap b bonded to an end of the axis molecule, and the polyrotaxane A and the polyrotaxane B are bonded via a host-guest interaction and / or an inclusion interaction between the cap a and the cap b. Figure 3 is a schematic diagram of a ternary complex comprising the polyrotaxane A, polyrotaxane B, and the second biological material. As shown in the schematic diagram of Figure 3, in the ternary complex, the core of the polyion complex of polyrotaxane B and the second biological material is sterically shielded by polyrotaxane A, which is thought to improve the stability of the ternary complex in an in vivo environment (e.g., in the presence of serum). As shown in Figure 3, the complex is a ternary complex consisting of polyrotaxane A, polyrotaxane B, and the second biological material, and can deliver the second biological material as an active ingredient to the target receptor due to the target receptor-directing ability of polyrotaxane A and the polyion complex-forming ability of polyrotaxane B. In other words, the ternary complex can function as a delivery agent (delivery carrier) to the target receptor.

[0080] Specific and preferred examples of the cap a and cap b (combination) for linking (binding) polyrotaxane A and polyrotaxane B to form a complex include those described above for the first aspect. The molar ratio of polyrotaxane A to polyrotaxane B in the ternary complex is not particularly limited, but may be 1:0.25 to 1:0.75. The molar ratio of the second biological material to polyrotaxane B (and / or polyrotaxane A) in the ternary complex is not particularly limited, but may be, for example, 1:0.25 to 0.25:1, with 1:0.5 to 0.5:1 being preferred.

[0081] Specific examples and preferred examples of polyrotaxane A, polyrotaxane B, second biological material, and target receptor include those described above for the first aspect. In particular, as described above for the first aspect, in polyrotaxane B, at least a portion of the macrocyclic molecules b preferably have an amine-containing group b that has a monovalent proton at neutral pH and a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)). This can promote the release of the ternary complex taken up into an intracellular endosome by disrupting the endosome into the cytoplasm. In other words, the ternary complex can function not only as a delivery agent (delivery carrier) to the target receptor but also as a cell introduction agent (cell introduction carrier) for the second biological material. Specific examples and preferred examples of the amine-containing group b include those described above for the first aspect.

[0082] <<Method for Producing a Composite (Ternicomposite)>> A third aspect of the present invention is a method for producing the composite (ternary composite) according to the second aspect, which includes mixing polyrotaxane A and polyrotaxane B, and mixing a second biological material. There are no particular limitations on the order of mixing polyrotaxane A and polyrotaxane B and mixing polyrotaxane B and the second biological material, but examples include mixing polyrotaxane A and polyrotaxane B first and then further mixing the second biological material. There are no particular limitations on the molar ratio of polyrotaxane A to polyrotaxane B, but examples include 1:0.25 to 1:0.75. There are no particular limitations on the molar ratio of the second biological material to polyrotaxane B (and / or polyrotaxane A), but examples include 1:0.25 to 0.25:1, and preferably 1:0.5 to 0.5:1.

[0083] Polyrotaxane A and polyrotaxane B can be mixed by stirring at room temperature in an aqueous solution, buffer solution, or medium. The mixing time can be appropriately set depending on the polyrotaxane A and polyrotaxane B and their concentrations, but is usually about 5 minutes to overnight, 5 minutes to 6 hours, 5 to 180 minutes, 5 to 120 minutes, 5 to 60 minutes, or 5 to 30 minutes.

[0084] The second biological material and polyrotaxane B (and / or polyrotaxane A) can be mixed by stirring in an aqueous solution, buffer solution, or culture medium at room temperature. The mixing time can be appropriately set depending on the polyrotaxane B and second biological material and their concentrations, but is usually about 5 minutes to overnight, 5 minutes to 6 hours, 5 to 180 minutes, 5 to 120 minutes, 5 to 60 minutes, or 5 to 30 minutes.

[0085] The polyion complex can be formed by mixing the biological material with the polyrotaxane B. Mixing in a container can be performed by stirring in an aqueous solution, buffer solution, or culture medium at room temperature. The mixing time can be appropriately set depending on the polyrotaxane B and second biological material, their concentrations, etc., but is usually performed for about 5 minutes to overnight, 5 minutes to 6 hours, 5 to 180 minutes, 5 to 120 minutes, 5 to 60 minutes, or 5 to 30 minutes.

[0086] <<Pharmaceutical Composition>> A fourth aspect of the present invention is a pharmaceutical composition comprising the polyrotaxane A according to the first aspect, or a pharmaceutical composition comprising the following polyrotaxane A, the following polyrotaxane B, and a second biological material, wherein the biological material is capable of forming a polyion complex with the polyrotaxane B. [Polyrotaxane A: a polyrotaxane comprising a plurality of macrocyclic molecules a, an axis molecule a penetrating the rings of the macrocyclic molecules a, and a cap a bonded to an end of the axis molecule a, wherein at least a part of the macrocyclic molecules a have one or more ligand groups, and the one or more ligand groups possessed by the macrocyclic molecules a are recognized by a target receptor that recognizes the one or more ligand groups, and the number of the ligand groups possessed by the macrocyclic molecules a is smaller than the number of the ligand groups recognized by the target receptor, or the number of the ligand groups possessed by the macrocyclic molecules a is equal to or greater than the number of the ligand groups recognized by the target receptor. ] [Polyrotaxane B: the polyrotaxane having a plurality of macrocyclic molecules b, an axis molecule b penetrating the rings of the macrocyclic molecules, and a cap b bonded to the end of the axis molecule, and having the ability to form a polyion complex with the second biological material, wherein the cap b has a host-guest interaction and / or an inclusion interaction with the cap a.] When the pharmaceutical composition according to the fourth aspect is a pharmaceutical composition containing polyrotaxane A according to the first aspect, as described above in the first aspect, it is preferable that the polyrotaxane A has an arbitrary drug group directly or via a linker. Specific examples and preferred examples of the drug group and linker include those described above in the polyrotaxane A according to the first aspect. This allows the polyrotaxane A itself to function as an active ingredient and be delivered to a target receptor as the active ingredient. The pharmaceutical composition according to the fourth aspect can form the ternary complex consisting of polyrotaxane A, polyrotaxane B, and a second biological material in the pharmaceutical composition, and can deliver the active ingredient to a target receptor. That is, the pharmaceutical composition according to the fourth aspect is preferably a pharmaceutical composition for delivery of a biological material to a target receptor.

[0087] In the pharmaceutical composition, the molar ratio of polyrotaxane A to polyrotaxane B is not particularly limited and may be 1:0.25 to 1:0.75, for example, 1:0.25 to 1:0.75. In the pharmaceutical composition, the molar ratio of second biological material to polyrotaxane B (and / or polyrotaxane A) is not particularly limited and may be, for example, 1:0.25 to 0.25:1, for example, 1:0.5 to 0.5:1 is preferred. Specific examples and preferred examples of polyrotaxane A, polyrotaxane B, biological material, and target receptor include those described above for the first aspect. Specific examples and preferred examples of cap a and cap b (combination) for linking (binding) polyrotaxane A and polyrotaxane B to form a complex include those described above for the first aspect. In particular, as described above in relation to the first aspect, in polyrotaxane B, at least a portion of the macrocyclic molecules b preferably have an amine-containing group b that has a monovalent proton at neutral pH and a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)). This can promote the ternary complex taken up into an intracellular endosome to be released into the cytoplasm by disrupting the endosome. In other words, the pharmaceutical composition according to the fourth aspect can function not only as a pharmaceutical composition for delivering a biological material to a target receptor, but also as a pharmaceutical composition for introducing a biological material into a cell. Specific and preferred examples of the amine-containing group b include those described above in relation to the first aspect.

[0088] The pharmaceutical composition according to the fourth aspect may be a pharmaceutical composition for oral or parenteral administration, and may be, for example, an injection such as an injection for intravenous injection, an injection for subcutaneous administration, an injection for intramuscular injection, or an infusion. Alternatively, the pharmaceutical composition according to the fourth aspect may be a cell treatment agent for cell therapy. When administered to a subject such as a mammal (including model animals such as mice and humans), the above-mentioned formulation may be administered orally, or an injection or infusion may be administered into the blood (into a vein or an artery).

[0089] The pharmaceutical composition according to the fourth aspect may or may not contain a pharmacologically acceptable carrier (formulation additive). The type of formulation additive used in the preparation of the pharmaceutical composition, the ratio of the formulation additive to the active ingredient, and the method of preparation of the pharmaceutical composition can be appropriately selected by those skilled in the art depending on the form of the composition. Formulation additives can generally be incorporated in an amount of 1% to 99% by weight of the active ingredient. Here, various organic or inorganic carrier substances commonly used as formulation materials are used as pharmacologically acceptable carriers, and are incorporated as excipients, lubricants, binders, disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid preparations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed. Suitable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, gum arabic, dextrin, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, etc. Suitable examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Suitable examples of binders include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc. Preferable examples of disintegrants include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, light anhydrous silicic acid, low-substituted hydroxypropylcellulose, etc. Preferable examples of solvents include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, etc.Suitable examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate. Suitable examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, and polyoxyethylene hydrogenated castor oil. Suitable examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, and glucose. Suitable examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Suitable examples of soothing agents include benzyl alcohol.

[0090] Suitable examples of preservatives include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Suitable examples of antioxidants include sulfites, ascorbic acid, etc. Suitable examples of coloring agents include water-soluble food tar dyes (e.g., food dyes such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, Food Blue No. 1 and No. 2, water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), natural dyes (e.g., β-carotene, chlorophyll, red iron oxide, etc.), etc. Suitable examples of sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, etc.

[0091] Dosage forms of pharmaceutical compositions include oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions, and suspensions; and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and intravitreal injections), drip infusions, topical preparations (e.g., intranasal preparations, transdermal preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, drip infusions, and sustained-release preparations, each of which can be safely administered orally or parenterally. Pharmaceutical compositions can be prepared by methods commonly used in the pharmaceutical technology field, such as those described in the Japanese Pharmacopoeia. Specific preparation methods for formulations are described in detail below.

[0092] For example, injections are produced by dissolving, suspending, or emulsifying the above-mentioned protein or polypeptide as an active ingredient in an aqueous solvent (e.g., distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (e.g., vegetable oils such as olive oil, sesame oil, cottonseed oil, and corn oil, propylene glycol, etc.) together with dispersants (e.g., polysorbate 80, polyoxyethylene hydrogenated castor oil 60, etc.), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), preservatives (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol, etc.), isotonicity agents (e.g., sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.). In this case, additives such as solubilizing agents (e.g., sodium salicylate, sodium acetate, etc.), stabilizers (e.g., human serum albumin, etc.), and soothing agents (e.g., benzyl alcohol, etc.) may be used, if desired.

[0093] Oral preparations are produced by adding, for example, excipients (e.g., lactose, sucrose, starch, D-mannitol, etc.), disintegrants (e.g., carboxymethylcellulose calcium, etc.), binders (e.g., pregelatinized starch, gum arabic, carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.), or lubricants (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.) to the above-mentioned protein or polypeptide as an active ingredient, followed by compression molding, and then coating with a coating base by a method known per se, as needed, for the purposes of taste masking, enteric coating, or sustained release. Examples of such coating bases include sugar coating bases, water-soluble film coating bases, enteric film coating bases, and sustained-release film coating bases. Sucrose is used as the sugar coating base, and one or more of talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax, etc. may also be used in combination. Examples of water-soluble film coating bases include cellulose polymers such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, and methylhydroxyethyl cellulose; synthetic polymers such as polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E (Eudragit E (trade name), Rohm Pharma Co., Ltd.), and polyvinylpyrrolidone; and polysaccharides such as pullulan.

[0094] Examples of enteric film coating bases include cellulose-based polymers such as hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; acrylic acid-based polymers such as methacrylic acid copolymer L [Eudragit L (trade name), Rohm Pharma Co., Ltd.], methacrylic acid copolymer LD [Eudragit L-30D55 ​​(trade name), Rohm Pharma Co., Ltd.], and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co., Ltd.]; and natural products such as shellac. Examples of sustained-release film coating bases include cellulose-based polymers such as ethyl cellulose; and acrylic acid-based polymers such as aminoalkyl methacrylate copolymer RS ​​[Eudragit RS (trade name), Rohm Pharma Co., Ltd.] and ethyl acrylate-methyl methacrylate copolymer suspension [Eudragit NE (trade name), Rohm Pharma Co., Ltd.]. Two or more of the above-mentioned coating bases may be mixed in an appropriate ratio. In addition, a light-shielding agent such as titanium oxide or iron sesquioxide may be used during coating.

[0095] The dosage and administration frequency of the pharmaceutical composition according to the fourth aspect vary depending on the subject, administration route, target disease, symptoms, etc., but those skilled in the art can appropriately select an appropriate dosage. For example, when administered into human blood, the daily dose can be 0.001 to 100 g, or 0.01 to 1000 mg / kg. The daily dose may also be divided and administered several times. The administration frequency can be daily, weekly, every two weeks, monthly, or once every few months. The administration period can be determined appropriately based on the improvement of symptoms, and can be one month, several months, six months, one year, several years, five years, or ten years. For example, when administered into human blood, the daily dose can be 0.001 to 100 g, or 0.01 to 1000 mg / kg. The daily dose may also be divided and administered several times. The administration frequency can be daily, weekly, every two weeks, monthly, or once every few months. The administration period can be appropriately determined based on the improvement of symptoms, and can be one month, several months, six months, one year, several years, five years, or ten years.

[0096] As described above, polyrotaxane A, polyrotaxane B, and the biological material have different functions. Therefore, polyrotaxane A, polyrotaxane B, and the biological material are preferably used as a combination drug (combined drug) or as a kit product (described later). In this case, polyrotaxane A, polyrotaxane B, and the biological material can be used simultaneously or at intervals. Furthermore, the administration routes of polyrotaxane A, polyrotaxane B, and the biological material may be the same or different. When these drugs are used in combination, each drug can be formulated by mixing it with a pharmacologically acceptable carrier, excipient, binder, diluent, etc., either separately or simultaneously, and administered orally or parenterally as a pharmaceutical composition. When drugs are formulated separately, the separate formulations can be mixed with a diluent or the like at the time of use and administered. However, the separate formulations may also be administered simultaneously or at intervals to the same subject. The pharmaceutical composition according to the fourth aspect also includes kit products in which separately formulated drugs are mixed with a diluent or the like at the time of use and administered (for example, an injection kit containing ampoules containing individual drugs in powder form and a diluent or the like for mixing and dissolving two or more drugs at the time of use), and kit products in which separately formulated drugs are administered simultaneously or separately at staggered times to the same subject (for example, a tablet kit in which tablets containing individual drugs are placed in the same or separate bags and, if necessary, have a column for writing the time at which the drugs are administered, for administering two or more types of tablets simultaneously or separately at staggered times).

[0097] The present invention also relates to a method for treating or ameliorating a disease or condition in a patient, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition according to the fourth aspect or a conjugate according to the second aspect. "Treatment" is an approach for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilized (i.e., not worsening) disease, delayed or slowed disease progression, amelioration or remission of the disease state, and remission (partial or total). "Treatment" may also mean increasing life expectancy relative to the expected life expectancy if not treated. An "effective amount" is an amount sufficient to achieve beneficial or desired clinical results, including clinical outcomes. An effective amount can be administered in one or more doses. Treated subjects include mammals such as humans, cows, horses, dogs, cats, pigs, sheep, etc., and preferably humans.

[0098] The present invention will be explained in more detail below by showing examples of the present invention, but the present invention is not limited to these examples and various applications are possible within the scope of the technical idea of ​​the present invention.

[0099] <Production Example 1: Preparation of polyrotaxane having caps a at both ends of the axis molecule (hereinafter simply referred to as "Ad-cap-PRX")> "Ad-cap-PRX" was prepared according to the following scheme. (Production Example 1-1) Preparation of 20 kDa polyethylene glycol (PEG) aminated at both ends (hereinafter simply referred to as "PEG-DAT") PEG (20 kDa, manufactured by Sigma) (56 g, 2.8 mmol) was dissolved in tetrahydrofuran (THF) (200 mL), and N,N-carbonyldiimidazole (CDI) (2.0 g, 12.4 mmol) was added. The mixture was stirred at 50°C for 18 hours under nitrogen purging. The reaction solution was added dropwise to ethylenediamine (6.0 mL, 88 mmol), and the mixture was stirred at 50°C for 2 hours under nitrogen purging. Ethanol (200 mL) was added to the reaction solution, and the mixture was allowed to stand at -20°C for 2 hours. The mixture was then centrifuged and the supernatant was removed. The precipitate was washed several times with cold ethanol and dried under reduced pressure to obtain PEG-DAT. Yield: 52.7 g, 94% yield (Production Example 1-2) Preparation of polypseudorotaxane having PEG-DAT (20 kDa) as the axis molecule a (hereinafter simply referred to as "PEG-DAT (20 kDa) / α-CyDPPRX") The above PEG-DAT (1.5 g) was added to a 12% (w / v) aqueous solution of α-cyclodextrin (α-CyD) (50 mL). After stirring overnight at 4°C, the mixture was centrifuged, the supernatant was removed, and PEG-DAT (20 kDa) / α-CyDPPRX was obtained by lyophilization. (Production Example 1-3) Adamantane acetic acid (2.45 g, 12.6 mmol), peptide condensation agent hexafluorophosphate (BOP reagent) (5.25 g, 11.8 mmol), 1-hydroxybenzotriazole (HOBt) (1.75 g, 11.4 mmol), and N-ethyldiisopropylamine (EDIPA) (2.28 mL, 13.2 mmol) were dissolved in DMF (100 mL), and the PEG-DAT / α-CyD PPRX (14 g) was added. After stirring at 4°C for 48 hours, the mixture was centrifuged and the supernatant was removed. The precipitate was washed twice with an equal volume mixed solvent of methanol / DMF (N,N-dimethylformamide) and twice with methanol, then suspended in DMSO (dimethyl sulfoxide) and precipitated by dropping into cold water under stirring. The above washing procedure was repeated three times, and Ad-cap-PRX was obtained by lyophilization. Yield: 10.23 g, Yield: 82% (based on PEG)

[0100] <Production Example 2: Preparation of Ligand Group> (Production Example 2-1) Preparation of GalNAc-triethylene glycol-ethylenediamine (hereinafter simply referred to as "GalNAc-TEG-EDA") GalNAc-TEG-EDA represented by the above formula was prepared as follows. First, GAPA-TEG (triethylene glycol)-Cl was prepared using galactosamine pentacetate (GAPA) as the starting material. GAPA (2.4 g, 6.16 mmol) dried under reduced pressure was dispersed in 20 mL of anhydrous dichloromethane (hereinafter simply referred to as "DCM"). Molecular sieves (4 Å) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) (3.78 mL, 21 mmol) were added, and the mixture was stirred at 50°C under nitrogen reflux for 18 hours. After adding 1.6 mL of triethylamine (TEA) to the reaction mixture on ice, 200 mL of saturated sodium bicarbonate solution was added. After washing, the organic layer was recovered. 200 mL of water was added to the collected organic layer, which was then washed. The organic layer was then concentrated and dried under reduced pressure to obtain GAPA-oxazoline as a crude product. The resulting GAPA-oxazoline was redissolved in 34 mL of anhydrous DCM, and molecular sieves (4 Å) and 2-[2-(2-chloroethoxy)ethoxy]ethanol (TEG-Cl; 1.38 mL, 9.5 mmol) were added. TMSOTf (0.62 mL, 3.42 mmol) was then added to the reaction mixture, which was then stirred at room temperature (23°C) for 18 hours. On ice, 1.6 mL of TEA was added to the reaction mixture, followed by 200 mL of saturated sodium bicarbonate solution. After washing, the organic layer was collected. 200 mL of water was added to the collected organic layer, which was then washed. Unnecessary by-products were removed from the organic layer by centrifugation and filtration, followed by concentration and drying under reduced pressure to obtain GAPA-TEG (triethylene glycol)-Cl as a crude product. The obtained GAPA-TEG-Cl was dissolved in 10 mL of DMSO and slowly added dropwise to ethylenediamine (EDA) (8 mL, 60.16 mmol) at 50°C while stirring. After stirring at 50°C for 18 hours, the precipitated salt was removed by centrifugation, and the supernatant was added dropwise to diethyl ether under ice cooling. After centrifuging the mixture, the lower layer was recovered and washed three times each with diethyl ether and acetone. The obtained precipitate was dissolved in water and concentrated using an evaporator. Redissolution in water and concentration were repeated five times, followed by drying under reduced pressure to obtain GalNAc-TEG-EDA.

[0101] Example 1 Preparation 1 of Polyrotaxane A Having One Ligand Group in Macrocyclic Molecule a Polyrotaxane A having one ligand group in macrocyclic molecule a (hereinafter simply referred to as "monoGalNAc-PRX") was prepared as follows. (Example 1-1) Preparation of Fluorescein-Labeled Hydroxypropyl Polyrotaxane (hereinafter simply referred to as "FAM-HP-PRX") FAM-HP-PRX was prepared according to the above scheme. (Preparation of fluorescein-labeled polyrotaxane (hereinafter simply referred to as "FAM-RRX")) 5(6)-carboxyfluorescein (FAM-COOH) (340 mg, 0.9 mmol), peptide condensation agent hexafluorophosphate (BOP reagent) (375 mg, 0.84 mmol), and N-ethyldiisopropylamine (EDIPA) (163 μL, 0.94 mmol) were dissolved in DMF (7.14 mL), and PEG-DAT (20 kDa) / α-CyDPPRX (1 g) obtained in Production Example 1-2 above was added. After stirring overnight on ice, the mixture was centrifuged and the supernatant was removed. The precipitate was washed twice with an equal volume mixed solvent of methanol / DMF and twice with methanol, then dissolved in DMSO, dialyzed (Spectra / Por (registered trademark) Membrane, molecular weight cut-off (MWCO): 6-8 kDa, solvent: water), and centrifuged to recover the precipitate. The precipitate was further washed five times with acetone and five times with water, and freeze-dried to obtain FAM-RRX. Yield: 666 mg, yield: 90% (based on PEG).

[0102] (Preparation of FAM-HP-PRX) The above FAM-PRX (250 mg) was dissolved in 1N NaOH (50 mL), and propylene oxide (9 mL) was added dropwise. After stirring overnight on ice, the reaction solution was dialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water) and lyophilized to obtain FAM-HP-PRX. Yield: 259 mg (96%). Note that the hydroxypropyl group (HP) was introduced to improve solubility.

[0103] (Example 1-2) Preparation of polyrotaxane A having one ligand group in macrocyclic molecule a (hereinafter simply referred to as "monoGalNAc-PRX") MonoGalNAc-PRX was prepared according to the above scheme. The above FAM-HP-PRX (20 mg, 249 nmol, α-cyclodextrin (α-CyD): 12.2 μmol) was dissolved in DMSO (1.2 mL), and N,N-carbonyldiimidazole (CDI) (21.6 mg, 134.4 μmol) was added. After stirring overnight at room temperature under nitrogen purging, the reaction solution was added to the above GalNAc-TEG-EDA (160 mg, 403 μmol) solution (2 mL of DMSO), and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and GalNAc-TEG-EDA were removed by dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), and GalNAc was deprotected by adding 10 N NaOH (final concentration: 1 N) and stirring on ice for 2 hours. After redialysis (Spectra / Por (registered trademark) Membrane, molecular weight cutoff (MWCO): 25 kDa, solvent: water), monoGalNAc-PRX (corresponding to polyrotaxane A) was obtained by lyophilization. Yield: 19.2 mg (77%). The monoGalNAc-PRX obtained is as follows: Number of α-CyDs: 49 Penetration rate (coverage): 21.6% GalNAc / α-CyD: 0.9 GalNAc / polymer: 44 Molecular weight: 100 kDa In order to avoid discrepancies in the cellular uptake efficiency test described below, the GalNAc modification rate per polymer was adjusted so that the total number of modifications as monoGalNAc was approximately equal.

[0104] Example 2: Preparation 2 of polyrotaxane A having one ligand group in macrocyclic molecule a (hereinafter simply referred to as "FA-PRX") Folic acid (FA)-PRX was prepared as described in the above scheme. The above FAM-HP-PRX (20 mg, 249 nmol, α-CyD: 12.2 μmol) was dissolved in DMSO (0.6 mL), and CDI (7.5 mg, 44 μmol) was added. After stirring overnight at room temperature under nitrogen purging, the reaction solution was added to a 1,2-bis(2-aminoethoxy)ethane (BAEE) (65 μL, 440 μmol) solution (DMSO 0.2 mL), and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and BAEE were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water), and BAEE-FAM-HP-PRX was obtained by lyophilization. Folic acid (FA) (88.2 mg, 0.2 mmol) and NHS (69 mg, 0.6 mmol) were dissolved in 5 mL of DMSO under heating, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (115 mg, 0.6 mmol) was added, followed by stirring at room temperature for 4 hours. BAEE-FAM-HP-PRX (20 mg) and 10 μL of TEA were dissolved in DMSO (0.6 mL) and added to the activated FA solution. After stirring for an additional 24 hours at room temperature, unreacted EDC, NHS, and FA were removed by dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: 1 M aqueous ammonia), and the mixture was redialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water) and then lyophilized to obtain FA-PRX (corresponding to polyrotaxane A). Yield: 20.5 mg (76%). The obtained FA-PRX had the following properties: α-CyD number: 49, penetration rate (coverage): 21.6%, FA / α-CyD: 0.94, FA / polymer: 46, molecular weight: 108 kDa.

[0105] Example 3 Preparation 3 of Polyrotaxane A Having One Ligand Group in Macrocyclic Molecule a (Hereinafter, Simply Referred to as “Ad-cap-GalNAc-PRX”) Ad-cap-GalNAc-PRX represented by the above formula was prepared as follows. Example 3-1 Preparation of 35 kDa Polyethylene Glycol (PEG) Aminated at Both Ends (hereinafter simply referred to as "PEG-DAT") As in Production Example 1-1, PEG (35 kDa, manufactured by Sigma) (56 g, 2.8 mmol) was dissolved in tetrahydrofuran (THF) (200 mL), and N,N-carbonyldiimidazole (CDI) (2.0 g, 12.4 mmol) was added. The mixture was stirred at 50°C for 18 hours under nitrogen purging. The reaction solution was added dropwise to ethylenediamine (6.0 mL, 88 mmol), and the mixture was stirred at 50°C for 2 hours under nitrogen purging. Ethanol (200 mL) was added to the reaction solution, and the mixture was allowed to stand at -20°C for 2 hours. The mixture was then centrifuged and the supernatant was removed. The precipitate was washed several times with cold ethanol and dried under reduced pressure to obtain 35 kDa PEG-DAT aminated at both ends. Yield: 52.7 g, 94% yield

[0106] (Example 3-2) Preparation of polypseudorotaxane having PEG-DAT (35 kDa) as the axis molecule a (hereinafter simply referred to as "PEG-DAT (35 kDa) / α-CyDPPRX") The above PEG-DAT (1.5 g) was added to a 12% (w / v) aqueous solution of α-cyclodextrin (α-CyD) (50 mL). After stirring overnight at 4°C, the mixture was centrifuged, the supernatant was removed, and PEG-DAT (35 kDa) / α-CyDPPRX was obtained by lyophilization.

[0107] Example 3-3: Adamantane acetic acid (2.45 g, 12.6 mmol), peptide condensation agent hexafluorophosphate (BOP reagent) (5.25 g, 11.8 mmol), 1-hydroxybenzotriazole (HOBt) (1.75 g, 11.4 mmol), and N-ethyldiisopropylamine (EDIPA) (2.28 mL, 13.2 mmol) were dissolved in DMF (100 mL), and the above PEG-DAT / α-CyD PPRX (14 g) was added. After stirring at 4°C for 48 hours, the mixture was centrifuged and the supernatant was removed. The precipitate was washed twice with an equal volume mixture of methanol and DMF and twice with methanol, then suspended in DMSO and precipitated by dropping into cold water with stirring. The above washing procedure was repeated three times, and Ad-cap-PRX was obtained by lyophilization. Yield: 10.23 g, Yield: 82% (based on PEG)

[0108] (HP of Ad-cap-PRX) The above Ad-cap-PRX (250 mg) was dissolved in 1N NaOH (50 mL), and propylene oxide (9 mL) was added dropwise. After stirring overnight on ice, the reaction solution was dialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water) and lyophilized to HP Ad-cap-PRX. Yield: 259 mg (96%). The hydroxypropyl group (HP) was introduced to improve solubility.

[0109] Example 3-4 Preparation of the Ad-cap-GalNAc-PRX The HP-modified Ad-cap-PRX (20 mg, 249 nmol, α-cyclodextrin (α-CyD): 12.2 μmol) was dissolved in DMSO (1.2 mL), and N,N-carbonyldiimidazole (CDI) (21.6 mg, 134.4 μmol) was added. After stirring overnight at room temperature under nitrogen purging, the reaction solution was added to the GalNAc-TEG-EDA (160 mg, 403 μmol) solution (DMSO 2 mL), and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and GalNAc-TEG-EDA were removed by dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), and GalNAc was deprotected by adding 10 N NaOH (final concentration: 1 N) and stirring on ice for 2 hours. After redialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), Ad-cap-GalNAc-PRX (corresponding to polyrotaxane A) was obtained by lyophilization. Yield: 19.2 mg (77%). The Ad-cap-GalNAc-PRX obtained is as follows: Number of α-CyDs: 86 Penetration rate (coverage): 21.5% Ligand / α-CyD: 1.7 Ligand / polymer: 147 Molecular weight: 210 kDa

[0110] Example 4 Preparation 4 of Polyrotaxane A Having One Ligand Group in Macrocyclic Molecule a (Hereinafter, Simply Referred to as “Ad-cap-FA-PRX”) Ad-cap-FA-PRX represented by the above formula was prepared as follows. The above-mentioned HP-conjugated Ad-cap-PRX (20 mg, 249 nmol, α-CyD: 12.2 μmol) was dissolved in DMSO (0.6 mL), and CDI (7.5 mg, 44 μmol) was added. After stirring overnight at room temperature under nitrogen purging, the reaction solution was added to a 1,2-bis(2-aminoethoxy)ethane (BAEE) (65 μL, 440 μmol) solution (DMSO 0.2 mL), and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and BAEE were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water), and Ad-cap-PRX after BAEE conjugation and HP-conjugation was obtained by lyophilization. FA (88.2 mg, 0.2 mmol) and NHS (69 mg, 0.6 mmol) were dissolved in 5 mL of DMSO under heating, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (115 mg, 0.6 mmol) was added, followed by stirring at room temperature for 4 hours. BAEE- and HP-conjugated Ad-cap-PRX (20 mg) and 10 μL of TEA were dissolved in DMSO (0.6 mL) and added to the activated FA solution. After stirring for an additional 24 hours at room temperature, unreacted EDC, NHS, and FA were removed by dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: 1 M aqueous ammonia), and the mixture was redialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water) and then lyophilized to obtain Ad-cap-FA-PRX (equivalent to polyrotaxane A). Yield: 20.5 mg (76%). The properties of the obtained Ad-cap-FA-PRX are as follows: Number of α-CyDs: 86 Penetration rate (coverage): 21.5% Ligand / α-CyD: 1.1 Ligand / polymer: 95 Molecular weight: 204 kDa

[0111] Example 5: Preparation 1 of polyrotaxane A having three ligand groups in macrocyclic molecule a> (Example 5-1) Preparation of a fluorescein-labeled hydroxypropyl polyrotaxane backbone having three carboxy groups to which ligand groups can be bound (hereinafter simply referred to as "triPRXbackbone") TriPRX backbon was prepared according to the above scheme. The above FAM-HP-PRX (20 mg, 249 nmol, α-CyD: 12.2 μmol) was dissolved in DMSO (1.2 mL), and N,N-carbonyldiimidazole (CDI) (15.6 mg, 96.72 μmol) was added. After stirring overnight at room temperature under nitrogen purging, Tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (hereinafter also referred to as "tri") (147 mg, 0.29 mmol) was added to the reaction solution, and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and tri were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: DMSO), and 1 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 1 hour to deprotect the carboxylic acid. The isobutene produced by deprotection and unreacted tri were removed by evaporating under reduced pressure and dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: DMSO). Further dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water) was performed, and then lyophilization was performed to obtain triPRX backbone. Yield: 18.4 mg (85%).

[0112] (Example 5-2) Preparation of polyrotaxane A having three ligand groups in macrocyclic molecule a (hereinafter simply referred to as "triGalNAc-PRX") TriGalNAc-PRX was prepared as described in the above scheme. The triPRX backbone (20 mg, 249 nmol, α-CyD: 12.2 μmol) was dissolved in DMSO (2.0 mL), and N-hydroxysuccinimide (NHS) (8 mg, 67 μmol) and EDC (13 mg, 67 μmol) were added. After stirring at room temperature for 3 hours, the reaction solution was added to a solution of GalNAc-TEG-EDA (160 mg, 134.4 μmol) and TEA (201.15 μmol) in DMSO (2 mL), and the mixture was stirred at room temperature for an additional 6 hours. Unreacted EDC, NHS, and GalNAc-TEG-EDA were removed by dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), and GalNAc was deprotected by adding 10 N NaOH (final concentration: 1 N) and stirring on ice for 2 hours. After redialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), triGalNAc-PRX (corresponding to polyrotaxane A) was obtained by lyophilization. Yield: 18.8 mg (79%). The properties of the obtained triGalNAc-PRX are as follows: Number of α-CyDs: 49 Penetration rate (coverage): 21.6% GalNAc / α-CyD: 0.9 GalNAc / polymer: 44 tri / α-CyD: 0.34 tri / polymer: 16 Molecular weight: 103 kDa In order to avoid discrepancies in the cellular uptake efficiency test described below, the GalNAc modification rate per polymer was adjusted so that the total number of modifications as monoGalNAc was approximately equal.

[0113] <Production Example 3: Preparation of polyrotaxane B capable of forming a polyion complex with a biological material and having caps b at both ends of the axis molecule (hereinafter simply referred to as "β-CyD-cap-35k LDS")> "β-CyD-cap-35k LDS" was prepared according to the following scheme. (Production Example 3-1) Preparation of 35 kDa polyethylene glycol (PEG) aminated at both ends (hereinafter simply referred to as "PEG-DAT") PEG (35 kDa, manufactured by Sigma) (56 g, 2.8 mmol) was dissolved in tetrahydrofuran (THF) (200 mL), CDI (2.0 g, 12.4 mmol) was added, and the mixture was stirred at 50°C for 18 hours under nitrogen purging. The reaction solution was added dropwise to ethylenediamine (6.0 mL, 88 mmol), and the mixture was stirred at 50°C for 2 hours under nitrogen purging. Ethanol (200 mL) was added to the reaction solution, and the mixture was allowed to stand at -20°C for 2 hours, followed by centrifugation and removal of the supernatant. The precipitate was washed several times with cold ethanol and dried under reduced pressure to obtain 35 kDa PEG-DAT. Yield: 52.7 g, 94% yield

[0114] (Production Example 3-2) Preparation of polypseudorotaxane having PEG-DAT (35 kDa) as the axis molecule b (hereinafter simply referred to as "PEG-DAT (35 kDa) / α-CyDPPRX") The above PEG-DAT (1.5 g) was added to a 12% (w / v) aqueous α-CyD solution (50 mL). After stirring overnight at 4°C, the mixture was centrifuged, the supernatant was removed, and PEG-DAT (35 kDa) / α-CyDPPRX was obtained by lyophilization.

[0115] (Production Example 3-3) Preparation of polyrotaxane having caps b at both ends of axis molecule b (hereinafter simply referred to as "β-CyD-cap-PRX") Glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) (1345.5 mg, 0.9 mmol; manufactured by Ensui-ko Sugar Refining Co., Ltd.), BOP reagent (375 mg, 0.84 mmol), and EDIPA (0.163 mL, 0.94 mmol) were dissolved in DMF (28.57 mL), and the above-mentioned PEG-DAT (35 kDa) / α-CyD PPRX (1 g) was added. After stirring at room temperature for 48 hours, the mixture was centrifuged and the supernatant was removed. The precipitate was washed twice with an equal volume mixed solvent of methanol / DMF and twice with methanol, then dissolved in DMSO, dialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 10 kDa, solvent: water), and centrifuged to recover the precipitate. The precipitate was washed five times with water and lyophilized to obtain β-CyD-cap-PRX. Yield: 444 mg, yield: 59% (based on PEG).

[0116] (Production Example 3-4) Preparation of Polyrotaxane B (β-CyD-cap-35kLDS) Capped at Both Ends of Axle Molecule b and Capped with Biological Materials: The above β-CyD-cap-PRX(PEG 35kDa) (50 mg, 0.37 μmol, α-CyD: 38 μmol) was dissolved in DMSO (3 mL), CDI (49 mg, 0.3 mmol) was added, and the mixture was stirred at room temperature for 24 hours under nitrogen atmosphere. Cystamine dihydrochloride (685 mg, 3.0 mmol) was dissolved in DMSO (10 mL), TEA (843 μL, 6.1 mmol) was added, and the mixture was desalted by stirring at room temperature for 30 minutes. Diethylenetriamine (DET) (330 μL, 3.0 mmol) was added to the desalted cystamine solution, and the mixture was stirred at room temperature for 30 minutes. The PRX solution was added dropwise, and the mixture was further stirred overnight at room temperature under nitrogen purging. The reaction solution was then added dropwise to cold ethanol while stirring to cause precipitation. After centrifugation and removal of the supernatant, the precipitate was washed five times with ethanol, and the ethanol was removed using an evaporator. The precipitate was then redissolved in water and lyophilized to obtain β-CyD-cap-35kLDS. Yield: 48.8 mg, yield: 81% The obtained β-CyD-cap-35kLDS has the following properties: α-CyD number: 98, penetration rate (coverage): 24.6%, Cys, DET / α-CyD: 0.5, 0.5, molecular weight: 166 kDa

[0117] Comparative Preparation Example 1: Preparation of a dextran compound (monoGalNAc-DEX) in which the ligand group is fluorescently modified with one FAM. A dextran compound (monoGalNAc-DEX) in which the ligand group is fluorescently modified with one FAM was prepared for use as a comparative control in the comparative tests described below. (Comparative Preparation Example 1-1) Preparation of FAM-HP-DEX Dextran 70 (DEX) (700 mg, 0.01 mmol) was dissolved in DMSO (20 mL), and CDI (20 mg, 0.124 mmol) was added. After stirring overnight at room temperature under a nitrogen atmosphere, the reaction solution was added dropwise to ethylenediamine (EDA) (165 μL, 1.24 mmol), and the mixture was further stirred overnight at room temperature under a nitrogen atmosphere. Unreacted CDI and EDA were removed by dialysis (Spectra / Por® Membrane, MWCO: 6-8 kDa, solvent: water), and EDA-DEX was obtained by lyophilization. 400 mg of EDA-DEX was dissolved in DMSO (20 mL), and FAM-COOH (27 mg, 70.8 μmol) activated with 2 equivalents of EDC / NHS was added. After stirring overnight at room temperature, unreacted FAM-COOH was removed by dialysis (Spectra / Por® Membrane, MWCO: 6-8 kDa, solvent: water), and FAM-DEX was obtained by lyophilization. FAM-DEX (215 mg) was dissolved in 1 N NaOH (43 mL), and propylene oxide (7.74 mL) was added dropwise. After stirring overnight on ice, the reaction solution was dialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water) and lyophilized to obtain FAM-HP-DEX. Yield: 251 mg (87%)

[0118] Comparative Preparation Example 1-2: Preparation of a dextran compound (monoGalNAc-DEX) fluorescently modified with one FAM ligand group. The above-mentioned FAM-HP-DEX (20 mg, 213 nmol) was dissolved in DMSO (1.2 mL), and CDI (18.5 mg, 115 μmol) was added. After stirring overnight at room temperature under nitrogen purging, the reaction solution was added to a GalNAc-TEG-EDA (136 mg, 345 μmol) solution (2 mL of DMSO), and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and GalNAc-TEG-EDA were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water), and 10 N NaOH (final concentration: 1 N) was added. The mixture was stirred on ice for 2 hours to deprotect the GalNAc. After redialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), monoGalNAc-DEX was obtained by lyophilization. Yield: 18.7 mg (78%). The properties of the obtained monoGalNAc-DEX are as follows: GalNAc / polymer: 44; Molecular weight: 110 kDa. To avoid discrepancies in the cellular uptake efficiency test described below, the GalNAc modification rate per polymer was adjusted so that the total number of monoGalNAc modifications was approximately the same.

[0119] Comparative Preparation Example 2: Preparation of a dextran compound (FA-DEX) fluorescently modified with one FAM ligand group. A dextran compound (FA-DEX) fluorescently modified with one FAM ligand group was prepared for use as a comparative control in the comparative tests described below. The above FAM-HP-DEX (20 mg, 213 nmol) was dissolved in DMSO (0.6 mL), and CDI (6.4 mg, 38 μmol) was added. After stirring overnight at room temperature under nitrogen purging, the reaction solution was added to a BAEE (55 μL, 377 μmol) solution (DMSO 0.2 mL), and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and BAEE were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water), and BAEE-FAM-HP-DEX was obtained by lyophilization. Folic acid (FA) (88.2 mg, 0.2 mmol) and NHS (69 mg, 0.6 mmol) were dissolved in 5 mL of DMSO under heating, and EDC (115 mg, 0.6 mmol) was added. The mixture was then stirred at room temperature for 4 hours. BAEE-FAM-HP-DEX (20 mg) and 10 μL of TEA were dissolved in DMSO (0.6 mL) and added to the activated FA solution. After stirring at room temperature for an additional 24 hours, unreacted EDC, NHS, and FA were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: 1 M aqueous ammonia). The mixture was then redialyzed (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water) and lyophilized to obtain FA-DEX. Yield: 18.1 mg (70%) The properties of the obtained FA-DEX are as follows: FA / polymer: 45 Molecular weight: 120 kDa To avoid discrepancies in the cellular uptake efficiency test described below, the FA modification rate per polymer was adjusted to be approximately the same.

[0120] Comparative Preparation Example 3: Preparation of a dextran compound (triGalNAc-DEX) in which the ligand groups are fluorescently modified with three FAMs. A dextran compound (triGalNAc-DEX) in which the ligand groups are fluorescently modified with three FAMs was prepared for use as a comparative control in the comparative tests described below. (Comparative Preparation Example 3-1) Preparation of triDEX backbone The above-mentioned FAM-HP-DEX (20 mg, 213 nmol) was dissolved in DMSO (1.2 mL), and CDI (13.3 mg, 82.99 μmol) was added. After stirring overnight at room temperature under nitrogen purging, Tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (tri) (125 mg, 0.25 mmol) was added to the reaction solution, and the mixture was further stirred overnight at room temperature under nitrogen purging. Unreacted CDI and tri were removed by dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: DMSO), and 1 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 1 hour to deprotect the carboxylic acid. The isobutene produced by deprotection and unreacted tri were removed by evaporating under reduced pressure and dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: DMSO), and the product was further dialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 25 kDa, solvent: water), followed by lyophilization to obtain triDEX backbone. Yield: 19 mg (89%)

[0121] Comparative Preparation Example 3-2: Preparation of a dextran compound (triGalNAc-DEX) fluorescently modified with three FAM ligand groups. The triDEX backbone (20 mg, 213 nmol) was dissolved in DMSO (2.0 mL), and NHS (7 mg, 58 μmol) and EDC (12 mg, 58 μmol) were added. After stirring at room temperature for 3 hours, the reaction solution was added to a solution of GalNAc-TEG-EDA (136 mg, 134.4 μmol) and TEA (172.5 μmol) (DMSO 2 mL), and the mixture was stirred at room temperature for an additional 6 hours. Unreacted EDC, NHS, and GalNAc-TEG-EDA were removed by dialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water), and GalNAc was deprotected by adding 10 N NaOH (final concentration: 1 N) and stirring on ice for 2 hours. After redialysis (Spectra / Por® Membrane, MWCO: 25 kDa, solvent: water), triGalNAc-PRX was obtained by lyophilization. Yield: 16.5 mg (71%). The properties of the obtained triGalNAc-DEX are as follows: GalNAc / polymer: 44 tri / polymer: 16 Molecular weight: 117 kDa To avoid differences in the cellular uptake efficiency test described below, the GalNAc modification rate per polymer was adjusted so that the total number of modifications as monoGalNAc was approximately equal.

[0122] Example 10: Test of cellular uptake efficiency of polyrotaxane A (monoGalNAc-PRX) having one ligand group in the macrocyclic molecule a and polyrotaxane A (triGalNAc-PRX) having three ligand groups in the macrocyclic molecule a The cellular uptake efficiency of polyrotaxane A (monoGalNAc-PRX) having one ligand group in the macrocyclic molecule a obtained in Example 1 and polyrotaxane A (triGalNAc-PRX) having three ligand groups in the macrocyclic molecule a obtained in Example 5 was tested using HepG2 cells (ASGPR-positive cells) as a hepatic parenchymal cell model, by detecting fluorescence derived from labeled FAM with a flow cytometer and confocal laser scanning microscope (CLSM). The above-mentioned monoGalNAc-DEX and triGalNAc-DEX were used as comparative control compounds.

[0123] HepG2 cells were cultured in a 24-well plate at 1.0 × 10 ５ The cells were seeded at a density of 100 cells / well and cultured for 24 hours in a medium containing 10% (v / v) FBS (fetal bovine serum). After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing various polymers (monoGalNAc-PRX, triGalNAc-PRX, monoGalNAc-DEX, and triGalNAc-DEX) (final concentrations of 0.78-200 nM) was added and incubated at 37°C and 5% CO. ２ The cells were incubated under a 5% CO2 concentration for 1.5-24 hours. The cells were washed twice with Hank's balanced salt solution (HBSS) and detached by the trypsin-EDTA method. The cells were collected by centrifugation (3,000 rpm), dispersed in 1 mL of HBSS containing 10% FBS, passed through a nylon mesh, and then analyzed at 1.0 x 10 s by centrifugation using a BD Accuri C6 flow cytometer (BD Bioscience). ４ HepG2 cells were plated in a 35 mm glass-bottom dish at a density of 4.0 × 10 cells. ４ The cells were seeded at a cell density of 100 cells / well and cultured in a medium containing 10% (v / v) FBS for 24 hours. After washing the cells twice with serum-free medium, 200 μL of serum-free medium containing various polymers (final concentration: 0.78-200 nM) was added, and the cells were incubated at 37°C and 5% CO２ The cells were incubated for 1.5 to 24 hours under a 5% concentration of HCl. The cells were washed twice with HBSS, and 100 μL of 4% paraformaldehyde was added. After incubation at room temperature for 10 minutes, fixation was performed. Nuclei were stained by adding 100 μL of Hoechst 33342 solution (2 μg / mL) and incubating at room temperature for 10 minutes. After washing twice more with phosphate-buffered saline (PBS), FAM-derived fluorescence was observed using a Leica confocal laser microscope (Leica TCS SP5). The results are shown in Figure 4.

[0124] 4 shows the results of a test of the cellular uptake efficiency of monoGalNAc-PRX, triGalNAc-PRX, monoGalNAc-DEX, and triGalNAc-DEX. In FIG. 4, values ​​are expressed as the mean ± standard error (n=3 for each group), and the significance level for monoGalNAc-DEX is *: p<0.001, and the significance level for triGalNAc-DEX is †: p<0.001.

[0125] As is clear from the results shown in Figure 4(A), after treatment of HepG2 cells, monoGalNAc-PRX, monoGalNAc-DEX, triGalNAc-PRX, and triGalNAc-DEX all showed treatment concentration-dependent cellular uptake efficiencies. As is clear from the results shown in Figure 4(B), after treatment of HepG2 cells, monoGalNAc-PRX, monoGalNAc-DEX, triGalNAc-PRX, and triGalNAc-DEX all showed treatment time-dependent cellular uptake, and the increase in cellular uptake was linear up to at least 24 hours. This linear increase is presumed to support the rapid internalization and re-presentation of ASGPR interacting with each of the GalNAc-modified polymers.

[0126] As is clear from the results shown in Figures 4(A) and (B), the cellular uptake efficiency of the PRX system was significantly higher than that of the DEX system under various treatment concentrations and time conditions. This suggests that the mobility of each GalNAc-PRX may be able to promote multivalent interactions while avoiding (resolving) spatial mismatch between the modified GalNAc and ASGPR. Notably, triGalNAc-DEX exhibited significantly higher cellular uptake efficiency than monoGalNAc-DEX, whereas triGalNAc-PRX and monoGalNAc-PRX exhibited similar cellular uptake efficiencies. This suggests that the monovalent GalNAc modified on monoGalNAc-PRX may be presented as a trivalent form, conforming to the structure of ASGPR.

[0127] In general, it is known that drugs, drug carriers, etc. bound to trivalent GalNAc exhibit uptake into hepatic parenchymal cells at a rate 10 times or more higher than that of monovalent GalNAc. (Prakash T.P., Graham M.J., Yu J., Carty R., Low A., Chappell A., Schmidt K., Zhao C., Aghajan M., Murray H.F., Riney S.,Booten S.L.,Murray S.F.,Gaus H.,Crosby J.,Lima W.F.,Guo S.,Monia B.P.,Swayze E.E.,Seth P.P.,Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. , 42, 8796-8807 (2014). , Prakash T. P. , Yu J. , Migawa M. T. , Kinberger G. A. , WanW. B. , Ostergaard M. E. , Carty R. L. , Vasquez G. , Low A. , Chappell A. , Schmidt K. , Aghajan M. , Crosby J. , Murray H. M. , Booten S. L. , Hsiao J. , Soriano A. , Machemer T. , County P. , Burel S. A. , Murray S. F. , Gaus H. , Graham M. J. , Swayze E. E. , Seth P. P. , Comprehensive Structure-Activity Relationship of Triantennary N-Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes. J. Med. Chem. , 59, 2718-2733 (2016). However, even in the DEX system where a difference in internalization efficiency was observed as in the above results, the difference was only about 3 to 4 times.The reason for this is that the GalNAc-modified polymer used above has approximately 44 GalNAc groups per molecule, and it is speculated that monoGalNAc-DEX was able to interact strongly to some extent even if it did not match the trimer structure of ASGPR. However, the significant difference in cellular uptake efficiency between monoGalNAc-DEX and triGalNAc-DEX strongly suggests the importance of trivalent GalNAc design for ASGPR. In addition, compared to triGalNAc-DEX, monoGalNAc-PRX and triGalNAc-PRX showed significantly higher cellular uptake, suggesting that the resolution of spatial mismatch due to the dynamic properties of PRX may be very important in ensuring highly efficient multivalent interactions between ligands and receptors.

[0128] Example 11: ASGPR competitive inhibition test using a competitive inhibitor (free GalNAc) for the cellular uptake of polyrotaxane A (monoGalNAc-PRX) having one ligand group in the macrocyclic molecule a and polyrotaxane A (triGalNAc-PRX) having three ligand groups in the macrocyclic molecule a Polyrotaxane A (monoGalNAc-PRX) having one ligand group in the macrocyclic molecule a obtained in Example 1 and polyrotaxane A (triGalNAc-PRX) having three ligand groups in the macrocyclic molecule a obtained in Example 5 were tested for ASGPR competitive inhibition using HepG2 cells (ASGPR-positive cells) as a liver parenchymal cell model and free GalNAc as a competitive inhibitor. The above-mentioned monoGalNAc-DEX and triGalNAc-DEX were used as comparative control compounds.

[0129] HepG2 cells were cultured in a 24-well plate at 1.0 × 10 ５ The cells were seeded at a cell density of 100 cells / well and cultured in a medium containing 10% (v / v) FBS for 24 hours. After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing free GalNAc (final concentration: 6.25-200 mM) was added, and the cells were incubated at 37°C and 5% CO ２After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing free GalNAc and various polymers (monoGalNAc-PRX, triGalNAc-PRX, monoGalNAc-DEX, and triGalNAc-DEX) (final free GalNAc concentration: 6.25 to 200 mM, final polymer concentration: 25 nM) was added, and the cells were incubated at 37°C and 5% CO ２ The cells were incubated for 1.5 hours under a high concentration of 0.1% HCl. The cells were washed twice with HBSS and detached by the Trypsin-EDTA method. The cells were collected by centrifugation (3,000 rpm), dispersed in 1 mL of HBSS containing 10% FBS, passed through a nylon mesh, and then analyzed at 1.0 × 10 s by centrifugation using a BD Accuri C6 flow cytometer (BD Bioscience). ４ The results are shown in Figure 5.

[0130] 5 shows the results of an ASGPR competitive inhibition test using a competitive inhibitor (free GalNAc) of the cellular uptake of monoGalNAc-PRX, triGalNAc-PRX, monoGalNAc-DEX, and triGalNAc-DEX. In FIG. 5 , values ​​are expressed as mean ± standard error (n=3 per group), with * representing significance for monoGalNAc-PRX, p<0.001 for +triGalNAc-PRX, † representing significance for +triGalNAc-DEX, and ‡ representing significance for +triGalNAc-DEX. The inhibition rate was calculated by expressing the cellular uptake as a relative value to the cellular uptake in the absence of a competitive inhibitor.

[0131] As is clear from the results shown in Figure 5, the cellular uptake of monoGalNAc-PRX, monoGalNAc-DEX, triGalNAc-PRX, and triGalNAc-DEX was inhibited in an inhibitor concentration-dependent manner, suggesting the involvement of ASGPR-mediated endocytosis. However, while the cellular uptake of monoGalNAc-DEX was inhibited by approximately 80% even at low concentrations (6.25 mM) of the inhibitor, the cellular uptake of monoGalNAc-PRX was less inhibited in the order monoGalNAc-PRX = triGalNAc-PRX >>> triGalNAc-DEX > monoGalNAc-DEX, with triGalNAc-PRX and monoGalNAc-PRX in particular only being inhibited by approximately 70% even at high concentrations (200 mM) of the inhibitor.

[0132] Such differences in competitive inhibition rates are thought to be due not to nonspecific adsorption or uptake into HepG2 cells, but to differences in the strength of the interaction between GalNAc modified with various polymers and ASGPR. That is, in the DEX system of immobile polymers, the interaction between trivalent GalNAc and ASGPR is stronger than that of monovalent GalNAc, so the interaction by the trivalent GalNAc is less likely to be inhibited by free GalNAc. Furthermore, it is presumed that GalNAc modified with PRX of mobile polymers avoids ASGPR whose binding region is blocked by free GalNAc, and recognizes and interacts with ASGPR that is not blocked by the inhibitor. In fact, when the uptake of various polymers was examined in HeLa cells, which hardly express ASGPR, it was suggested that almost none of the polymers was taken up into the cells (not shown). That is, it was suggested that the low competitive inhibition rate in HepG2 cells is not due to nonspecific interaction, but is due to the strength of specific interaction. These findings suggest that the order of strength of interaction with ASGPR is monoGalNAc-PRX≧triGalNAc-PRX>triGalNAc-DEX>monoGalNAc-DEX.

[0133] Furthermore, although no significant differences were observed in the cellular uptake efficiency or competitive inhibition efficiency between monoGalNAc-PRX and triGalNAc-PRX, monoGalNAc-PRX exhibited a slightly higher cellular uptake efficiency in both cases, suggesting a tendency for it to be less susceptible to competitive inhibitors. As described above, polyrotaxane A, in which macrocyclic molecule a has one or more ligand groups, is shown to be target receptor selective (preferably target receptor selective) due to highly efficient multivalent interactions between the ligand and receptor resulting from the elimination of spatial mismatches due to the dynamic properties of RX. As mentioned above, the above trimer model of ASGPR is merely a model of an oligomer that primarily exists, and it is known that it actually exists as a trimer or higher, up to a hexamer. (Stockert R.J., The asialoglycoprotein receptor:relationships between structure, function, and Physiol. Rev., 75, 591-609 (1995). Biochemistry, 29, 10009-10018 (1990). That is, while triGalNAc-PRX has the flexibility of a trivalent GalNAc, its valency can only be changed by a multiple of three, whereas monoGalNAc-PRX is expected to be able to freely present monovalent GalNAc and appropriately change its valency. Therefore, monoGalNAc-PRX may be highly efficiently recognized by the minor oligomers mentioned above, and may have slightly high interaction and internalization efficiency. Thus, it was suggested that monoGalNAc-PRX may be modified to match the structure of ASGPR on the hepatic parenchymal cell membrane, thereby 1) presenting GalNAc with a valency corresponding to the oligomeric structure of ASGPR, and 2) achieving multivalent interactions with multiple ASGPR oligomers while avoiding spatial mismatch, resulting in highly efficient uptake into hepatic parenchymal cells.

[0134] Example 12: Test of the uptake efficiency of polyrotaxane A (FA-PRX) having one ligand group in the macrocyclic molecule a into cancer cells. The highly efficient interaction achieved by eliminating spatial mismatch is also effective against monovalent receptors that do not form oligomers, such as ASGPR. The uptake efficiency of polyrotaxane A (FA-PRX) having one ligand group in the macrocyclic molecule a obtained in Example 2 into cancer cells was tested by using HeLa cells as the cancer cells and detecting fluorescence derived from the labeled FAM with a flow cytometer. The above-mentioned FA-DEX was used as a comparative control compound.

[0135] HeLa cells were cultured in a 24-well plate at 3.75 × 10 ４ The cells were seeded at a cell density of 100 cells / well and cultured for 24 hours in a medium containing 10% (v / v) FBS. After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing various polymers (final concentration: 62.5 to 1000 nM) was added, and the cells were incubated at 37°C and 5% CO ２ The cells were incubated for 3 hours under a high concentration of 0.1% HCl. The cells were washed twice with HBSS and detached by the Trypsin-EDTA method. The cells were collected by centrifugation (3,000 rpm), dispersed in 1 mL of HBSS containing 10% FBS, passed through a nylon mesh, and then analyzed at 1.0 × 10 s by centrifugation using a BD Accuri C6 flow cytometer (BD Bioscience). ４ The results are shown in Figure 6.

[0136] 6 shows the results of a test on the cellular uptake efficiency of FA-PRX and FA-DEX. In FIG. 6, values ​​are expressed as the mean ± standard error (n=4 for each group), and the significance level for FA-DEX is *: p<0.001.

[0137] As is clear from the results shown in Figure 6, FA-PRX and FA-DEX were taken up into cells in a concentration-dependent manner, and furthermore, it was suggested that the cellular uptake efficiency of FA-PRX was significantly higher than that of FA-DEX. This suggests that FA-PRX, due to its dynamic properties, efficiently presents FA to FR-α on the cancer cell membrane while avoiding spatial mismatch, and through multivalent interactions, is highly efficiently internalized into cancer cells. As described above, it was suggested that a dynamic targeting strategy mediated by PRX is useful for both oligomeric receptors such as ASGPR and monovalent receptors such as FR-α.

[0138] Example 13: Target cell uptake and stability tests of Ad-cap-GalNAc-PRX / β-CyD-cap-35kLDS / Cas9RNP and Ad-cap-FA-PRX / β-CyD-cap-35kLDS / Cas9RNP As shown in the schematic diagram of Figure 3, the ligand-modified PRX / 35kLDS / Cas9RNP sterically shields the cations in the 35kLDS / Cas9RNP core, improving the stability of the ternary complex in vivo (particularly in the presence of serum). Furthermore, by reducing cation-mediated cellular uptake, it is possible to improve the efficiency of targeting by the ligand-modified PRX. We tested whether targeting of Cas9RNP can be imparted by mixing Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX with β-CyD-cap-35kLDS and then mixing with Cas9RNP to form a ternary complex. Here, Cas9RNP was prepared from recombinant Streptococcus pyogenes Cas9 protein (N-terminally modified with a nuclear localization signal peptide (NLS); manufactured by Takara Bio Inc.), sgRNA, and crRNA (manufactured by Integrated DNA Technologies Japan). Therefore, we tested the uptake of this complex into target cells in the presence of serum, and its stability and targeting ability were examined. In the following examples, a system in which Ad-GalNAc-PRX and β-CyD-cap-35kLDS are mixed will be referred to as the "GalNAc system," and a system in which Ad-cap-FA-PRX and β-CyD-cap-35kLDS are mixed will be referred to as the "FA system."

[0139] The GalNAc system and FA system were prepared by adding β-CyD-cap-35kLDS in solid form to an aqueous solution of Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX, followed by vortexing. To modify both termini of β-CyD-cap-35kLDS, the molar ratio of Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX to β-CyD-cap-35kLDS was adjusted to 2:1 (final concentration: 1 mg / mL (in vitro) or 2 mg / mL (in vivo) of β-CyD-cap-35kLDS). The uptake of the ternary complex of the GalNAc system or FA system and Cas9RNP (29.2 nM) into target cells (HepG2 cells or HeLa cells) was measured using a flow cytometer as follows.

[0140] (Uptake of GalNAcsystem / Cas9RNP into HepG2 cells) HepG2 cells were cultured in a 24-well plate at 1.0 × 10 ５ The cells were seeded at a cell density of 100 cells / well and cultured for 24 hours in a 10% (v / v) FBS-containing medium. After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing GalNAcsystem / Cas9RNP (mixing ratio 125% of imprinting) or 10% FBS-containing medium (final concentration 29.2 nM) was added, and the cells were incubated at 37°C and 5% CO. ２ The cells were incubated for 4 hours under a 5% (v / v) FBS concentration. The cells were washed twice with serum-free medium, and 500 μL of 10% (v / v) FBS-containing medium was added, followed by further culture for 5 days. The cells were washed twice with HBSS and detached by pipetting with 500 μL of HBSS. The cells were collected by centrifugation (3,000 rpm), dispersed in 1 mL of HBSS containing 10% FBS, passed through a nylon mesh, and then analyzed at 1.0 × 10 s by centrifugation using a BD Accuri C6 flow cytometer (BD Bioscience). ４ HepG2 cells were plated in a 24-well plate at 1.0 × 10 ５The cells were seeded at a cell density of 100 cells / well and cultured in a medium containing 10% (v / v) FBS for 24 hours. After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing various polymers (final concentrations of 0.78 to 200 nM) was added, and the cells were incubated at 37°C and 5% CO ２ The cells were incubated for 1.5-24 hours under a high concentration of 0.1% EDTA. The cells were washed twice with HBSS and detached by the Trypsin-EDTA method. The cells were collected by centrifugation (3,000 rpm), dispersed in 1 mL of HBSS containing 10% FBS, passed through a nylon mesh, and then analyzed at 1.0 x 10 s by centrifugation using a BD Accuri C6 flow cytometer. ４ Cells were analyzed.

[0141] (Uptake of FAsystem / Cas9RNP into HeLa cells) 3.75 × 10 HeLa cells were placed in a 24-well plate. ４ The cells were seeded at a cell density of 100 cells / well and cultured for 24 hours in a 10% (v / v) FBS-containing medium. After washing the cells twice with serum-free medium, 500 μL of serum-free medium containing FAsystem / Cas9RNP (mixing ratio 125% of imprinting) or 10% FBS-containing medium (final concentration 29.2 nM) was added, and the cells were incubated at 37°C and 5% CO. ２ The cells were incubated for 4 hours under a 5% (v / v) FBS concentration. The cells were washed twice with serum-free medium, and 500 μL of 10% (v / v) FBS-containing medium was added, followed by further culture for 5 days. The cells were washed twice with HBSS and detached by pipetting with 500 μL of HBSS. The cells were collected by centrifugation (3,000 rpm), dispersed in 1 mL of HBSS containing 10% FBS, passed through a nylon mesh, and then analyzed at 1.0 × 10 s by centrifugation using a BD Accuri C6 flow cytometer. ４ Cells were analyzed.

[0142] The results are shown in Figures 7 and 8. Figure 7 shows the results of uptake of the ternary complex of GalNAc system and Cas9RNP into target cells (HepG2 cells). In the figure, values ​​are the mean ± standard error (n = 3 for each group). (A) shows the results in the absence of 10% FBS. The significance level relative to administration of Cas9RNP alone is *: p < 0.05, and the significance level relative to +GalNAc system is †: p < 0.05. (B) shows the results in the presence of 10% FBS. The significance level relative to administration of Cas9RNP alone is *: p < 0.05, and the significance level relative to +β-CyD-cap-35kLDS is †: p < 0.05. (C) shows the relative cellular uptake efficiency in the presence of 10% FBS compared to the absence of 10% FBS, and the significance level for +β-CyD-cap-35kLDS is *: p<0.05.

[0143] Figure 8 shows the results of uptake of the ternary complex of FAsystem and Cas9RNP into target cells (HeLa cells). (D) shows the results in the absence of 10% FBS. The significance level for administration of Cas9RNP alone is *: p<0.05, and the significance level for +FAsystem is †: p<0.05. (E) shows the results in the presence of 10% FBS. The significance level for administration of Cas9RNP alone is *: p<0.05, and the significance level for +β-CyD-cap-35kLDS is †: p<0.05. (F) shows the relative cellular uptake efficiency in the presence of 10% FBS compared to the absence of 10% FBS. The significance level for +β-CyD-cap-35kLDS is *: p<0.05.

[0144] As is clear from the results shown in Figure 7(A) and Figure 8(D), in the absence of serum, both GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP showed significantly lower cellular uptake than β-CyD-cap-35kLDS / Cas9RNP.

[0145] On the other hand, as is clear from the results shown in Figures 7(B) and (C) and Figures 8(E) and (F), it was suggested that the cellular uptake of both GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP in the presence of serum (10% FBS) was significantly higher than that of β-CyD-cap-35kLDS / Cas9RNP.

[0146] The reason for the significant decrease in cellular uptake of β-CyD-cap-35kLDS / Cas9RNP in the presence of serum may be that the β-CyD-cap-35kLDS / Cas9RNP complex dissociated due to electrostatic competition with proteins contained in serum, or that serum proteins adsorbed to the β-CyD-cap-35kLDS / Cas9RNP surface, thereby attenuating cation-mediated uptake. In contrast, both GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP maintained cellular uptake in the presence of serum, and the maintenance rate was suggested to be 100% or higher.

[0147] Example 14: Test of GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP uptake into target cells under competitive inhibition conditions. A test of GalNAcsystem / Cas9RNP uptake into HepG2 cells or FAsystem / Cas9RNP uptake into HeLa cells was performed using a 10% FBS-containing medium containing free GalNAc (final concentration 100 mM) or free FA (final concentration 4 mM) as a competitive inhibitor. The results are shown in Figure 9.

[0148] Figure 9 shows the results of a test on the uptake of GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP into target cells under competitive inhibition conditions. In (A), the significance level for GalNAc(-) is *: p<0.05. In (B), the significance level for FA(-) is *: p<0.05. As is clear from the results shown in Figure 9(A), the uptake of GalNAcsystem / Cas9 RNP into HepG2 cells was significantly reduced in the presence of free GalNAc. This suggests that GalNAcsystem / Cas9RNP is taken up into HepG2 cells via ASGPR.

[0149] Similarly, as is clear from the results shown in Figure 9 (B), the uptake of FAsystem / Cas9RNP into HeLa cells was significantly reduced in the presence of free FA. This suggests that FAsystem / Cas9RNP is taken up into HeLa cells via FR-α. These results suggest that GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP are sterically shielded from the cations in the 35k LDS / Cas9RNP core, improving the stability of the complex in the presence of serum, and further suppressing cation-mediated cellular uptake, suggesting that they are selectively taken up into target cells.

[0150] Example 15: Testing in vivo genome editing effect in target tissue (liver) after single administration of GalNAcsystem / Cas9RNP into mouse tail vein The previous examples suggested that GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP may have excellent stability and targeting ability in the presence of serum. Therefore, the in vivo genome editing effect in target tissue (liver) after single administration of GalNAcsystem / Cas9RNP into mouse tail vein was tested.

[0151] 500 μL of HBSS containing GalNAcsystem / Cas9RNP (415 pmol) was administered into the tail vein of healthy mice (Balb / c, 4-week-old, male). sgTTR, targeting atypical transthyretin (TTR), was used as the single-stranded guide RNA (sgRNA). Seven days after administration, the liver and other organs (heart, lungs, kidneys, and spleen) were collected by perfusion. After collection, 1 mL of TRIzol was added to the rapidly frozen liver tissue, and total RNA was extracted after homogenization. Expression of TTR mRNA relative to GAPDH was quantified using the real-time PCR method described above. It is known that hereditary ATTR amyloidosis develops when atypical transthyretin (TTR) produced in the liver forms insoluble amyloid fibrils and deposits throughout the body.

[0152] The results are shown in Figure 10. Figure 10 shows the results of an in vivo genome editing effect test in the target tissue (liver) after a single administration of GalNAcsystem / Cas9RNP into the tail vein of mice. In Figure 10, values ​​are expressed as the mean ± standard error of n = 3 for each group, and the significance level relative to administration of Cas9RNP alone or sgCont is *: p < 0.05.

[0153] As is clear from the results shown in Figure 10 (a), GalNAcsystem / Cas9RNP, Cas9RNP alone, β-CyD-cap-35k LDS / Cas9RNP, etc. It was suggested that it is possible to induce about 20% of TTR knockout, that is, genome editing effect, which is highly efficient compared to. Note that Figure 10 (b) shows the results confirmed that GalNAcsystem / Cas9RNP prepared using non-targeting sgRNA (sgCont) does not show TTR knockout.

[0154] Example 16: Test of in vivo genome editing effect in target tissue (cancer) after single administration of FAsystem / Cas9RNP into mouse tail vein The in vivo genome editing effect in target tissue (cancer) after single administration of FAsystem / Cas9RNP into mouse tail vein was tested. HeLa / GFP cell suspension (1 x 10 ６Cells / 100 μL) were inoculated into the left hind leg of BALB / c nu / nu mice. Approximately four days later, mice with tumors reaching a major axis of 5 mm were administered 500 μL of HBSS containing FAsystem / Cas9RNP (415 pmol) into the tail vein. GFP-targeting sgRNA (sgGFP) was used. Five days after administration, perfusion was performed and the tumor and other organs (liver, heart, lungs, kidneys, and spleen) were collected. After collection, RIPA (Radioimmunoprecipitation) buffer (1 mL per 50 mg of tumor) was added according to the weight of the flash-frozen tumor and then homogenized. After centrifugation at 5,000 rpm for 10 minutes, 200 μL of the supernatant was centrifuged again. 50 μL of the supernatant was collected, and the GFP fluorescence intensity was measured using a fluorescent plate reader. The results are shown in Figure 11.

[0155] 11 shows the results of an in vivo genome editing effect test in target tissues (cancer) after a single administration of FAsystem / Cas9RNP into the tail vein of mice. In FIG. 11, values ​​are expressed as mean ± standard error for n = 3 in each group, and the significance level relative to administration of Cas9RNP alone is *: p < 0.05.

[0156] As is clear from the results shown in Figure 11 (a), FAsystem / Cas9RNP, Cas9RNP alone, β-CyD-cap-35kLDS / Cas9RNP It was suggested that it is possible to induce about 60% of GFP knockout, that is, genome editing effect, which is highly efficient compared to. Note that Figure 11 (b) shows the results confirmed that FAsystem / Cas9RNP prepared using non-targeting sgRNA (sgCont) does not show GFP knockout.

[0157] Conventional LNP carriers for Cas9RNP required large doses of Cas9RNP (2.5 mg / kg x 3 doses of sgRNA) to induce approximately 50% genome editing efficacy in the liver after intravenous administration to mice. (Wei T., Cheng Q., Min Y.L., Olson E.N., Siegwart D. J., Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue-specific genome editing. Nat. Commun., 11, 3232 (2020)). In contrast, the GalNAcsystem and FAsystem of the present invention suggest the possibility of inducing a genome editing effect of about 20% in the liver and about 60% in cancer tissues, despite a single administration of only 415 pmol of Cas9RNP (about 0.67 mg / kg as sgRNA). In addition, the DET group disrupts endosomes, releasing GalNAcsystem / Cas9RNP and FAsystem / Cas9RNP into the cytoplasm, and furthermore, nuclear localization of Cas9RNP by the nuclear localization signal peptide (NLS) has been demonstrated.

[0158] This suggests that the GalNAc system and FA system of the present invention, which have the ability to control intracellular dynamics in addition to their stability in the presence of serum and highly efficient targeting due to the dynamic properties of PRX, may be useful for systemic delivery of Cas9RNP. In this example, GalNAc and FA were used as modified ligands, but by constructing PRXs modified with other ligands, it is expected that they can also be used for targeting tissues other than the liver and cancer tissues.

[0159] Example 17 Complex Formation Test of Polyrotaxane A (Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX) with Polyrotaxane B (5G 35k CD-cap) The Ad-cap-GalNAc-PRX prepared in Example 3 or the Ad-cap-FA-PRX prepared in Example 4 was mixed with polyrotaxane B (5G 35k CD-cap) at a molar ratio of 2:1. Here, polyrotaxane B (5G 35k CD-cap) is polyrotaxane B that has the ability to form a polyion complex with biological materials and has β-CyD as caps b at both ends of the axis molecule, and is the same as "β-CyD-cap-35kLDS" prepared in Production Example 3. After dilution with HBSS to a final concentration of 0.3 mg / mL, complex formation was confirmed by measuring particle size by dynamic light scattering using a Zetasizer Pro device (Malvern Instruments). The results are shown in Figure 12. In Figure 12, values ​​are expressed as mean ± standard error for n = 3 in each group.

[0160] As is clear from the results shown in Figure 12(A), an increase in particle size was confirmed by mixing polyrotaxane A (Ad-cap-GalNAc-PRX) and polyrotaxane B (5G 35k CD-cap), which suggests that a complex (ABA complex) was formed in which polyrotaxane A was linked to both ends of polyrotaxane B via host-guest interaction (inclusion interaction).

[0161] As is clear from the results shown in Figure 12(B), an increase in particle size was confirmed by mixing polyrotaxane A (Ad-cap-FA-PRX) and polyrotaxane B (5G 35k CD-cap). This indicates that a complex (ABA complex) was formed in which polyrotaxane A was linked to both ends of polyrotaxane B via host-guest interaction (inclusion interaction).

[0162] Example 18: Ternary complex (polyion complex) formation test by further mixing Cas9RNP with polyrotaxane A (Ad-cap-GalNAc-PRX or Ad-cap-FA-PRX) and polyrotaxane B (5G 35k CD-cap). Ad-cap-GalNAc-PRX prepared in Example 3 or Ad-cap-FA-PRX prepared in Example 4 was mixed with polyrotaxane B (5G 35k CD-cap) in a molar ratio of 2:1, and then further mixed with Cas9RNP to form a ternary complex (polyion complex). A binary complex (polyion complex) prepared by mixing polyrotaxane B (5G 35k CD-cap) and Cas9RNP without mixing with polyrotaxane A was used as a control. Here, Cas9RNP was prepared from recombinant Streptococcus pyogenes Cas9 protein (N-terminally modified with a nuclear localization signal peptide (NLS); manufactured by Takara Bio Inc.), sgRNA and crRNA (manufactured by Integrated DNA Technologies Japan). In the following examples, a system in which Ad-GalNAc-PRX and polyrotaxane B (5G 35k CD-cap) are mixed is referred to as the "GalNAc system", and a system in which Ad-cap-FA-PRX and polyrotaxane B (5G 35k CD-cap) are mixed is referred to as the "FA system". In the "GalNAc system", the macrocyclic molecule a has one GalNAC, which is less than the number of GalNAC ligands that are multivalently recognized by the target receptor ASGPR. In the "FA system," macrocyclic molecule a has one FA, the same number of ligands FA recognized by the target folate receptor monovalently. After diluting the ternary complex (sgRNA: 1.0 μg) with HBSS to 1 mL, the particle size and ζ potential were measured by dynamic light scattering using a Zetasizer Pro device (Malvern Instruments), and ternary complex formation was confirmed. In addition, the ternary complex (sgRNA: 0.2 μg, 10 μL) was confirmed by agarose gel electrophoresis. The results are shown in Figures 13(A) to (C). In Figure 13, values ​​are expressed as the mean ± standard error of n = 3 for each group, *: p < 0.05.

[0163] As is clear from the electrophoresis results shown in FIG. 13(A), it was confirmed that a polyion complex was formed in both the GalNAc system and the FA system.

[0164] As is clear from the results shown in Figure 13(B), both the GalNAc system and the FA system showed significantly smaller particle sizes than the polyion complex with polyrotaxane B (5G 35k CD-cap), which was used as a comparison control. This is thought to be due to compaction and monodispersion caused by the ternary complex. As is clear from the results shown in Figure 13(C), both the GalNAc system and the FA system showed significantly lower zeta potentials than the polyion complex with polyrotaxane B (5G 35k CD-cap), which was used as a comparison control. This is thought to be due to the fact that polyrotaxane B (5G 35k CD-cap) has a zeta potential due to its multiple amino groups, whereas complexation with polyrotaxane A, which has no or a low zeta potential, reduces the zeta potential of the entire complex. In other words, it is thought to be due to the formation of a ternary complex.

[0165] Example 19: Pharmacokinetics (2 hours after intravenous administration) test of ternary complex (polyion complex) of GalNAc system and Cas9RNP ５５０ The ternary complex (polyion complex) formed by mixing the 5G 35k CD-cap and Cas9RNP (CRISPR RNA (crRNA) and ATTO™) was administered into the tail vein of Balb / c mice (male, 4 weeks old) at a concentration of 100 pmoles / 200 μL. As a control, polyrotaxane B (5G 35k CD-cap) and Cas9RNP (CRISPR RNA (crRNA) and ATTO™) were administered into the tail vein of Balb / c mice (male, 4 weeks old). ５５０A binary complex (polyion complex) of labeled tracrRNA was also administered in the same manner. Two hours after the administration, the mice were perfused with PBS and 4% paraformaldehyde / phosphate buffer (4% PFA), and then various organs were collected and observed using an IVIS Imaging System (PerkinElmer). The results are shown in Figure 14. In Figure 14, H indicates the heart, Lu indicates the lungs, Li indicates the liver, S indicates the stomach, and K indicates the kidneys.

[0166] As is clear from the observation results shown in Figure 14, the ternary complex (polyion complex) using the GalNAc system accumulates in the liver and kidney more than the binary complex (polyion complex) using polyrotaxane B (5G 35k CD-cap). It is believed that the GalNAc contained in the GalNAc system is recruited by ASGPR expressed on the surface of liver cells and kidney cells, resulting in the accumulation of the ternary complex. This demonstrates the in vivo accumulation of the ternary complex at its target receptor (e.g., organ-selective and / or stable accumulation). Partial accumulation in the liver and kidneys was also observed for the binary complex (polyion complex) with polyrotaxane B (5G 35k CD-cap). This is thought to be because the binary complex is cationic and therefore easily taken up (e.g., non-selectively) by various cells, and also because vascular endothelial cells in the liver and kidneys (e.g., glomeruli) are fenestrated, resulting in the binary complex being taken up through the blood vessels and partially accumulating.

[0167] Example 20: Pharmacokinetics (2 hours after intravenous administration) test of ternary complex (polyion complex) of FAsystem and Cas9RNP A HeLa / GFP cell suspension (100 μL, 1 × 10 ６ Four days after transplantation, Cas9RNP (CRISPR RNA (crRNA) and ATTO™) were added to the FAsystem. ５５０The ternary complex (polyion complex) formed by mixing the 5G 35k CD-cap and Cas9RNP (CRISPR RNA (crRNA) and ATTO™) was administered into the tail vein of the mice at a concentration of 100 pmoles / 200 μL. As a control, polyrotaxane B (5G 35k CD-cap) and Cas9RNP (CRISPR RNA (crRNA) and ATTO™) were administered into the tail vein of the mice. ５５０ A binary complex (polyion complex) of labeled tracrRNA was also administered in the same manner. Two hours after the administration, the mice were perfused with PBS and 4% paraformaldehyde / phosphate buffer (4% PFA), and then various organs were collected and observed using an IVIS Imaging System (PerkinElmer). The results are shown in Figure 15. In Figure 15, H indicates the heart, Lu indicates the lung, Li indicates the liver, S indicates the stomach, K indicates the kidney, and T indicates the tumor.

[0168] As is clear from the observation results shown in Figure 15, the ternary complex (polyion complex) produced by the FA system accumulated in tumors more than the binary complex (polyion complex) produced by polyrotaxane B (5G 35k CD-cap). This demonstrates the in vivo accumulation of the ternary complex at its target receptor. Partial accumulation in tumors was also observed for the binary complex (polyion complex) produced by polyrotaxane B (5G 35k CD-cap). This is thought to be due to partial accumulation of the binary complex resulting from fragile blood vessels around the tumor.

[0169] Example 21 Preparation of Polyrotaxane A Having an Antibody on Cap a and One Ligand Group on Macrocyclic Molecule a (Hereinafter referred to as "monoGalNAc-PRX-antibody" (LYTAC)) As described below, monoGalNAc-PRX having a DBCO terminal was prepared, and N ３ The monoGalNAc-PRX-antibody (LYTAC) was prepared by mixing with the monoGalNAc-PRX-antibody. (Example 21-1) Preparation of HP-PRX in which caps a (adamantyl groups) at both ends have DBCO groups (hereinafter simply referred to as "DBCO-HP-PRX") It was prepared according to the following scheme. (1) 1.0 g of polypseudorotaxane (PEG: 20 kDa) was added to 597 mg (2.4 mmol) of 1,3-adamantanediacetic acid, BOP reagent (656 mg, 1.47 mmol), and EDIPA (0.285 mL, 1.65 mmol) in 3.57 mL of DMF and reacted overnight at 4°C. The precipitate was collected by centrifugation (4°C, 8,000 rpm, 10 min) and washed four times with methanol / DMF and three times with methanol. The precipitate was dissolved in DMSO, incubated for 1 hour, and then dialyzed against water for 2 days (cutoff value: 8 kDa). The precipitate was collected by centrifugation (4°C, 8,000 rpm, 10 min), washed 10 times with water, and lyophilized to obtain COOH-capped PRX (yield: 723 mg). (2) To 100 mg of the above COOH-capped PRX in 1 N aqueous sodium hydroxide, propylene oxide (3 g, 3.61 mL) was added dropwise and stirred overnight on ice. The mixture was then dialyzed against water for 2 days (cutoff value: 50 kDa) to obtain COOH-HP-PRX (yield: 102 mg). (3) *20 mg scale: To 20 mg (215 nmol) of the above COOH-HP-PRX in 0.6 mL of DMSO, 2.49 mg (6.45 μmol) of BOP, 1.2 μL (6.45 μmol) of EDIPA, and 1.8 mg (6.45 μmol) of DBCO-amine were added dropwise and stirred overnight at room temperature. After acetone precipitation, the mixture was washed five times with acetone to obtain DBCO-HP-PRX (yield: 16.5 mg).

[0170] (Example 21-2) Preparation of monoGalNAc-PRX having a DBCO terminus (hereinafter simply referred to as "DBCO-GalNAc-PRX (20k)") The "20k" above means that the PEG moiety is 20 kDa. This was prepared according to the following scheme using the DBCO-HP-PRX as a starting material.

[0171] (*10 mg scale) 16.1 mg (99 μmol) of CDI was added dropwise to 10 mg of the above DBCO-HP-PRX (106 nmol, CD: 6.68 μmol) in 0.6 mL of DMSO and stirred overnight at 25°C under a nitrogen atmosphere. Then, 100.8 mg (253.5 μmol) of GalNAc-TEG-EDA was added dropwise to 0.5 mL of DMSO and stirred at 25°C under a nitrogen atmosphere for 6 hours. After dialysis (MWCO: 50 kDa) for 2 days, DBCO-GalNAc-PRX(20k) was obtained by lyophilization. The obtained DBCO-GalNAc-PRX(20k) is as follows: DBCO: 1-1.5 / PRX GalNAc / CD: ~1.0 Penetration rate (coverage): 27.8% Molecular weight: 134 kDa Yield: 12 mg (84% yield)

[0172] (Example 21-3) Preparation of monoGalNAc-PRX-antibody (LYTAC) (Test to confirm the reactivity of the above DBCO-GalNAc-PRX (20k)) ３ -Anti-BSA (bovine serum albumin) antibody and FAM-TEG-N represented by the following formula ３ (Tokyo Chemical Industry Co., Ltd.) as a starting material, a monoGalNAc-PRX-antibody (LYTAC) was prepared according to the scheme shown in Figure 16 while confirming the reactivity of the DBCO-GalNAc-PRX (20k). ３ The anti-BSA antibody was prepared by modifying the sugar chain selectively with N-glycosylation of anti-BSA antibody (PROTEINTECH) using Siteclick™ Antibody Azido Modification Kit (Invitrogen). ３ was obtained by labeling. As shown in FIG. 16(A), FAM-TEG-N ３ DBCO-GalNAc-PRX(20k) reacted with N emits fluorescence based on FAM. ３ - Reacts with anti-BSA antibody and DBCO-GalNAc-PRX(20kJ) is FAM-TEG-N ３Based on the above scheme, the monoGalNAc-PRX-antibody (LYTAC) was prepared while confirming the reactivity of the DBCO-GalNAc-PRX(20k).

[0173] (*10 mg scale) N ３ 1 μg of anti-BSA antibody and an equimolar amount of the above DBCO-GalNAc-PRX (20kJ) were mixed and stirred overnight at 25°C. ３ 1 μL of (0.5 mg / mL) was added and stirred overnight at 25°C. After ultrafiltration using an Amicon Ultra (50 kDa; manufactured by Merck), the fluorescence of the filtrate was measured using a fluorescence plate reader. The results are shown in Figure 17. In Figure 17, values ​​are expressed as the mean ± standard error for n = 3 in each group, and * indicates "DBCO-GalNAc-PRX(20 kDa) + FAM-TEG-N ３ " p<0.05.

[0174] As is clear from the results shown in FIG. 17, "DBCO-GalNAc-PRX(20kJ)+FAM-TEG-N ３ " to "N ３ -Anti-BSA antibody + DBCO-GalNAc-PRX (20k) + FAM-TEG-N ３ " has a significant decrease in fluorescence intensity, and N ３ It can be said that the anti-BSA antibody reacted with DBCO-GalNAc-PRX (20k), and the target monoGalNAc-PRX antibody (LYTAC) was prepared.

[0175] Example 22: In vitro test of the ability of monoGalNAc-PRX-antibody (LYTAC) to deliver a target substance (e.g., an antigen) The antigen delivery ability (ability to take up antigen into HepG2 cells) of monoGalNAc-PRX-antibody (LYTAC) was compared in vitro with the previously reported triGalNAc-LYTAC (G. Ahn et al., Nat. Chem. Biol., 17, 937-946 (2021)). Here, the previously reported triGalNAc-LYTAC is a compound of triGalNAc-DBCO and N ３- LYTAC conjugated by click reaction with anti-BSA antibody. (Preparation of FITC-BSA) FITC (fluorescein isothiocyanate)-BSA used as an antigen in this in vitro test was prepared as follows. (*200 mg scale) 200 mg (300 nmol) of BSA was dissolved in PBS (pH 7.4) by inversion, and 3.5 mg (900 nmol) of FITC in 0.2 mL of DMF was slowly added. The solution was left in the dark (25°C, 24 hours). Dialysis (MWCO: 10 kDa, against PBS, 1 day) and dialysis (MWCO: 10 kDa, against water, 3 days) were performed (yield: 204 mg).

[0176] FIG. 18(A) is a diagram showing an outline of an in vitro test of the antigen delivery ability of monoGalNAc-PRX-antibody (LYTAC), and FIG. 18(B) is a diagram showing the results of the in vitro test of delivery ability.

[0177] As shown in FIG. 18(A), a mixture of various LYTACs and FITC-BSA was added to HepG2 cells and incubated for 4 hours, after which the antigen uptake ability into HepG2 cells was evaluated by flow cytometry.

[0178] (LYTAC preparation) N ３ Anti-BSA antibody was mixed with equimolar DBCO-GalNAc-PRX or triGalNAc-DBCO overnight at 25°C to prepare the antibody. (Reaction with antigen) FITC-BSA and various LYTACs were mixed at a molar ratio of 10:1 at room temperature for 20 minutes to react. (Treatment of cells) HepG2 cells (1.0 x 10) were seeded on a 24-well plate. ５ After washing twice with serum-free medium, 500 mL of each of the following samples 1) to 4) was added and incubated at 37°C for 24 hours. 1) FITC-BSA alone (final concentration: 100 nM) 2) N ３- Anti-BSA antibody (final concentration: 10 nM) + FITC-BSA (final concentration: 100 nM) mixed solution 3) GalNAc-PRX-LYTAC (final concentration: 10 nM) + FITC-BSA (final concentration: 100 nM) mixed solution 4) triGalNAC-LYTAC (final concentration: 10 nM) + FITC-BSA (final concentration: 100 nM) mixed solution

[0179] The results are shown in Figure 18(B). In Figure 18(B), values ​​are expressed as mean ± standard error for n=6 in each group, * indicates p<0.05 compared to FITC-BSA alone, and † indicates "FITC-BSA + N" ３ - p<0.05 vs. "anti-BSA antibody"; ‡ p<0.05 vs. "FITC-BSA+triGalNAC-LYTAC."

[0180] In GalNAc-PRX-LYTAC, macrocyclic molecule a has one GalNAC, which is fewer than the number of GalNAC ligands recognized by the target receptor ASGPR in a multivalent manner. As is clear from the results shown in Figure 18(B), GalNAc-PRX-LYTAC has the ability to deliver a target substance (e.g., an antigen) to in vitro target cells with a LYTAC efficiency that is significantly higher (preferably significantly higher) than that of the previously reported technology (triGalNAC-LYTAC).

[0181] Example 23 In Vivo Test of the Ability of monoGalNAc-PRX-Antibody (LYTAC) to Deliver a Target Substance (e.g., an Antigen) to a Target Cell (e.g., a Hepatocyte, Preferably, a Lysosome in a Hepatocyte) In comparison with triGalNAc-LYTAC, which has been previously reported (G. Ahn et al., Nat. Chem. Biol., 17, 937-946 (2021)), the ability of monoGalNAc-PRX-antibody (LYTAC) to deliver an antigen to a target cell (e.g., a hepatocyte, preferably, a lysosome in a hepatocyte) was tested in vivo for comparison.

[0182] FIG. 19(A) shows an outline of an in vivo test of the ability of monoGalNAc-PRX-antibody (LYTAC) to deliver an antigen to target cells (for example, hepatocytes, preferably lysosomes in hepatocytes).

[0183] (Procedure) The procedure for the in vivo antigen delivery test is described below with reference to Figure 19(A). As shown in Figure 19(A), 133 µg (50 µL) of FITC-BSA was administered into the tail vein of a Balb / c mouse (male, 4 weeks old). Immediately (approximately 30 seconds) after the administration into the tail vein, 50 µL of each type of LYTAC (10 equimolar amount to BSA) was intravenously administered into the retro-orbital venous plexus of the mouse's left eye. This allows the various types of LYTAC to capture the antigen FITC-BSA in the blood.

[0184] (Isolation of target cells (hepatocytes)) After 4 hours, the cells were cultured in HBSS (Ca ２＋ The liver was perfused with HCl (+) and collagenase buffer, and the liver was collected and cut with scissors while immersed in collagenase buffer. After homogenization using tweezers and pipetting, the homogenate was filtered through a nylon mesh and centrifuged (800 rpm, 5 minutes), after which the supernatant was removed. The liver was redispersed in 1% BSA, centrifuged (800 rpm, 5 minutes), the supernatant was removed, and the liver parenchymal cells were isolated by centrifuging again in 1% BSA (800 rpm, 5 minutes), the supernatant was removed, and the liver was redispersed in HCl (+) and centrifuged (800 rpm, 5 minutes), the supernatant was removed, and the liver was isolated by centrifuging again in HCl (+) and collagenase buffer.

[0185] (Lysosome isolation) Lysosomal fractions were extracted using the Minute™ Lysosome Isolation Kit (manufactured by Invent Biotechnologies), and 100 μL of Minute™ Non-Denatured Protein Solubilization Reagent was added. Measurements were then performed using a fluorescent plate reader. The results are shown in Figure 19(B). Figure 19(B) shows the quantification of FITC-BSA in lysosomes of hepatocytes in an in vivo antigen delivery test.

[0186] In Figure 19(B), the amount of LYTAC administered was 1 / 10 the molar amount of FITC-BSA, so the theoretical maximum accumulation amount was 10%. In Figure 19(B), values ​​are expressed as mean ± standard error for n = 4 in each group, * indicates p < 0.05 compared to saline, † indicates p < 0.05 compared to FITC-BSA alone, and ‡ indicates "FITC-BSA + N" ３ - p<0.05 for "anti-BSA antibody", # p<0.05 for "FITC-BSA+triGalNAC-LYTAC".

[0187] In GalNAc-PRX-LYTAC, macrocyclic molecule a has one GalNAC, which is fewer than the number of GalNAC ligands recognized by the target receptor ASGPR in a multivalent manner. As is clear from the results shown in Figure 19(B), GalNAc-PRX-LYTAC has a significantly higher (preferably more pronounced) ability to deliver a target substance (e.g., an antigen) to the lysosomes of hepatic parenchymal cells than the previously reported technology (triGalNAC-LYTAC).

[0188] 20 is a diagram summarizing the test results for the ability of monoGalNAc-PRX-antibody (LYTAC) to deliver a target substance (e.g., an antigen) to a target cell (e.g., a hepatocyte, preferably a lysosome in a hepatocyte) in Examples 22 and 23. Due to its dynamic properties, monoGalNAc-PRX-antibody (LYTAC) can interact with ASGPR more efficiently than the previously reported technology (triGalNAC-LYTAC), and can be taken up into the lysosome in the hepatocyte via ASGPR-mediated endocytosis and delivered.

Claims

1. A polyrotaxane A having a plurality of macrocyclic molecules a, an axial molecule a that passes through the rings of the macrocyclic molecules a, and caps a that are bonded to the ends of the axial molecules a, wherein at least some of the macrocyclic molecules a have one or more ligand groups, and the one or more ligand groups possessed by the macrocyclic molecules a are recognized by a target receptor that recognizes the one or more ligand groups, and the number of ligand groups possessed by the macrocyclic molecules a is either less than the number of ligand groups recognized by the target receptor, or the number of ligand groups possessed by the macrocyclic molecules a is equal to or greater than the number of ligand groups recognized by the target receptor.

2. Polyrotaxane A according to claim 1, wherein the number of the ligand groups possessed by the macrocyclic molecule a is less than the number of the ligand groups recognized by the target receptor.

3. The polyrotaxane A according to claim 1, wherein the number of the ligand groups possessed by the macrocyclic molecule a is equal to or greater than the number of the ligand groups recognized by the target receptor.

4. The polyrotaxane A according to claim 1, wherein the macrocyclic molecule a has two or more of the ligand groups, and the two or more ligand groups are covalently bonded to the macrocyclic molecule a via a linking group, and the linking group has a ligand bonded to each of two or more ends of a branched hydrocarbon chain having 5 to 80 carbon atoms which may contain a keto group, an ether bond, an amide bond, a urethane bond, and / or a divalent amino group.

5. The polyrotaxane A according to claim 1, wherein the cap a has a drug group directly or via a linker.

6. Polyrotaxane A according to claim 5, wherein the drug group is an antibody or an antibody fragment.

7. The polyrotaxane A according to claim 1, for use in a composition containing the following polyrotaxane B. [Polyrotaxane B: a polyrotaxane having a plurality of macrocyclic molecules b, an axial molecule b passing through the rings of the macrocyclic molecules, and a cap b attached to the end of the axial molecule, and capable of forming a polyion complex with a biological material, wherein the cap b has a host-guest interaction and / or an inclusion interaction with the cap a.] 8. The polyrotaxane A according to claim 7, wherein at least a portion of the macrocyclic molecules b in the polyrotaxane B has an amine-containing group b, and the amine-containing group b has a monovalent proton at neutral pH and a divalent proton at acidic pH.

9. The polyrotaxane A according to claim 8, wherein at least some of the macrocyclic molecules b further have an amino group via an intracellularly degradable bond in addition to the amine-containing group b.

10. The polyrotaxane A according to claim 7, wherein the composition further comprises a biological material, and the biological material has the ability to form a polyion complex with the polyrotaxane B.

11. The polyrotaxane A according to claim 10, wherein the biological material is a nucleic acid molecule or a complex of Cas9 protein and guide RNA (Cas9 RNP).

12. A complex comprising the polyrotaxane A, polyrotaxane B and a biological material according to claim 1, wherein the polyrotaxane B and the biological material form a polyion complex, wherein the polyrotaxane B has a plurality of macrocyclic molecules b, an axial molecule b that passes through the rings of the macrocyclic molecules, and caps b that are bonded to the ends of the axial molecules, and the polyrotaxane A and the polyrotaxane B are bonded via host-guest interactions and / or inclusion interactions between the caps a and the caps b.

13. A pharmaceutical composition comprising the polyrotaxane A according to claim 5.

14. A pharmaceutical composition comprising the following polyrotaxane A, the following polyrotaxane B, and a biological material, wherein the biological material is capable of forming a polyion complex with the polyrotaxane B. [Polyrotaxane A: a polyrotaxane having a plurality of macrocyclic molecules a, an axis molecule a penetrating the rings of the macrocyclic molecules a, and a cap a bonded to an end of the axis molecule a, wherein at least a portion of the macrocyclic molecules a have one or more ligand groups, wherein the one or more ligand groups possessed by the macrocyclic molecules a are recognized by a target receptor that recognizes the one or more ligand groups, and wherein the number of ligand groups possessed by the macrocyclic molecules a is less than the number of ligand groups recognized by the target receptor, or the number of ligand groups possessed by the macrocyclic molecules a is equal to or greater than the number of ligand groups recognized by the target receptor. [Polyrotaxane B: the polyrotaxane having a plurality of macrocyclic molecules b, an axis molecule b penetrating the rings of the macrocyclic molecules, and a cap b bonded to an end of the axis molecule, and having the ability to form a polyion complex with the biological material, and the cap b having a host-guest interaction and / or an inclusion interaction with the cap a.] 15. A pharmaceutical composition according to claim 13 or 14, for the introduction of said biological material into cells and for delivery to a target.

16. A method for producing the complex of claim 12, comprising mixing polyrotaxane A and polyrotaxane B, and mixing a biological material.

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