Polycatenane, biological material intracellular delivery agent containing same, polyion complex containing same, composition containing same, and method for delivering biological materials into cells

Abstract

The present invention provides: as suitable carriers for intracellular delivery of biological materials such as nucleic acids and proteins, a polycatenane, a biological material intracellular delivery agent containing the same, a polyion complex containing the same, and a composition containing the same; and a method for delivering biological materials into cells. This polycatenane has a plurality of macrocyclic molecules and a cyclic axle molecule threaded through the rings of the macrocyclic molecules, at least some of the macrocyclic molecules in the polycatenane having an amine-containing group.

Description

Polycatenane, cell introduction agent for biological material containing same, polyion complex containing same, composition containing same, and method for introducing biological material into cells

[0001] The present invention relates to a polycatenane suitable as a carrier for introducing biological materials such as nucleic acids and proteins into cells, a cell introduction agent for biological materials containing the polycatenane, a polyion complex containing the polycatenane, a composition containing the polycatenane, and a method for introducing biological materials into cells.

[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 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. This carrier has demonstrated higher intracellular delivery efficiency and safety than Lipofectamine CRISPRMAX, the most commonly used carrier among commercially available Cas9RNP delivery reagents (Patent Document 1).

[0004] On the other hand, so-called interlocked supramolecules called catenanes are known, which consist of a macrocyclic molecule and an axial molecule that pierces the rings of the macrocyclic molecule in a skewer-like manner and that itself has a larger cyclic structure than the macrocyclic molecule. In particular, catenanes that consist of a plurality of macrocyclic molecules (e.g., four or more) and a axial molecule that pierces the rings of the macrocyclic molecule in a skewer-like manner and that itself has a larger cyclic structure than the macrocyclic molecule are known as polycatenanes. The present inventors have successfully produced polycatenanes using polypseudorotaxanes (e.g., Patent Document 2).

[0005] International Publication No. 2022 / 163729A1 International Publication No. 2018 / 164225A1

[0006] The present invention has been made in consideration of the above-mentioned problems of the conventional technology, and aims to provide polycatenanes suitable as carriers for introducing biological materials such as nucleic acids and proteins into cells, cell introduction agents for biological materials containing the same, polyion complexes containing the same, compositions containing the same, and methods for introducing biological materials into cells.

[0007] As a result of extensive research into better carriers, the present inventors have found that polycatenanes, which have amine-containing groups on the macrocyclic molecule and a cyclic axial molecule, can efficiently form and stabilize polyion complexes simply by mixing with biological materials such as proteins and nucleic acids, and are efficiently taken up into cells. The present invention has been completed based on this finding. Specifically, the present invention is as follows.

[0008] <1> A polycatenane having a plurality of macrocyclic molecules and a cyclic axis molecule penetrating the rings of the macrocyclic molecules, wherein at least some of the macrocyclic molecules in the polycatenane have an amine-containing group. <2> The polycatenane according to <1>, which has the ability to form a polyion complex with a biological material. <3> The polycatenane according to <2>, wherein the biological material is a nucleic acid molecule or a complex of a Cas9 protein and a guide RNA (Cas9 RNP). <4> The polycatenane according to <1>, wherein the amine-containing group has a monovalent proton at at least one point between pH 7.0 and 7.6 and a divalent proton at at least one point between pH 4.0 and 6.5. <5> The polycatenane according to <1>, wherein at least some of the macrocyclic molecules further have an amino group, in addition to the amine-containing group, via an intracellularly degradable bond. <6> The polycatenane according to <1>, having a penetration rate (the coverage rate of the cyclic axis molecule by the macrocyclic molecule) of 1 to 21%. <7> An agent for introducing biological materials into cells, comprising the polycatenane according to <1> above. <8> A polyion complex of a biological material and the polycatenane according to <2> above. <9> A composition comprising the polycatenane according to <2> above and a biological material, or comprising the polyion complex according to <8> above. <10> A method for introducing a biological material into cells, comprising contacting the composition according to <9> above with cells and incorporating the polyion complex into the cells.

[0009] According to the present invention, it is possible to provide polycatenanes suitable as carriers for introducing biological materials such as nucleic acids and proteins into cells, cell introduction agents for biological materials containing the same, polyion complexes containing the same, compositions containing the same, and methods for introducing biological materials into cells.

[0010] 1 is a schematic diagram of a polyion complex composed of a polycatenane and a biological material. 2 is a schematic diagram showing an outline of an example of polycatenane production. 3 is a diagram showing the results of a test for polyion complex formation with siRNA (electrophoresis). 4 is a diagram showing the results of a test for polyion complex formation with siRNA (DLS). 5 is a diagram showing the results of a test for polyion complex formation with Cas9RNP (electrophoresis). 6 is a diagram showing the results of a test for polyion complex formation with Cas9RNP (DLS). 7 is a diagram showing the results of an in vitro test for RNAi effect. 8 is a diagram showing the results of an in vitro test for genome editing effect. 9 is a diagram showing the results of a test for polyplex stability (under electrostatic competition). 10 is a diagram showing the results of a test for polyplex stability (under electrostatic competition). 11 is a diagram showing the results of a test for polyplex stability (in the presence of FBS). 12 is a diagram showing the results of an in vitro test for genome editing effect by polyplex with Cas12a (Cpf1) RNP. 13 is a diagram showing the results of an in vitro test for knockdown effect by polyplex with gapmer. 1 is a diagram showing the results of an in vivo RNAi effect test (antitumor activity test). 2 is a diagram showing the results of an in vivo RNAi effect test (antitumor activity test). 3 is a diagram showing the results of an in vivo genome editing effect test (BACE1 expression suppression test). 4 is a diagram showing the results of HeLa cell uptake of DET-PCn / siRNA. 5 is a diagram showing the results of HeLa cell uptake of DET-PCn / Cas9RNP. 6 is a diagram showing the results of a zeta potential measurement test of DET-PCn / siRNA under different pH environments. 7 is a diagram showing the results of a zeta potential measurement test of DET-PCn / Cas9RNP under different pH environments. 8 is a diagram showing the results of a membrane disruption performance test of DET-PCn under an intraendosomal pH environment. 9 is a diagram showing the results of a cell safety comparison test of DET-PCn / siRNA. 10 is a diagram showing the results of a cell safety comparison test of DET-PCn / Cas9RNP.

[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 each term used in this specification are as follows. In this specification, a "polycatenane" is a so-called interlocked supramolecule consisting of a plurality of (e.g., three or more, four or more, five or more) macrocyclic molecules and an axial molecule that pierces the rings of the macrocyclic molecules in a skewer-like manner and that itself has a larger cyclic structure than the macrocyclic molecules. As described below, a "polycatenane" can be produced using, for example, the polypseudorotaxane shown below. Similarly, a "polyrotaxane" is also a so-called interlocked supramolecule, consisting of a plurality of macrocyclic molecules and an axial molecule (not cyclic, for example, linear) that pierces the rings of the macrocyclic molecules and has caps on both ends. A polypseudorotaxane has a plurality of macrocyclic molecules and an axial molecule (not cyclic, for example, linear) that pierces the rings of the macrocyclic molecules, but does not have a cap structure. Here, the term "cap" refers to a bulky substituent bonded to the terminals (preferably both terminals) of a polyrotaxane to prevent the macrocyclic molecules from detaching from the axial molecule. For example, the cap may include a group having a cyclodextrin (eg, α-, β-, or γ-cyclodextrin), an adamantyl group, or the like.

[0013] As used herein, a "macrocyclic molecule" refers to a cyclic molecule having an internal opening (cavity) large enough for the axis molecule to pass through 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 "amine-containing group" 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. 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).

[0014] As used herein, the term "axial molecule" refers to an axial molecule that pierces the rings of a macrocyclic molecule in a skewer-like manner, and the axial molecule itself is a cyclic axial molecule that has a larger cyclic structure than the macrocyclic molecule, as described above. The "axial molecule" is usually a polymer, and examples thereof include a polymer of a single monomer (homopolymer) and a copolymer of two or more types of monomers. The term "polymer" refers to a macromolecule formed by repeatedly bonding many of one or more monomers. A monomer is usually a molecule having one carbon-carbon double bond or a molecule having at least two functional groups per molecule. The copolymer may be a random copolymer, an alternating copolymer, and / or a block copolymer, etc. When the axial molecule is a homopolymer, examples of the polymer include polyalkylene oxide, polyethylene glycol (PEG), polypropylene glycol (PPG), 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, a polypropylene glycol-polysaccharide graft copolymer, etc. The axial molecule may also be a branched multi-arm polymer, such as star-shaped polyethylene glycol, hyperbranched polyether, hyperbranched oligoethylene glycol, or 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, about 1,000 to 36,000 Da, about 1,200 to 30,000 Da, etc. can be used.

[0015] As used herein, "intracellularly degradable bond" refers to a bond that is easily degraded under conditions in the intracellular environment that differ from those outside the cell, for example, physicochemical conditions such as pH or biological conditions such as intracellular enzymes, and intracellularly degradable bond is preferably a bond that is not easily degraded outside the cell. Here, the terms "degradable" and "not easily degradable" do not have an absolute meaning and do not mean completely degraded or not at all degraded, respectively.

[0016] "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.

[0017] In polycatenane, the axial molecule penetrates the macrocyclic molecule by passing through the opening of the macrocyclic molecule. Polycatenane has a plurality of macrocyclic molecules sewn to the axial molecule in this way. The number of macrocyclic molecules in one polycatenane molecule can be set depending on the purpose, but can be, for example, an average of about 4 to about 200, about 5 to about 100, or about 8 to about 50. 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.

[0018] <Polycatenane> A first aspect of the present invention is a polycatenane having a plurality of macrocyclic molecules and a cyclic axis molecule that passes through the rings of the macrocyclic molecules, wherein at least some of the macrocyclic molecules in the polycatenane have an amine-containing group.

[0019] FIG. 1 is a schematic diagram of a polyion complex composed of a polycatenane and a biological material. As shown in FIG. 1, the polycatenane according to the first embodiment has an amine-containing group on the macrocyclic molecule and a cyclic axial molecule. Therefore, simply mixing the polycatenane with a biological material such as a protein or nucleic acid efficiently forms a stabilized polyion complex, which can be efficiently taken up into cells. Furthermore, as is clear from the examples described below, the polycatenane can have higher intracellular introduction efficiency and stability than PRX. Because the polycatenane according to the first embodiment can efficiently form a polyion complex, it can contribute to a reduction in dosage, which in turn can contribute to improved long-term safety, reduced drug costs, reduced side effects, and the like.

[0020] The polycatenane according to the first aspect preferably has the ability to form a polyion complex with a biological material that is an active ingredient (e.g., an ingredient having pharmacological activity in the body of an animal, including a human, or an ingredient that, when contacted with other substances such as microbial contaminants, causes 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.). In the present invention, the term "polyion complex" (polyplex) refers to the formation or actual formation of a complex between polymers having multiple charges on their surfaces through electrostatic interaction in an aqueous solution, buffer solution, etc.

[0021] In the case of PRX, it has been reported that a high penetration rate of 30-60% and a high amino group modification rate efficiently form complexes (polyplexes) with biological materials and are well taken up into cells (Tamura A., Yui N., Cellular internalization and gene silencing of siRNA polyplexes by cytocleavable cationic polyrotaxanes with tailored rigid backbones. Biomaterials, 34, 2480-2491 (2013)). Furthermore, it has been reported that complexes of highly penetrating aminated PRXs and biological materials form lamellar structures, resulting in excellent colloidal stability and cellular uptake (Badwaik VD, Aicart E., Mondjinou YA, Johnson MA, Bowman VD, Thompson DH, Structure-property relationship for in vitro siRNA delivery performance of cationic 2-hydroxypropyl-β-cyclodextrin: PEG-PPG-PEG polyrotaxane vectors. Biomaterials, 84, 86-98 (2016)). In contrast, as will be apparent from the Examples described below, the present inventors have found that polycatenanes, despite their lower penetration rate compared to PRXs, are superior in terms of efficient polyion complex formation and cellular uptake. This is presumably due to the cyclic structure of the axial molecule. In the first aspect, the coverage (penetration rate) of the cyclic axis molecule with the macrocyclic molecule is not particularly limited, but from the viewpoint of efficient formation of a polyion complex, it is preferably 1 to 21%, more preferably 2 to 20%, even more preferably 3 to 19%, particularly preferably 4 to 19%, and especially preferably 5 to 19%.

[0022] In the first aspect, specific examples and preferred examples of the macrocyclic molecule include the same specific examples and preferred examples as those described above for the "macrocyclic molecule." Specific examples and preferred examples of the cyclic axis molecule include the same specific examples and preferred examples as those described above for the "axis molecule." From the viewpoint of skewering through the rings of the macrocyclic molecule, when the macrocyclic molecule is α-cyclodextrin, the polymer constituting the cyclic axis molecule preferably contains PEG. From the same viewpoint, when the macrocyclic molecule is β-cyclodextrin, the polymer constituting the cyclic axis molecule preferably contains PPG. For example, when polycatenane is produced using the polypseudorotaxane described below (particularly when produced in accordance with the Examples described in paragraphs 0164 to 0198 of Patent Document 2 (WO 2018 / 164225 A1)), the polymer constituting the cyclic axis molecule may be PEG-PPG-PEG (Pluronic (registered trademark) copolymer), thereby preventing detachment of β-cyclodextrin from the polypseudorotaxane.

[0023] In the first aspect, at least a part of the macrocyclic molecules has an amine-containing group, which can contribute to the formation of a polyion complex with a biological material. The amine-containing group is preferably a group having a secondary amino group or a primary amino group, and -L １ -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 -, -CH２ 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 includes a diethylenetriamine group (hereinafter, also simply referred to as "DET").

[0024] In the polycatenane according to the first aspect, at least a portion of the macrocyclic molecules preferably have an amine-containing group that has a monovalent proton at at least one point between pH 7.0 and 7.6 (neutral pH; more specifically, pH 7.4) and a divalent proton at at least one point between pH 4.0 and 6.5 (weakly acidic pH; preferably pH 4.3 to 6.3, more preferably pH 4.5 to 6.0, even more preferably pH 5.0 to 5.8, more specifically, pH 5.5). The amine-containing group is safe for biological components because it has a monovalent proton at at least one point between pH 7.0 and 7.6, while it can disrupt endosomal membranes because it has a divalent proton at at least one point between pH 4.0 and 6.5, which is the intraendosomal environment. This facilitates the release of a complex with a biological material taken up into an intracellular endosome via endosome disruption into the cytoplasm.

[0025] In the polycatenane, at least some of the macrocyclic molecules preferably further have, in addition to the amine-containing group, a group having an amino group (hereinafter simply referred to as "intracellularly degradable amino group") bonded via an intracellularly degradable bond. In other words, the amine-containing group and the "intracellularly degradable amino group" are preferably different groups. In this case, the amine-containing group is preferably bonded to the macrocyclic molecule via a bond that is not degraded or is difficult to degrade intracellularly. This allows the amino group of the amine-containing group to maintain the bond between the polycatenane and the biological material (polyion complex formation), even if some of the bonds of the intracellularly degradable amino group are degraded and the amino group dissociates from the macrocyclic molecule before release into the cell. Furthermore, after release into the cytoplasm, most of the bonds of the intracellularly degradable amino group are cleaved, thereby weakening the bond between the biological material and the polycatenane (polyion complex formation) and promoting dissociation of the biological material from the polycatenane.

[0026] 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. 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 -NH ２ , -(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.

[0027] For example, when the macrocyclic molecule has a hydroxyl group such as cyclodextrin, it is preferable that the hydroxyl group is substituted with the amine-containing group 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 cyclodextrin may be a hydroxyl group in 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 and the oxygen atom constituting the hydroxyl group of the α-cyclodextrin is 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:

[0028]

[0029] Also preferred as the polycatenane is a polycatenane consisting of a plurality of macrocyclic molecules and a cyclic axis molecule that penetrates the rings of the macrocyclic molecules, in which an amine-containing group is bonded to at least some of the macrocyclic molecules and a group having an amino group is bonded to at least some of the macrocyclic molecules via a bond that can be decomposed in cells.

[0030] In the present invention, the term "biological material" refers to a natural or artificial substance consisting of amino acids or nucleic acids that is to be introduced into cells as an active ingredient, and is not particularly limited as long as it has the ability to form a polyion complex with the polycatenane (is able to form a polyion complex).

[0031] More specifically, the "biological material" may include, for example, nucleic acid molecules, vectors, proteins, peptides, fusion products thereof, or complexes 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), decoys, CpG oligos, miRNA, antisense DNA (e.g., gapmer-type antisense nucleic acids), antisense RNA, aptamers, mRNA, and nucleic acid vaccines (e.g., mRNA vaccines, DNA vaccines, etc.).

[0032] 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)).

[0033] 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)).

[0034] 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 groups (amine-containing groups and / or intracellularly degradable amino groups) bound to the macrocyclic molecules of the polycatenane to the maximum number of cationic monomers that can be bound to the 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)).

[0035] For example, if the group bonded to the macrocyclic molecule 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 on the polycatenane. 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.

[0036] The polycatenane according to the first aspect may or may not have any label (e.g., fluorescent label, ultraviolet label, radioactive label, etc.), but from the viewpoint of any therapeutic or diagnostic use, it is preferable that it has any label, more preferably has any labeling group, and even more preferably that the "macrocyclic molecule substituent" has any labeling group. The labeling group may be 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), a radioactive labeling group (e.g., １８ and groups containing radioactive isotopes such as F (fluoro).

[0037] (Method for Producing Polycatenane) FIG. 2 is a diagram showing an outline of an example of a method for producing polycatenane. There are no particular limitations on the method for producing polycatenane, but it can be produced, for example, as follows. As shown in FIG. 2 , first, any solution (e.g., an aqueous solution) containing both an axial molecule (not cyclic, for example, linear) having amino groups at both ends and an axial molecule (not cyclic, for example, linear) having carboxyl groups at both ends can be mixed with any solution (e.g., an aqueous solution) containing macrocyclic molecules to obtain a polypseudorotaxane solution (i.e., a mixed solution of polypseudorotaxane 1 consisting of a plurality of macrocyclic molecules and an axial molecule (not cyclic, for example, linear) that penetrates the rings of the macrocyclic molecules and has amino groups at both ends, and polypseudorotaxane 2 consisting of a plurality of macrocyclic molecules and an axial molecule (not cyclic, for example, linear) that penetrates the rings of the macrocyclic molecules and has carboxyl groups at both ends). Next, the obtained polypseudorotaxane is dispersed in an arbitrary liquid (e.g., DMF), and the amino group and the carboxyl group are condensed by an arbitrary reaction (e.g., peptide condensing agent hexafluorophosphate (BOP reagent) / 1-hydroxybenzotriazole (HOBt) / N-ethyldiisopropylamine (EDIPA)). As a result, the polypseudorotaxane 1 and the polypseudorotaxane 2 are cyclized, whereby a polycatenane can be produced.

[0038] It can also be produced by a method known to those skilled in the art in accordance with the examples described in paragraphs 0164 to 0198 of Patent Document 2 (WO 2018 / 164225A1).

[0039] <Cell introduction agent> A second aspect of the present invention is a cell introduction agent for biological materials, comprising the polycatenane according to the first aspect. The polycatenane according to the first aspect is efficiently taken up by endosomes (endocytosis (e.g., clathrin-independent carriers / glycosylphosphatedylinositol-anchored-protein-enriched endosomal compartment (CLIC / GEEC) endocytosis, clathrin endocytosis, caveolae endocytosis, etc.)), released from endosomes within cells, and dissociated from the biological material in the cytoplasm after release, and therefore can efficiently introduce biological materials into the cytoplasm or nucleus, in particular. In other words, the polycatenane according to the first aspect can be used as a cell introduction agent (cell introduction carrier) for biological materials, in particular as a cytoplasmic introduction agent or nuclear introduction agent. In addition to the polycatenane, the cell introduction agent may contain a buffer solution, a stabilizer, etc., as necessary. Specific examples and preferred examples of the polycatenane include those similar to those described above for the first aspect. Specific examples and preferred examples of the amine-containing group include those similar to those described above for the first aspect. In particular, as described above for the first aspect, in the polycatenane, at least a portion of the macrocyclic molecules preferably have an amine-containing group having a monovalent proton at at least one point between pH 7.0 and 7.6 and a divalent proton at at least one point between pH 4.0 and 6.5.

[0040] <Polyion Complex (Polyplex)> The third aspect of the present invention is a polyion complex of the polycatenane according to the first aspect and a biological material. As shown in the schematic diagram of Figure 1, the core of the polyion complex of the polycatenane and the biological material is sterically shielded by the polycatenane, which is thought to improve the stability of the complex in an in vivo environment (e.g., in the presence of serum). As a result, the biological material as an active ingredient can be delivered.

[0041] In the polyion complex, the molar ratio of the biological material to the polycatenane is not particularly limited, but may be, for example, 1:0.25 to 0.25:1, preferably 1:0.5 to 0.5:1.

[0042] As described above for the first aspect, in the polycatenane, at least a portion of the macrocyclic molecules preferably have an amine-containing group that has a monovalent proton at at least one point between pH 7.0 and 7.6 and a divalent proton at at least one point between pH 4.0 and 6.5. This can promote the polyion complex taken up into an intracellular endosome to disrupt the endosome and be released into the cytoplasm. In other words, the polyion complex can function as a cell introduction agent (cell introduction carrier) for biological materials.

[0043] The polyion complex can be formed by mixing the biological material with the polycatenane. 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 polycatenane and biological material, their concentrations, etc., but is typically 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.

[0044] <Composition> A fourth aspect of the present invention is a composition containing the polycatenane (capable of forming a polyion complex with a biological material) according to the first aspect and a biological material, or the polyion complex according to the third aspect. The composition according to the fourth aspect can function as a composition for introducing a biological material into a cell.

[0045] In the composition, the molar ratio of the biological material to the polycatenane (capable of forming a polyion complex with the biological material) is not particularly limited, but may be, for example, 1:0.25 to 0.25:1, and preferably 1:0.5 to 0.5:1.

[0046] The composition according to the fourth aspect may be a pharmaceutical composition, and may be a pharmaceutical composition for oral or parenteral administration, such as an injection for intravenous injection, subcutaneous injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracerebral injection, or infusion. Alternatively, the 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).

[0047] The composition according to the fourth aspect may or may not contain a pharmacologically acceptable carrier (a pharmaceutical additive). The type of pharmaceutical additive used in the preparation of the composition, the ratio of the pharmaceutical additive to the active ingredient, or the method for preparing the composition can be appropriately selected by those skilled in the art depending on the form of the composition. Pharmaceutical 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 pharmaceutical ingredients are used as pharmaceutical ingredients, 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. Pharmaceutical 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.

[0048] 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.

[0049] Dosage forms of the composition 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, intratumoral injections, intraperitoneal injections, intracerebral injections, and intravitreal injections), infusions, topical preparations (e.g., intranasal preparations, transdermal preparations, ointments, etc.), suppositories (e.g., rectal suppositories, vaginal suppositories, etc.), pellets, infusions, and sustained-release preparations, each of which can be safely administered orally or parenterally. The composition can be prepared by conventional methods in the pharmaceutical technology field, such as those described in the Japanese Pharmacopoeia. Specific preparation methods for the formulations are described in detail below.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The dosage and administration frequency of the composition according to the fourth aspect will vary depending on the subject, administration route, target disease, symptoms, etc., but those skilled in the art will be able to 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 administered in divided doses. The administration frequency can be daily, weekly, every two weeks, monthly, or once every several 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.

[0054] As mentioned above, because the polycatenane and the biological material have different functions, it is also preferable that the polycatenane and the biological material be used as a combination drug (compound drug) or as a kit product (described below). In this case, the polycatenane and the biological material can be used together simultaneously or at an interval. Furthermore, the administration routes of the polycatenane and the biological material may be the same or different. When these drugs are used in combination, each drug can be formulated separately or simultaneously with a pharmacologically acceptable carrier, excipient, binder, diluent, etc., and administered orally or parenterally as a composition. When drugs are formulated separately, the separately formulated drugs can be mixed with a diluent or the like at the time of use and administered, or the separately formulated individual formulations can be administered simultaneously or at intervals to the same subject. The 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).

[0055] The present invention also relates to a method for treating or ameliorating a disease or condition in a subject (e.g., a patient), comprising administering an effective amount of a composition according to the fourth aspect to a subject (e.g., a patient) in need thereof. Examples of such diseases or conditions include cancer (e.g., colon cancer) and neurodegenerative diseases (e.g., Alzheimer's disease). Neurodegenerative diseases refer to a group of diseases characterized by various degenerative changes primarily affecting nerve cells. "Treatment" may refer to either pathological or clinical treatment. "Treatment" refers to an approach to achieving 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 disease, stabilization (i.e., non-worsening) of disease, delay or slowing of disease progression, amelioration or remission of the disease state, and remission (partial or total). "Treatment" may also refer to extending 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. Treatment subjects include mammals such as humans, cows, horses, dogs, cats, pigs, and sheep, with humans being preferred. The mode of administration is not particularly limited and can be oral or parenteral, including intravenous, subcutaneous, intramuscular, intratumoral, intraperitoneal, intracerebral, and infusion. Intracerebral injections include stereotactic intracerebral injections into specific brain regions (e.g., hippocampus, striatum, cerebellum, prefrontal cortex, other cerebral cortices, thalamus, hypothalamus, pineal gland, pituitary gland, midbrain, hindbrain, choroid plexus, etc.), intracerebral transplantation, intraventricular injection, and intrathecal injection. The administration route can be appropriately selected by those skilled in the art. The dosage, frequency, and duration of administration can also be appropriately determined by those skilled in the art based on the type, sex, age, and symptoms of the subject. The daily dose can be 0.001 to 100 g, or 0.01 to 1000 mg / kg. The daily dose can be divided into several doses and administered. 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.

[0056] <<Method for Introducing Biological Material into Cells>> A fifth aspect of the present invention is a method for introducing a biological material into cells, comprising contacting the composition according to the fourth aspect with cells and incorporating the polyion complex into the cells. The cells may be in vitro or in vivo. Contact with the in vivo cells may be achieved via intravenous administration, subcutaneous administration, intramuscular injection, intratumoral administration, intraperitoneal administration, intracerebral administration, or the like. This method may optionally include mixing the biological material with the polycatenane according to the first aspect to form a polyion complex, and contacting the polyion complex with cells to incorporate the polyion complex into the cells. Furthermore, this method can be used as a method for genome editing by using a complex of Cas9 protein and guide RNA as the biological material. Therefore, in one aspect, the present disclosure relates to a method for genome editing, comprising contacting a polyion complex of a Cas9 protein and guide RNA (Cas9RNP) with a polycatenane according to the first aspect with cells, thereby incorporating the polyion complex into the cells. The method may optionally include producing a polyion complex by mixing the Cas9 protein and guide RNA (Cas9RNP) with the polycatenane according to the first aspect, and contacting the polyion complex with cells to incorporate the polyion complex into the cells. The contact between the polyion complex and the cells can be achieved by adding the polyion complex to a cell culture solution and culturing the cells. The medium and culture conditions used can be appropriately set depending on the cells used, but are typically cultured at 37°C, 5% CO ２The incubation period can be 1 to overnight, 1 to 12 hours, 2 to 6 hours, or 3 to 5 hours. If necessary, the cells may be washed to remove the polyion complex, and then further incubated for 1 to 4 days or more, and up to 6 to 10 days.

[0057] 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.

[0058] Example 1 Preparation of Polycatenane in Which Macrocyclic Molecules Have Amine-Containing Groups (Preparation of Polypseudorotaxane (hereinafter simply referred to as "PPRX")) A 2 kDa PEG having amino groups at both ends (hereinafter simply referred to as "PEG-NH ２ ") PEO amine (manufactured by Kawaken Fine Chemicals Co., Ltd.) was used. ２ was prepared as follows. PEG (20 kDa, manufactured by Sigma) was dissolved in tetrahydrofuran (THF), N,N-carbonyldiimidazole (CDI) was added, and the mixture was stirred at 50°C for 18 hours under nitrogen purging. The reaction solution was added dropwise to ethylenediamine, and the mixture was stirred at 50°C for 2 hours under nitrogen purging. Ethanol was added to the reaction solution, and the mixture was allowed to stand at -20°C for 2 hours, after which it was centrifuged and the supernatant was removed. The precipitate was washed several times with cold ethanol and dried under reduced pressure to obtain 20 kDa PEG-NH with amino groups at both ends. ２ was prepared.

[0059] 2 kDa PEG having a carboxyl group at the end (hereinafter simply referred to as "PEG-COOH") was prepared as follows: PEG-NH ２ (2 kDa) (10 g, 0.5.0 mmol) and succinic anhydride (30 g, 1300 mmol) were dissolved in DMSO (350 mL) and stirred at 55°C for 18 hours under nitrogen purging. Ethanol (350 mL) was added, and the mixture was allowed to stand at -20°C for 2 hours. The precipitate was then collected by centrifugation, washed several times with cold ethanol, and dried under reduced pressure.

[0060] 20 kDa PEG-COOH with a terminal carboxyl group was prepared as follows: PEG (20 kDa) (10 g, 0.5 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl (100 mg, 0.64 mmol), sodium bromide (NaBr; 100 mg, 0.97 mmol), and sodium hypochlorite (NaClO; available chlorine concentration >5%, 10 mL) were dissolved in water and stirred at room temperature for 15 minutes. To quench the reaction, ethanol (10 mL) was added to the reaction mixture, and the reaction mixture was deionized by adding 1 N HCl to adjust the pH to <2. After dialysis against water using a Spectra / Por membrane (MWCO: 10 kDa), the sample was concentrated using an evaporator and lyophilized to dryness.

[0061] 2 kDa PEG-NH ２ A mixture of equal amounts (1:1 molar ratio) of 2 kDa PEG-COOH was prepared. The mixture was added to an aqueous solution of α-cyclodextrin (α-CyD) at the 1:1 molar ratio. After stirring overnight at 4°C, the mixture was centrifuged, the supernatant was removed, and then freeze-dried to obtain PPRX (2 kDa) with α-CyD as the macrocyclic molecule and PEG (2 kDa) as the axial molecule. Similarly, 20 kDa PEG-NH ２ After preparing a mixture of equal amounts (molar ratio 1:1) of α-CyD and 20 kDa PEG-COOH, PPRX (20 kDa) was obtained, with α-CyD as the macrocyclic molecule and PEG (20 kDa) as the axial molecule.

[0062] (Condensation and Cyclization) The peptide condensation agent hexafluorophosphate (BOP reagent) (14.42 g) and 1-hydroxybenzotriazole (HOBt) (5.38 g) were dissolved in N,N-dimethylformamide (DMF; 22 mL), and N-ethyldiisopropylamine (EDIPA) (6.264 mL) was added. The above-mentioned PPRX (2 kDa) (7.0 g) having an α-CyD macrocyclic molecule and a PEG axial molecule was gradually added. After stirring for 48 hours at 4°C under a nitrogen atmosphere, 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 suspended in dimethyl sulfoxide (DMSO) and precipitated by adding dropwise to cold water with stirring. The above washing procedure was repeated three times, followed by two washes with distilled water. The mixture was then dialyzed (Spectra / Por (registered trademark) Membrane, MWCO: 10 kDa, solvent: water) and freeze-dried to obtain a PEG 2 kDa (total 4 kDa) polycatenane (hereinafter simply referred to as "α-CD PCn"). A PEG 20 kDa (total 40 kDa) polycatenane was also obtained in the same manner. The results are shown in Table 1 below. While it is possible to achieve a lower penetration rate than the polyrotaxane (PRX) of Comparative Example 1 described later, it is possible to achieve higher intracellular introduction efficiency and stability than PRX, as will be apparent from the examples described later.

[0063] (Amination) Polycatenane in which the macrocyclic molecule has an amine-containing group was prepared according to the following scheme.

[0064] The above α-CD PCn (20 mg) containing 2 kDa PEG (4 kDa in total) was dissolved in DMSO, and CDI (24 mg) was added. The mixture was stirred at room temperature (23°C) for 18 hours under nitrogen purging. The mixture was added dropwise to an excess amount of diethylenetriamine (DET) DMSO solution, and the mixture was stirred at room temperature for 18 hours under nitrogen purging. After dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 8 kDa, solvent: water), polycatenane (hereinafter simply referred to as "DET-PCn") in which macrocyclic molecules have DET groups was obtained by lyophilization. The results are shown in Table 2 below.

[0065] Comparative Example 1 Preparation of Polyrotaxane (PRX) Having Amine-Containing Groups in Macrocyclic Molecules (Preparation of Polyrotaxane Having Adamantyl Group Caps on Both Ends of the Axle Molecule (hereinafter simply referred to as "Ad-cap-PRX")) "Ad-cap-PRX" was prepared according to the following scheme.

[0066] The above-mentioned 2 kDa PEG-NH ２ The above was added to an aqueous α-CyD solution. After stirring overnight at 4°C, the mixture was centrifuged, the supernatant was removed, and then lyophilized to obtain PPRX with α-CyD as the macrocyclic molecule and PEG (2 kDa) as the axial molecule. Adamantane acetic acid, peptide condensation agent hexafluorophosphate (BOP reagent), 1-hydroxybenzotriazole (HOBt), and N-ethyldiisopropylamine (EDIPA) were dissolved in DMF (100 mL), and the above PEG (2 kDa) PPRX was added. After stirring for 48 hours at 4°C, 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. (Amination) The above-mentioned Ad-cap-PRX of PEG 2 kDa was dissolved in DMSO, CDI was added, and the mixture was stirred at room temperature for 18 hours under nitrogen purging. The above mixture was added dropwise to a DMSO solution of an excess amount of DET, and the mixture was stirred at room temperature for 18 hours under nitrogen purging. After dialysis (Spectra / Por (registered trademark) Membrane, MWCO: 8 kDa, solvent: water), a polycatenane (2 kDa) (hereinafter simply referred to as "DET-PRX") in which the macrocyclic molecule has a DET group was obtained by lyophilization. The results are shown in Table 2 below.

[0067] Example 2: Polyion Complex Formation Test with siRNA (Electrophoresis) A polyion complex formation test with siRNA was performed using the DET-PCn obtained in Example 1 and the DET-PRX obtained in Comparative Example 1. Various concentrations of DET-PCn or DET-PRX, set to achieve an N / P ratio of 0.4 to 1.6, were mixed with 260 ng of siRNA in Hank's balanced salt buffer (HBSS buffer) at room temperature for 15 minutes to form a polyion complex. Here, the N / P ratio is the so-called charge ratio based on the primary amino group of the carrier (DET-PCn or DET-PRX) and the phosphate group of the siRNA. Subsequently, 2% agarose gel electrophoresis was performed at 100 V for 30 minutes and stained with ethidium bromide (EtBr). The results are shown in Figure 3.

[0068] (Results) As is clear from the results shown in Figure 3, with DET-PRX, a faint siRNA band remains even at an N / P ratio of 1.6, indicating that a faint amount of siRNA remains that has not formed a complex. On the other hand, with DET-PCn, the siRNA band completely disappears at an N / P ratio of 1.0 or more, indicating that all of the siRNA forms a complex at an N / P ratio of 1.0 or more. In other words, it is clear that DET-PCn forms a complex with siRNA more efficiently than DET-PRX.

[0069] (Dynamic Light Scattering) A polyion complex formation test with siRNA was performed for the DET-PCn obtained in Example 1 and the DET-PRX obtained in Comparative Example 1. Various concentrations of DET-PCn or DET-PRX, set to achieve N / P ratios of 0 to 120, were mixed with 1.36 μg of siRNA in 100 μL of Hank's balanced salt buffer (HBSS buffer) at room temperature for 15 minutes to form a polyion complex. The mixture was diluted to 1 mL with HBSS buffer (pH 7.4). The zeta potential, size, and polydispersity index (PDI) of the polyplexes were measured by dynamic light scattering using a Zetasizer Pro device (Malvern Instruments). The results are shown in Figure 4. In Figure 4, values ​​are expressed as mean ± standard error for n = 3 per group, and * indicates p < 0.05.

[0070] (Results) As is clear from the results shown in Figure 4(a), in the case of DET-PRX, the negative ζ potential of siRNA is inverted to positive at an N / P ratio of approximately 5. On the other hand, in the case of DET-PCn, the negative ζ potential of siRNA is inverted to positive at an N / P ratio of approximately 2.5. In other words, it is clear that DET-PCn forms a complex with siRNA more efficiently than DET-PRX.

[0071] As is clear from the results shown in Figures 4(b) and (c), with DET-PRX, the particle size was large at an N / P ratio of 10 or less, and polydispersity was observed at an N / P ratio of 5 or less, particle size compaction occurred at an N / P ratio of 15 or more, and monodispersity occurred at an N / P ratio of 10 or more. On the other hand, with DET-PCn, the particle size was small, compact, and monodispersity was observed at any N / P ratio. In other words, it was found that DET-PCn forms a complex with siRNA more efficiently than DET-PRX.

[0072] Example 3: Polyion Complex Formation Test with Cas9RNP (Electrophoresis) A polyion complex formation test with Cas9RNP was performed on the DET-PCn obtained in Example 1 and the DET-PRX obtained in Comparative Example 1. Various concentrations of DET-PCn or DET-PRX were mixed with the Cas9RNP (sgRNA: 500 ng) at room temperature for 15 minutes in Hanks' equilibrium buffer (HBSS buffer) to form a polyion complex, so that the imprinting rate was set to 2.0-10%. Here, the imprinting rate is the value calculated as 100% when the carrier is mixed so that there are 927 amines present at the terminal per Cas9RNP. 2% agarose gel electrophoresis was then performed at 100V for 40 minutes and stained with EtBr. The results are shown in Figure 5.

[0073] (Results) As is clear from the results shown in Figure 5, in DET-PRX, even at an imprinting rate of 7.5%, a faint Cas9RNP band remains, indicating that Cas9RNP that has not formed a complex remains faintly. On the other hand, in DET-PCn, the Cas9RNP band completely disappears at an imprinting rate of 5.0% or more, indicating that all Cas9RNP forms a complex at an imprinting rate of 5.0% or more. In other words, it can be seen that DET-PCn forms a complex with Cas9RNP more efficiently than DET-PRX.

[0074] (Dynamic Light Scattering) A polyion complex formation test with Cas9RNP was performed for the DET-PCn obtained in Example 1 and the DET-PRX obtained in Comparative Example 1. Various concentrations of DET-PCn or DET-PRX, set to achieve imprinting rates of 0-200%, were mixed with the Cas9RNP (sgRNA: 2.0 μg) in 100 μL of HBSS buffer at room temperature for 15 minutes to form a polyion complex. The mixture was diluted to 1 mL with HBSS buffer (pH 7.4). The zeta potential, size, and polydispersity index (PDI) of the polyplexes were measured by dynamic light scattering using a Zetasizer Pro device (Malvern Instruments). The results are shown in Figure 6. In FIG. 6, values ​​are expressed as mean ± standard error for n=3 in each group, and *: p<0.05.

[0075] (Results) As is clear from the results shown in Figure 6(a), in the case of DET-PRX, the negative ζ potential of Cas9RNP is inverted to positive at an imprinting rate of around 21%. On the other hand, in the case of DET-PCn, the negative ζ potential of Cas9RNP is inverted to positive at an imprinting rate of less than 10%. In other words, it can be seen that DET-PCn forms a complex with Cas9RNP more efficiently than DET-PRX.

[0076] As is clear from the results shown in Figures 6(b) and (c), with DET-PRX, the particle size is large and polydisperse at an imprinting rate of 50% or less, and compaction and monodispersion of particle size occur at an imprinting rate of 100% or more. On the other hand, with DET-PCn, the particle size is small, compact, and monodisperse at any imprinting rate. In other words, it is clear that DET-PCn forms a complex with siRNA more efficiently than DET-PRX.

[0077] Example 4: In vitro test of RNAi effect by polyplex with siRNA The following siGFP was used as the siRNA. (siGFP) sequence: 5'-GCAAGCUGACCCUGAAGUUCAUdTdT-3' (SEQ ID NO: 1) sequence: 5'-AUGAACUUCAGGGUCAGCUUGCCG-3' (SEQ ID NO: 2)

[0078] GFP-stably expressing HeLa cells (hereinafter simply referred to as "HeLa / GFP cells") (manufactured by Cell Biolabs) were seeded (3.75 x 10 ４ The cells were transfected in a 100-well plate (100 cells / well) at 37°C for 24 hours. After washing twice with serum-free medium, 300 μL of serum-free medium containing the following samples was added to the cells and transfected. (Samples) - siRNA (siGFP) alone (100 nM) - Polyplexes with DET-PCn or DET-PRX formed as polyion complexes with 100 nM siRNA to give N / P ratios of 5, 10, 15, or 20 - Polyplexes with DET-PCn or DET-PRX formed as polyion complexes with siCont (siRNA not targeting GFP) to give an N / P ratio of 20 - Control (saline)

[0079] After incubation in the presence of the sample at 37°C for 4 hours, the cells were washed twice with serum-free medium. 500 μL of DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (Fetalbovine Serum) was added, and the cells were incubated at 37°C for 68 hours. The RNAi effect was then evaluated by flow cytometry. The results are shown in Figure 7. In Figure 7, values ​​are expressed as the mean ± standard error for n = 3 in each group.

[0080] (Results) As is clear from the results shown in Figure 7, administration of siGFP alone did not result in a decrease in HeLa / GFP cells, indicating that siGFP alone is not taken up by cells. The polyplex of siCont (an siRNA not targeting GFP) with DET-PCn, or the polyplex of siCont with DET-PRX did not result in a decrease in HeLa / GFP cells, indicating that GFP is not targeted. The polyplex of siGFP with DET-PRX and the polyplex of siGFP with DET-PCn both resulted in a decrease in HeLa / GFP cells, indicating that siGFP was taken up by cells and that GFP was knocked down by siGFP. In particular, the polyplex with DET-PCn showed a higher RNAi effect than the polyplex with DET-PRX. Furthermore, it is presumed that after intracellular uptake, a series of steps, including endosomal escape, release of siRNA from DET-PCn, and recognition by the RNA-induced silencing complex (RISC), proceeds more smoothly in the polyplex with DET-PCn.

[0081] Example 5: In vitro test of genome editing effect by polyplex with Cas9RNP The following sgGFP and sgCont (sgRNA not targeting GFP) were used as sgRNA. sgGFP: 5'-GGGCGAGGAGCUGUUCACCG-3' (SEQ ID NO: 3) sgCont: 5'-AAAUGUGAGAUCAGAGUAAU-3' (SEQ ID NO: 4)

[0082] HeLa / GFP cells were seeded in a 24-well plate (3.75 × 10 ４The cells were then washed twice with serum-free medium, and 500 μL of serum-free medium containing the following samples was added to the cells. (Samples) - Cas9RNP (sgGFP) alone (116.8 nM) - Polyplexes with DET-PCn or DET-PRX formed as polyion complexes with 116.8 nM Cas9RNP to achieve imprinting rates of 10%, 20%, 50%, 75%, and 100% - Polyplexes with DET-PCn or DET-PRX formed as polyion complexes with Cas9RNP (sgCont) to achieve an imprinting rate of 100% - Control (saline)

[0083] After incubation in the presence of the sample at 37°C for 4 hours, the cells were washed twice with serum-free medium. 500 μL of DMEM containing 10% FBS was added, followed by incubation at 37°C for 44 hours. After daily medium changes, the cells were incubated for a total of 120 hours, after which the RNAi effect was evaluated by flow cytometry. The results are shown in Figure 8. In Figure 8, values ​​are expressed as the mean ± standard error for each group (n = 6-9).

[0084] (Results) As is clear from the results shown in Figure 8, administration of Cas9RNP (sgGFP) alone did not result in a decrease in HeLa / GFP cells, indicating that Cas9RNP alone is not taken up into cells. HeLa / GFP cells were not reduced with the polyplex of Cas9RNP (sgCont) and DET-PCn, or the polyplex of Cas9RNP (sgCont) and DET-PRX, indicating that GFP was not targeted. The polyplex of Cas9RNP and DET-PRX resulted in a small decrease in HeLa / GFP cells, indicating a small genome editing effect. It is presumed that the uptake efficiency was weak. On the other hand, it can be seen that the polyplex with DET-PCn achieved a higher genome editing effect than the polyplex with DET-PRX. Furthermore, it is presumed that after intracellular uptake, a series of steps including endosomal escape, release of Cas9RNP from DET-PCn, and translocation of Cas9RNP to the cell nucleus proceeds more smoothly in polyplexes with DET-PCn.

[0085] Example 6: Stability Test of Polyplexes with siRNA (During Electrostatic Competition) The stability of polyplexes in the presence of heparin (a negatively charged substance (polyanion)), which electrostatically disrupts polyplexes, was tested by a heparin competition assay. Polyion complexes were formed by 15 minutes of incubation with 260 ng of siRNA to achieve an N / P ratio of 10. The polyplexes with DET-PCn or DET-PRX were mixed with various concentrations of heparin sodium salt (Nacalai Tesque) solutions (heparin / siRNA mass ratios (w / w) of 0, 2.5, 5.0, 7.5, and 10) and incubated at room temperature for 10 minutes. Gel electrophoresis was performed on a 2% (w / v) agarose S gel at room temperature at 100 V for 30 minutes. The gel was stained with ethidium bromide. The results are shown in Figure 9.

[0086] 9, it is clear that the polyplex with DET-PRX dissociates from siRNA when the heparin / siRNA mass ratio (w / w) is 5.0 or more. On the other hand, the polyplex with DET-PCn does not dissociate from siRNA not only when the heparin / siRNA mass ratio (w / w) is 5.0 or more, but also when the heparin / siRNA mass ratio is 10, and the polyion complex is stable.

[0087] Example 7: Stability test of polyplexes with Cas9RNP (during electrostatic competition) Polyplexes with DET-PCn or polyplexes with DET-PRX were incubated for 15 minutes with Cas9RNP (sgRNA: 500 ng) to form polyion complexes so that the imprinting rate was 50%. The polyplexes were mixed with various concentrations of heparin sodium salt (Nacalai Tesque) solutions (heparin / sgRNA mass ratios (w / w) of 0, 2.5, 5.0, 7.5, and 10) and incubated at room temperature for 10 minutes. Gel electrophoresis was performed on a 2% (w / v) agarose S gel at room temperature at 100 V for 40 minutes. The gel was stained with ethidium bromide. The results are shown in FIG. 10.

[0088] (Results) As is clear from the results shown in Figure 10, polyplexes with DET-PRX dissociated from Cas9RNP at heparin / sgRNA mass ratios (w / w) of 2.5 or more. On the other hand, polyplexes with DET-PCn dissociated from Cas9RNP at heparin / sgRNA mass ratios (w / w) of 7.5 or more, indicating that the polyion complex was more stable than DET-PRX.

[0089] Example 8: Stability test of polyplexes with siRNA (in the presence of FBS) The stability of polyplexes in serum, simulating an in vivo environment, was tested in the presence of FBS. A polyion complex was formed by incubation of 520 ng of siRNA for 15 minutes to give an N / P ratio of 10. 20 μL of FBS (50% by mass (w / w)) was added to 20 μL of an aqueous solution of polyplexes with DET-PCn or DET-PRX, and the mixture was incubated at room temperature for 1 to 8 days. Gel electrophoresis was performed on a 2% (w / v) agarose S gel at room temperature at 100 V for 30 minutes. The gel was stained with ethidium bromide. The results are shown in FIG. 11.

[0090] (Results) As is clear from the results shown in FIG. 11, both the polyplex with DET-PCn and the polyplex with DET-PRX remained stable even after incubation in the presence of FBS for several days.

[0091] Example 9: In vitro test of genome editing effect by polyplex with Cas12a (Cpf1) RNP, a genome editing molecule different from Cas9RNP. The above-mentioned sgGFP was used as the sgRNA. HeLa / GFP cells (3.75 × 10 ４ The cells were then washed twice with serum-free medium, and 500 μL of serum-free medium containing the following samples was added to the cells. (Samples) - Cpf1RNP alone (58.4 nM) - Polyplexes with DET-PCn formed as polyion complexes with 58.4 nM Cpf1RNP at charge ratios N / (P+C) of 2, 5, and 10 - Control (physiological saline)

[0092] After incubation in the presence of the sample at 37°C for 4 hours, the cells were washed twice with serum-free medium. 500 μL of DMEM containing 10% FBS was added, followed by incubation at 37°C for 44 hours. After daily medium changes, the cells were incubated for a total of 120 hours, after which the RNAi effect was evaluated by flow cytometry. The results are shown in Figure 12. In Figure 12, values ​​are expressed as mean ± standard error for n = 3 per group.

[0093] (Results) As is clear from the results shown in Figure 12, administration of Cpf1RNP alone did not reduce the number of HeLa / GFP cells, indicating that Cas9RNP alone is not taken up by cells. On the other hand, polyplexes with DET-PCn showed genome editing effects at charge ratios N / (P+C) of 5 and 10.

[0094] Example 10: In vitro test of knockdown effect by polyplex with antisense oligonucleotide (ASO) gapmer. A GFP ASO gapmer (GAACTTCAGGGGTCAGC (SEQ ID NO: 5)) was used as the ASO gapmer for GFP knockdown. Three-base LNA (Locked Nucleic Acid) was placed at both ends of the ASO gapmer, and the phosphodiester bonds connecting each nucleotide were phosphorothioated.

[0095] HeLa / GFP cells were seeded in a 24-well plate (3.75 × 10 ４ The cells were transfected in 500 μL of serum-free medium containing the following samples after washing twice with serum-free medium. (Samples) ASO gapmer alone (300 nM) Polyplex with DET-PCn, in which polyion complexes were formed with 300 nM ASO gapmer at N / P ratios of 5, 10, 15, and 20 Control (physiological saline)

[0096] After incubation in the presence of the sample at 37°C for 4 hours, the cells were washed twice with serum-free medium. 500 μL of DMEM containing 10% FBS was added, and the cells were incubated at 37°C for 68 hours. The knockdown effect was then evaluated by flow cytometry. The results are shown in Figure 13. In Figure 13, values ​​are expressed as the mean ± standard error for n = 3 per group.

[0097] (Results) As is clear from the results shown in Figure 13, administration of ASO gapmer alone did not result in a decrease in HeLa / GFP cells, indicating that ASO gapmer alone is not taken up by cells. In both cases of polyplexes with DET-PCn, the number of HeLa / GFP cells decreased, indicating that ASO gapmer was taken up by cells and GFP was knocked down by the ASO gapmer.

[0098] Example 11: In vivo test of RNAi effect by polyplex with siRNA (antitumor activity test) Colon-26 cells are known as a mouse colon-derived murine colon carcinoma cell line. PLK1 is overexpressed in a wide variety of cancers, and its expression is known to often correlate with poor prognosis ("Cell cycle kinases in cancer", Current Opinion in Genetics & Development 17(1):60-5(2007)). It is known that silencing of PLK1 expression by RNAi in cultured cells results in the selective death of cells with oncogenic mutations in KRAS, while being harmless to normal cells ("Finding the weakness in cancer", The New England Journal of Medicine 361(9):922-924). "A genome-wide RNAi screen" Cell 137(5):835-48 (2009).

[0099] The following siPLK1 (mouse) was used as siRNA, and the following siGL2 (an siRNA sequence that knocks down the firefly luciferase GL2 gene) was used as siCont (an siRNA not targeting PLK1). (siPLK1) sequence: 5'-CCUUGAUGAAGAAGAUCACdTdT-3' (SEQ ID NO: 6) antisence: 5'-GUGAUCUUCUUCAUCAAGGdTdT-3' (SEQ ID NO: 7) (siCont) sequence: 5'-CGUACGCGGAAUACUUCGAdTdT-3' (SEQ ID NO: 8) antisence: 5'-UCGAAGUAUUCCGCGUACGdTdT-3' (SEQ ID NO: 9)

[0100] (Creation of Colon-26 Cancer Model Mice) Colon-26 cells (5 × 10 ５ The tumors were subcutaneously transplanted into the mice. After 7 days, the tumor length reached 8 mm in all mice. The mice were divided into 5 groups (n = 5 in each group) so that the average tumor volumes were approximately equal.

[0101] (Samples) ・ siRNA (siPLK1) alone (10 μg) ・ Polyplex with DET-PCn or polyplex with DET-PRX, which were formed as a polyion complex with 10 μg of siRNA (siPLK1) to give an N / P ratio of 10 ・ Polyplex with DET-PCn, which were formed as a polyion complex with siCont (siGL2) to give an N / P ratio of 10 ・ Control (physiological saline)

[0102] (Intratumoral Administration) Figure 14 shows the correlation between the number of days since the initial administration and body weight (g). As shown in Figure 14, 100 μL of each sample was intratumorally administered every two days for a total of three times. Tumor volume was monitored every two days. Tumors were harvested 14 days after the initial administration. The results are shown in Figures 15(a) and 15(b). In Figures 15(a) and 15(b), values ​​are expressed as mean ± standard error (n = 5 per group). The significance level relative to control administration is *: p<0.05, the significance level relative to administration of a polyplex of siCont (siGL2) and DET-PCn: †: p<0.05, and the significance level relative to administration of siPLK1 alone: ​​††: p<0.05.

[0103] (Results) As is clear from the results shown in Figures 15(a) and (b), the group administered with a polyplex of siCont and DET-PCn and the group administered with siPLK1 alone showed tumor volumes (mm ) comparable to those of the control group. ３ ) increased, and it can be seen that the RNAi effect (antitumor activity) was not obtained. Although there was no significant difference between the siPLK1 and DET-PRX polyplex administration groups, the tumor volume (mm ３ On the other hand, the group administered with the polyplex of siPLK1 and DET-PCn showed a significant increase in tumor volume (mm ) from 12 days after the initial administration, compared with both the control group and the polyplex of siCont and DET-PCn. ３ ) was significantly suppressed, indicating that a significant RNAi effect (antitumor activity) was obtained. This is presumably due to the high cellular uptake efficiency of DET-PCn.

[0104] Example 12: In vivo test of genome editing effect by polyplex with Cas9RNP (BACE1 expression suppression test) BACE1 (beta-secretase 1) is known as an aspartic acid protease important for the formation of myelin sheath in peripheral nerve cells, and in humans is encoded by the BACE1 gene ("Control of peripheral nerve myelination by the beta-secretase BACE1", Science 314(5799):664-6(2006)). The generation of the 40- or 42-amino acid long amyloid β peptide, which aggregates in the brains of Alzheimer's disease patients, requires two cleavage steps of amyloid precursor protein (APP). Cleavage of the extracellular domain of APP by BACE1 produces a soluble extracellular fragment and a membrane-bound fragment called C99. Cleavage of the transmembrane domain of C99 by γ-secretase liberates the intracellular domain of APP and produces amyloid-β. Initial cleavage of APP by α-secretase rather than BACE1 prevents the final production of amyloid-β. Drugs that block BACE1 (BACE inhibitors) could theoretically prevent the formation of amyloid-β and slow or halt the progression of Alzheimer's disease.

[0105] The following sgRNAs were used: sgBACE1 and sgCont (sgRNA not targeting BACE1). sgBACE1: 5'-CCCGGAUGGCUUUUGGCUA-3' (SEQ ID NO: 10)

[0106] (Samples) Polyplexes with DET-PCn or DET-PRX, in which a polyion complex was formed with 58 pmol of Cas9RNP to give an imprinting rate of 50% Polyplexes with DET-PCn, in which a polyion complex was formed with Cas9RNP (sgCont) to give an imprinting rate of 50% Control (saline)

[0107] (Stereotaxic Injection) Balb / c mice (male, 3 months old) were intraperitoneally administered a triple-anesthesia mixture. Each of the above samples was stereotaxically injected into the CA1 region of the hippocampus (1.12 mm temporal to bregma, 1.94 mm dorsal, 1.75 mm deep) to a concentration of 58 pmol of Cas9RNP. The mice were kept in a thermostatic chamber until they woke up, and housed for 14 days. However, after 12 days, one mouse died in each of the groups administered with a polyplex of Cas9RNP (sgBACE1) and DET-PRX and the polyplex of Cas9RNP (sgCont) and DET-PCn. After perfusion with phosphate-buffered saline (PBS) and 4% paraformaldehyde / phosphate buffer (4% PFA), the brains were collected and stored refrigerated in 4% PFA. After replacement with 15% sucrose, sections were prepared and subjected to immunofluorescence staining (red fluorescence indicates Base1, blue fluorescence indicates cell nuclei), and Base1 expression was measured based on fluorescence intensity. The results are shown in Figures 16(a) and (b). In Figure 16, values ​​are expressed as mean ± standard error (n = 3 for each group), and the significance level relative to control administration is *: p < 0.05.

[0108] (Results) As is clear from the results shown in Figure 16(a), BACE1 expression was suppressed in the group administered with a polyplex of Cas9RNP(sgBACE1) and DET-PRX, although there was no significant difference. On the other hand, BACE1 expression was significantly suppressed in the group administered with a polyplex of Cas9RNP(sgBACE1) and DET-PCn, compared to the control group. This is presumably due to the high cellular uptake efficiency of DET-PCn.

[0109] 16(b) shows that in the control administration group, red fluorescence 1 due to Base1 expression is more prominent in the photograph than blue fluorescence 2 due to cell nuclei. On the other hand, in the polyplex with DET-PRX administration group, red fluorescence 1 due to Base1 expression is reduced in the photograph compared to blue fluorescence 2 due to cell nuclei. In particular, in the polyplex with DET-PCn administration group, red fluorescence 1 due to Base1 expression is reduced in the photograph compared to blue fluorescence 2 due to cell nuclei, and is hardly visible.

[0110] Example 13: Cellular uptake test of polyplex with siRNA (DET-PCn / siRNA) 3.75 × 10 HeLa cells were cultured in a 24-well plate. ４ The cells were seeded at a cell density of 1000 cells / well and incubated at 37°C for 24 hours. After washing twice with serum-free medium, 300 μL of serum-free medium containing the following various samples was added to the cells for transfection. Polycations such as DET-PCn bind to the negatively charged cell surface and can be taken up into cells via endocytosis. (Samples) - FITC (fluorescein isothiocyanate) labeled siRNA (non-functional siRNA (scrambled sequence siRNA)) alone (100 nM) - Polyplexes with DET-PCn formed as polyion complexes with 100 nM FITC-labeled siRNA at N / P ratios of 5, 10, 15, and 20

[0111] After incubation in the presence of the sample at 37°C for 4 hours, the cells were washed twice with serum-free medium. After cell detachment, the cells were collected by centrifugation (3,000 rpm) and resuspended in HBSS. The mean fluorescent intensity based on FITC was measured by flow cytometry. The results are shown in Figure 17. Figure 17 shows the uptake of FITC-labeled siRNA and DET-PCn complexes (DET-PCn / siRNA) into HeLa cells. In Figure 17, values ​​are expressed as mean ± standard error (n = 4 per group).

[0112] (Results) As is clear from the results shown in Figure 17, the fluorescence intensity based on FITC increased as the N / P ratio increased, and it can be seen that the amount of siRNA taken up into HeLa cells via endocytosis increased as the N / P ratio increased.

[0113] Example 14: Cellular uptake test of polyplex with Cas9RNP (DET-PCn / Cas9RNP) 3.75 × 10 HeLa cells were cultured in a 24-well plate. ４The cells were seeded at a cell density of 1000 cells / well and incubated at 37°C for 24 hours. After washing twice with serum-free medium, 500 μL of serum-free medium containing the following samples was added to the cells for transfection. Polycations such as DET-PCn bind to the negatively charged cell surface and can be taken up into cells via endocytosis. (Sample) ATTO (trademark) ５５０ Labeled Cas9 RNP (non-functional sgRNA) alone (29.2 nM) ATTO™ ５５０ Polyplexes with DET-PCn formed as polyion complexes with labeled Cas9RNP (29.2 nM) to achieve imprinting rates of 20%, 50%, and 100%.

[0114] After incubation in the presence of the sample at 37°C for 4 hours, the cells were washed twice with serum-free medium. After cell detachment, the cells were collected by centrifugation (3,000 rpm), resuspended in HBSS, and analyzed by flow cytometry using ATTO™. ５５０ The fluorescence intensity based on the ATTO™ was measured. The results are shown in Figure 18. ５５０ 18 shows the results of HeLa cell uptake of a complex of labeled Cas9RNP and DET-PCn (DET-PCn / Cas9RNP). In Fig. 18, the values ​​are for n=1 in each group.

[0115] As is clear from the results shown in FIG. 18, as the imprinting rate increases, the ATTO™ ５５０ It can be seen that the fluorescence intensity based on the imprinting rate increases, and the amount of Cas9RNP taken up into HeLa cells via endocytosis increases as the imprinting rate increases.

[0116] Example 15: Zeta potential measurement test of DET-PCn / siRNA under different pH environments The zeta potential (mV) of DET-PCn / siRNA (siRNA: 1.36 μg; N / P ratio 10) was measured by dynamic light scattering in an HBSS buffer at pH 7.4 (in vivo environment) and in an HBSS buffer at pH 5.5 (endosomal environment). The results are shown in Figure 19. In Figure 19, values ​​are expressed as the mean ± standard error for n = 3 in each group.

[0117] As is clear from the results shown in Figure 19, the ζ potential is increased at pH 5.5 (endosomal environment) compared to pH 7.4 (in vivo environment), which indicates that the amine-containing group has a monovalent proton at pH 7.4 (in vivo environment) but a divalent proton at pH 5.5 (endosomal environment).

[0118] Example 16: Zeta potential measurement test of DET-PCn / Cas9RNP in different pH environments The zeta potential (mV) of DET-PCn / Cas9RNP (sgRNA: 2.0 μg; imprinting rate 50%) was measured by dynamic light scattering in HBSS buffer at pH 7.4 (in vivo environment) and in HBSS buffer at pH 5.5 (endosomal environment). The results are shown in Figure 20. In Figure 20, values ​​are expressed as the mean ± standard error for each group (n = 3).

[0119] As is clear from the results shown in Figure 20, the ζ potential is increased at pH 5.5 (endosomal environment) compared to pH 7.4 (in vivo environment), which indicates that the amine-containing group has a monovalent proton at pH 7.4 (in vivo environment) but a divalent proton at pH 5.5 (endosomal environment).

[0120] Example 17: Membrane disruption performance test of DET-PCn in an endosomal pH environment (4°C, LDH assay) To evaluate the endosomal membrane disruption performance of DET-PCn (membrane disruption performance in an endosomal pH environment), the endosomal membrane disruption performance of DET-PCn was measured at pH 7.4 (in vivo environment) and pH 5.5 (endosomal environment) using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories). The Cytotoxicity LDH Assay Kit-WST can measure membrane disruption performance by measuring the activity of lactate dehydrogenase (LDH) released from cells into the medium. LDH is an enzyme present in the cytoplasm that normally remains in the cytoplasm but leaks into the medium when the cell membrane is damaged. Because the released LDH is stable, it is widely measured as an indicator of the number of cells with damaged cell membranes. The results are shown in Figure 21.

[0121] 21, it is clear that LDH leakage is increased at pH 5.5 (endosomal environment) compared to LDH leakage at pH 7.4 (in vivo environment). This indicates that membrane-disrupting ability is weak at pH 7.4 (in vivo environment) but is increased at pH 5.5 (endosomal environment).

[0122] Example 18: Comparative test of cell safety of DET-PCn / siRNA HeLa cells were seeded (1.5 × 10 ４ The cells were transfected by adding 200 μL of serum-free medium containing the following samples to the cells after washing twice with serum-free medium. (Samples) - Polyplexes with DET-PCn or DET-PRX (2 kDa) formed as polyion complexes with 100 nM siRNA (non-functional siRNA) at N / P ratios of 5, 10, 15, and 20.

[0123] After incubation in the presence of the sample at 37°C for 4 hours, the plate was washed twice with serum-free medium. 200 μL of FBS-containing medium was added, and the plate was incubated at 37°C for 20 hours. The viable cell count was then measured using the WST-8 (Water-Soluble Tetrazolium Salt-8) method (Cell Counting Kit-8: Dojindo Laboratories) as follows. After washing twice with 200 μL of HBSS buffer, 100 μL of HBSS buffer and 10 μL of WST-8 were added to each well, followed by incubation at 37°C for 1 hour. The viable cell count was then determined by measuring absorbance at a wavelength of 450 nm (formazan) and 655 nm (background correction). The results are shown in Figure 22. In FIG. 22, values ​​are expressed as mean ± standard error for each group (n=7), and *: p<0.05.

[0124] As is clear from the results shown in Figure 22, there is almost no difference in cell viability between polyplexes with DET-PCn and polyplexes with DET-PRX at an N / P ratio of 15 or less. On the other hand, at an N / P ratio of 20 or more, the cell viability of polyplexes with DET-PRX decreases, whereas the cell viability of polyplexes with DET-PCn is significantly higher. In other words, it can be said that DET-PCn / siRNA is more cytosafe than DET-PRX / siRNA.

[0125] Example 19: Comparative cell safety test of DET-PCn / Cas9RNP HeLa cells were seeded (1.5 × 10) in a 96-well plate. ４ The cells were transfected with 200 μL of serum-free medium containing the following samples after washing twice with serum-free medium. (Samples) - Polyplexes with DET-PCn or DET-PRX (2 kDa) formed as polyion complexes with 29.2 nM Cas9RNP to achieve imprinting rates of 10%, 20%, 50%, 75%, 100%, and 200%.

[0126] After incubation in the presence of the sample at 37°C for 4 hours, the plate was washed twice with serum-free medium. 200 μL of FBS-containing medium was added, and the plate was incubated at 37°C for 20 hours. The viable cell count was then measured using the WST-8 method (Cell Counting Kit-8, manufactured by Dojindo Laboratories) as follows. After washing twice with 200 μL of HBSS buffer, 100 μL of HBSS buffer and 10 μL of WST-8 were added to each well and incubated at 37°C for 1 hour. The viable cell count was then determined by measuring absorbance at a wavelength of 450 nm (formazan) and 655 nm (background correction). The results are shown in Figure 23. In Figure 23, values ​​are expressed as the mean ± standard error of n = 7 per group, and * indicates p < 0.05.

[0127] As is clear from the results shown in Figure 23, there is almost no difference in cell viability between polyplexes with DET-PCn and polyplexes with DET-PRX at imprinting rates of 100% or less. On the other hand, at imprinting rates exceeding 100%, the cell viability of polyplexes with DET-PRX decreases, whereas the cell viability of polyplexes with DET-PCn is significantly higher. In other words, it can be said that DET-PCn / Cas9RNP has higher cell safety than DET-PRX / Cas9RNP.

Claims

1. A polycatenane having a plurality of macrocycle molecules and a cyclic axon molecule passing through the rings of the macrocycle molecules, wherein at least some of the macrocycle molecules in the polycatenane have an amine-containing group.

2. The polycatenane according to claim 1, which has the ability to form a polyion complex with a biological material.

3. The polycatenane of claim 2, wherein the biological material is a nucleic acid molecule or a complex of Cas9 protein and guide RNA (Cas9 RNP).

4. The polycatenane of claim 1, wherein the amine-containing group has a monovalent proton at at least one point between pH 7.0 and 7.6 and a divalent proton at at least one point between pH 4.0 and 6.

5.

5. The polycatenane of claim 1, wherein at least some of the macrocyclic molecules further have an amino group, separate from the amine-containing group, via an intracellularly degradable bond.

6. The polycatenane according to claim 1, wherein the penetration rate (the coverage rate of the cyclic axis molecules by the macrocyclic molecules) is 1 to 21%.

7. A cell introduction agent for biological materials, comprising the polycatenane of claim 1.

8. A polyion complex of a biological material and the polycatenane of claim 2.

9. A composition containing the polycatenane of claim 2 and a biological material, or containing the polyion complex of claim 8.

10. A method for introducing a biological material into a cell, comprising contacting the composition according to claim 9 with the cell and allowing the polyion complex to be incorporated into the cell.

Images