Biomolecule delivery system

Abstract

The present invention is a kit for delivering biomolecules to cell cytoplasm, by forming a complex with a biomolecule and administering the complex into a subject, the kit including: a complex of a biocompatible polymer-modified polyphenol, metal ions, and biomolecules; a biocompatible polymer-modified polyphenol; and a biocompatible polymer-modified polyphenol, and metal ions.

Description

Biomolecule Delivery Systems

[0001] The present invention relates to a biomolecule delivery system. More specifically, the present invention relates to a conjugate, a biocompatible polymer-modified polyphenol, and a kit. This application claims priority to Japanese Patent Application No. 2024-018594, filed February 9, 2024, the contents of which are incorporated herein by reference.

[0002] Biopharmaceuticals, which use proteins and other biomolecules as active ingredients, are attracting great expectations as groundbreaking treatments for intractable diseases such as cancer. For example, intracellular antibodies, which have been attracting attention in recent years, are expected to be new antibody drugs because they can bind to intracellular antigens and induce cell death and cell signaling.

[0003] However, biomolecules have low blood stability and retention, and do not exhibit the expected pharmacological activity. Furthermore, biopharmaceuticals have low cellular uptake and cytoplasmic transport, which limits their activity.

[0004] In response to this, chemical modification of biomolecules with biocompatible polymers such as polyethylene glycol (PEG) can improve the stability and retention of biomolecules in blood. However, this method raises concerns that chemical modification with PEG may reduce the pharmacological activity of biomolecules. Furthermore, PEGylation cannot improve the cellular uptake and cytoplasmic translocation of biomolecules.

[0005] In response to this, research is being conducted into the uptake of biomolecules into cells and their subsequent transfer into the cytoplasm by using cationic molecules such as cationic peptides, polymers such as polyethyleneimine, and liposomes (see, for example, Non-Patent Documents 1 to 3).

[0006] Chatin B., et al., Liposome-based Formulation for Intracellular Delivery of Functional Proteins, Mol Ther Nucleic Acids, 4, e244, 2015.Boussif O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl. Acad. Sci. USA, 92, 7297-7301, 1995.Akishiba M., et al., Cytosolic antibody delivery by lipid-sensitive endosomal peptide, Nature Chemistry, 9, 751-761, 2017.

[0007] Co-administration of a biological molecule, such as an intracellular antibody, and a cationic molecule allows the biological molecule to be taken up by cells and then disrupt the endosomal membrane, resulting in its transfer to the cytoplasm. However, cationic molecules can be highly toxic to living organisms. Furthermore, cationic molecules are easily recognized as foreign substances in the body. For this reason, methods using cationic molecules have not been able to fully utilize the activity of the biological molecule. The present invention aims to provide a technology for efficiently delivering biological molecules to the cytoplasm.

[0008] The present invention includes the following aspects: [1] A complex of a biocompatible polymer-modified polyphenol, a metal ion, and a biomolecule. [2] The complex according to [1], wherein the polyphenol is at least one selected from the group consisting of tannic acid, gallic acid, and derivatives thereof. [3] The complex according to [1] or [2], wherein the biocompatible polymer comprises at least one selected from the group consisting of polyethylene glycol, acrylic resin, polyamino acid, polyvinylamine, polyallylamine, polynucleotide, polyacrylamide, polyether, polyester, polyurethane, polysaccharide, and copolymers thereof. [4] The complex according to any one of [1] to [3], wherein the weight-average molecular weight of the biocompatible polymer is 1,000 to 50,000. [5] The complex according to any one of [1] to [4], wherein the metal ion is an iron(III) ion, a cobalt(II) ion, or a zirconium(IV) ion. [6] The conjugate according to any one of [1] to [5], wherein the biomolecule is an antibody or an antigen-binding fragment thereof, a physiologically active protein, a nucleic acid, a lipid nanoparticle, or an inorganic / organic microparticle. [7] A biocompatible polymer-modified polyphenol. [8] A kit for delivering the biomolecule to the cytoplasm by forming a conjugate with a biomolecule and administering the conjugate to a subject, the kit comprising a biocompatible polymer-modified polyphenol and a metal ion.

[0009] According to the present invention, a technique for efficiently delivering a biomolecule into the cytoplasm can be provided.

[0010] FIG. 1 shows the PEG-P[Lys(TFA)] measured in Experimental Example 1. ２５ of １ 2 is a 1 H NMR spectrum of PEG-P[Lys(TFA)] measured in Experimental Example 1. １０５ of １ 3 shows the H NMR spectrum of PEG-P[Lys(TFA)] measured in Experimental Example 1. ２５ 4 is a GPC curve of PEG-P[Lys] measured in Experimental Example 1. ２５ of １ 5 shows the H NMR spectrum of PEG-P[Lys] measured in Experimental Example 1.１０５ of １ 6 shows the H NMR spectrum of PEG-P[Lys １９ / (Lys-GA) ６ ]of １ 7 shows the H NMR spectrum of PEG-P[PEG-P[Lys ７２ / (Lys-GA) ３３ ]of １ 8 shows the H NMR spectrum of PEG-P[Lys １９ / (Lys-GA) ６ 9 is a GPC curve of PEG-P[PEG-P[Lys] measured in Experimental Example 1. ７２ / (Lys-GA) ３３ 10 is a GPC curve of PEG measured in Experimental Example 2. １０ｋ -TA's １ 11 is a 1 H NMR spectrum of PEG measured in Experimental Example 2. １０ｋ 12 is a GPC curve of PEG-TA measured in Experimental Example 2. ５ｋ -TA's １ 13 is a 1 H NMR spectrum of PEG measured in Experimental Example 2. ５ｋ 14 shows the GPC curve of GA / Fe measured in Experimental Example 3. ３＋ 15 shows the UV-vis spectrum of the GA / Fe composite at pH 7.4 and pH 4.0 measured in Experimental Example 3. ３＋ 16 shows the UV-vis spectrum of the complex of β-Gal / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ 17 is a graph showing the particle size measurement results of the antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ 18 is a graph showing the particle size measurement results of the MPN complex.ｎ / (Lys-GA) ｍ 19 is a graph showing the particle size measurement results of antibody-MPN conjugates by fluorescence spectroscopy correlation analysis when PEG-P[Lys ｎ / (Lys-GA) ｍ 20 is a transmission electron microscope (TEM) image of the antibody in Experimental Example 4. FIG. 21 is a graph showing the particle size measurement results of the antibody-MPN conjugate by fluorescence spectroscopy correlation method when antibody / PEG-P[Lys １１ / (Lys-GA) １４ 22 is a TEM image of antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ 23 is a TEM image of antibody / PEG-P[Lys ７２ / (Lys-GA) ３３ ] / Fe ３＋ 24 shows the TEM image of the antibody / PEG-TA / Fe complex by fluorescence spectroscopy correlation analysis in Experimental Example 4. ３＋ 25 is a graph showing the particle size measurement results of the antibody / PEG-TA / Fe complex by dynamic light scattering in Experimental Example 4. ３＋ 26 shows a size histogram of the MPN complex. ３＋ 27 is a graph showing the zeta potential of the MPN complex. ３＋ 28 is a graph showing the particle size measurement results of the MPN complex in Experimental Example 4. ３＋ 29 is a graph showing the particle size measurement results of the MPN complex. １０ｋ -TA / Zr ４＋ MPN complex, RNP / PEG １０ｋ -TA / Co ２＋30 is a graph showing the particle size measurement results of the MPN complex. ５ｋ -TA / Zr ４＋ MPN complex, RNP / PEG ５ｋ -TA / Co ２＋ 31 is a graph showing the particle size measurement results of the MPN complex. １０ｋ -TA / Fe ３＋ 32 is a graph showing the particle size measurement results of the MPN complex. ５ｋ -TA / Fe ３＋ 33 is a graph showing the particle size measurement results of the MPN complex. FIG. 34 is a TEM image of RNP in Experimental Example 5. FIG. 35 is a graph showing the particle size measurement results of the MPN complex. FIG. 36 is a TEM image of RNP in Experimental Example 5. FIG. 37 is a graph showing the particle size measurement results of the MPN complex. １０ｋ 35 shows a TEM image of RNP / PEG-TA in Experimental Example 5. １０ｋ -TA / Fe ３＋ FIG. 36 shows a TEM image of PEG-NH ２ , PEG １０ｋ -TA, PEG １０ｋ -TA / Fe ３＋ , Fe ３＋ 37 is a graph showing the results of evaluating the cytotoxicity of antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ Fig. 38 is a graph showing the colocalization efficiency of an antibody and lysosomes calculated from confocal microscope images in Experimental Example 7. Fig. 39 is a graph showing the intracellular distribution of anti-NPC antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ Fig. 40 is a graph showing the results of measuring the amount of cellular uptake of an antibody sample containing an antibody / PEG-TA / Fe complex in Experimental Example 7. Fig. 41 is a graph showing the amount of cellular uptake of an antibody / PEG-TA / Fe complex after 24 hours of incubation in Experimental Example 7. ３＋42 shows the intracellular distribution of antibody / PEG-TA / Fe after 48 hours of incubation in Experimental Example 7. ３＋ Fig. 43 is a graph showing the colocalization efficiency of the antibody and lysosomes after 24 hours and 48 hours of incubation, calculated from the confocal microscope images in Experimental Example 7. Fig. 44 is a graph showing the colocalization efficiency of the anti-NPC antibody / PEG-TA / Fe in Experimental Example 7. ３＋ Figure 45 shows a confocal microscope image showing the intracellular distribution of Cas9-sgRNA RNP / PEG in Experimental Example 8. １０ｋ -TA / Fe ３＋ 46 is a graph showing the results of evaluating the cytotoxicity of the MPN complex. １０ｋ -TA / Fe ３＋ 47 is a graph showing the results of evaluating the gene knockout efficiency of the MPN complex in Experimental Example 9, in a 4T1-Luc subcutaneous tumor mouse model. ３＋ , antibody / TA / Fe ３＋ 48 is a graph showing the results of comparing the organ accumulation of antibody, antibody / PEG-TA, antibody / PEG-TA / Fe in a 4T1-Luc subcutaneous tumor mouse model in Experimental Example 9. ３＋ , antibody / TA / Fe ３＋ 49 is a graph showing the results of comparing the blood retention of anti-NPC antibody, anti-NPC antibody / PEG-TA, and anti-NPC antibody / PEG-TA / Fe in Experimental Example 9. ３＋ , anti-NPC antibody / TA / Fe ３＋ 50 shows IVIS images of a 4T1 subcutaneous tumor mouse model administered with anti-NPC antibody, anti-NPC antibody / PEG-TA, and anti-NPC antibody / PEG-TA / Fe in Experimental Example 9. ３＋ , anti-NPC antibody / TA / Fe ３＋Figure 51 is a graph showing the results of quantifying the antibody-derived fluorescence intensity in the cell nuclei within the 4T1 subcutaneous tumor of Figure 50. Figure 52 is a fluorescent microscope image showing the distribution of anti-S100A4 antibody / PEG-TA / Fe in the 4T1-Luc subcutaneous tumor mouse model in Experimental Example 9. ３＋ 53 is a graph showing the results of measuring the antitumor effects of a comparative sample and an anti-S100A4 antibody / PEG-TA / Fe compound in a 4T1-Luc subcutaneous tumor mouse model in Experimental Example 9. ３＋ 54 is a graph showing the results of measuring the change in body weight when RNP / PEG-TA / Fe was administered to a 4T1-Luc subcutaneous tumor mouse model in Experimental Example 10. ３＋ 55 is a graph showing the results of measuring the Luc gene knockout efficiency when TA, TA (quinone), PEG, and the like were administered in Experimental Example 11. ５ｋ -TA (M ｎ = 6,700), PEG １０ｋ -TA (M ｎ 56 is a graph showing the UV absorption results of TA, PEG-NH (=11,700) measured by FT-IR in Experimental Example 11. ２ (Mn: 10k), PEG １０ｋ -TA, PEG-NH ２ 57 is a graph showing the spectrum of TA, PEG-NH (Mn: 10k) + TA measured by FT-IR in Experimental Example 11. ２ (Mn: 5k), PEG ５ｋ -TA, PEG-NH ２ 58 is a graph showing the spectrum of GalNac-PEG (Mn: 5k) + TA measured in Experimental Example 12. １０ｋ -NH ２ of １ 59 shows the H NMR spectrum of GalNac-PEG measured in Experimental Example 12. １０ｋ -TA's １ 60 shows the H NMR spectrum of PEG measured in Experimental Example 13. １０ｋ -TET's １61 shows the H NMR spectrum of PEG measured in Experimental Example 13. １０ｋ -COOH, PEG １０ｋ -TA, PEG １０ｋ -TET-TA, PEG １０ｋ 62 shows the SEC spectrum of PEG-TEP-TA measured in Experimental Example 13. １０ｋ -TEP's １ 63 shows the H NMR spectrum of PEG measured in Experimental Example 13. １０ｋ -S's １ 64 shows the H NMR spectrum of PEG measured in Experimental Example 13. １０ｋ Figure 65 shows the absorption spectrum of a mixture of polyphenol molecules and iron ions measured in Experimental Example 14. Figure 66 shows the SEC spectrum of β-Gal / PEG measured by fluorescence spectroscopic correlation spectroscopy in Experimental Example 15. １０ｋ -TA / Fe ３＋ MPN complex, β-Gal / GalNac-PEG １０ｋ -TA / Fe ３＋ 67 is a graph showing the particle size measurement results of the MPN complex. ５ｋ -TA, LNP / PEG ５ｋ -TA / Fe ３＋ 68 is a graph showing the particle size measurement results of PEG measured using Zetasizer Ultra (red) in Experimental Example 16. ５ｋ -TA and FeCl ３ ・6H ２ 69 is a graph showing the particle size measurement results of each LNP-MPN complex when the amount of O mixed is changed. ５ｋ -TA / Fe ３＋ , PS / PEG １０ｋ -TA / Fe ３＋ 70 is a graph showing the particle size measurement results of TUG1, TUG1 / PEG, and TUG1 / PEG in Experimental Example 16, measured by fluorescence spectroscopic correlation spectroscopy. １０ｋ-TA complex, TUG1 / PEG １０ｋ -TA / Zr ４＋ 71 is a graph showing the particle size measurement results of the complexes TUG1, TUG1 / PEG-P[Lys １１ / (Lys-GA) １４ ] complex, TUG1 / PEG-P[Lys １１ / (Lys-GA) １４ ] / Zr ４＋FIG. 72 is a graph showing the particle size measurement results of the complexes. FIG. 72 is a graph showing the particle size measurement results of the RNP-loaded MPN complexes prepared using PEG-TET-TA and PEG-TEP-TA in Experimental Example 17, measured by fluorescence spectroscopy correlation spectroscopy. FIG. 73 is a graph showing the particle size measurement results of the RNP-loaded MPN complexes prepared using PEG-S-TA in Experimental Example 17, measured by fluorescence spectroscopy correlation spectroscopy. FIG. 74 is a TEM image of an antibody-MPN complex sample in Experimental Example 18. FIG. 75 is the measurement results of the buffering effect of the antibody-MPN complex in Experimental Example 18. FIG. 76 is the measurement results of the blood stability of the antibody-loaded MPN complex in Experimental Example 18. FIG. 77 is the result of liver toxicity evaluation of the anti-S100A4 antibody-loaded MPN complex in Experimental Example 18. Figure 78 is a graph showing the change in tumor size over time in a breast cancer orthotopic model mouse administered with an anti-S100A4 antibody-loaded MPN complex in Experimental Example 18. Figure 79 is a graph showing the change in body weight over time in a breast cancer orthotopic model mouse administered with an anti-S100A4 antibody-loaded MPN complex in Experimental Example 18. Figure 80 shows the results of immunostaining for the tumor suppressor gene (p53) in a thin section of a tumor in a breast cancer subcutaneously transplanted model mouse administered with an anti-S100A4 antibody-loaded MPN complex, obtained in Experimental Example 18. Figure 81 is an image of a TUNEL assay of a thin section of a tumor in a breast cancer subcutaneously transplanted model mouse administered with an anti-S100A4 antibody-loaded MPN complex, obtained in Experimental Example 18. Figure 82 is a graph showing the results of measuring the blood retention of a β-Gal-loaded MPN complex in Experimental Example 19. Figure 83 is a graph showing the results of measuring the cellular uptake rate of β-Gal-loaded MPN complexes in the presence and absence of inhibitors in Experimental Example 20. Figure 84 is a graph showing the results of administering RNP-loaded MPN complexes to cancer-bearing model mice and measuring organ accumulation 6 hours later in Experimental Example 21. Figure 85 is a graph showing the results of administering RNP-loaded MPN complexes to cancer-bearing model mice and measuring tumor accumulation 6 hours later in Experimental Example 21. Figure 86 is a graph showing the results of administering RNP-loaded MPN complexes to cancer-bearing model mice and measuring blood retention in Experimental Example 21.Figure 87 is a graph showing the results of administering an RNP-loaded MPN complex to HeLa-Luc cells and measuring Luc luminescence intensity in Experimental Example 22. Figure 88 is a graph showing the results of measuring genome editing efficiency in the liver of Ai9 td Tomato mouse administered with an RNP-loaded MPN complex in Experimental Example 23. Figure 89 is a graph showing the results of measuring genome editing efficiency in the muscle of Ai9 td Tomato mouse administered with an RNP-loaded MPN complex in Experimental Example 23. Figure 90 is a graph showing the results of measuring genome editing efficiency in the brain of Ai9 td Tomato mouse administered with an RNP-loaded MPN complex in Experimental Example 23.

[0011] [Conjugate] In one embodiment, the present invention provides a conjugate of a biocompatible polymer-modified polyphenol, a metal ion, and a biomolecule. As described below in the Examples, administration of the conjugate of this embodiment allows the biomolecule to be taken up into cells, and then escape from the endosome and delivered to the cytoplasm. Therefore, the conjugate of this embodiment can be said to be a delivery system for delivering the biomolecule to the cytoplasm.

[0012] The complex of this embodiment may be added to a culture medium in vitro or administered to a living body in vivo. Examples of living bodies include humans and non-human animals. Non-human animals are not particularly limited, and include rodents such as mice, rats, and hamsters, non-human primates such as monkeys and chimpanzees, and mammals such as rabbits, sheep, cows, and pigs. Routes of administration to living bodies include intravenous injection.

[0013] In the conjugate of this embodiment, the biomolecule is preferably one that exerts its physiological activity when delivered to the cytoplasm and is preferably one that can be used as a biopharmaceutical. The biomolecule is not particularly limited as long as it has physiological activity, and examples thereof include antibodies or antigen-binding fragments thereof, physiologically active proteins, nucleic acids, lipid nanoparticles, inorganic / organic fine particles, etc.

[0014] The antibody may be a human antibody. Human antibodies have low immunogenicity when administered to humans, and therefore can suppress side effects such as anaphylactic shock. Examples of human antibodies include chimeric antibodies, humanized antibodies, and fully human antibodies.

[0015] A chimeric antibody refers to an antibody in which the variable region is derived from a non-human animal and at least a portion of the constant region is derived from a human. A humanized antibody refers to an antibody in which only the complementarity-determining regions (CDRs) of the heavy and light chains are derived from a non-human animal and the constant and framework regions are derived from a human. A fully human antibody refers to an antibody whose entire body, including the complementarity-determining regions, is derived from a human. Antigen-binding fragments of antibodies include F(ab') ２ , Fab', Fab, Fv, scFv and the like.

[0016] The physiologically active protein is not particularly limited as long as it has physiological activity, and includes enzymes, complexes of CRISPR / Cas proteins and gRNA (ribonucleoprotein, RNP), and the like.

[0017] The nucleic acid is not particularly limited as long as it has physiological activity, and examples thereof include DNA, RNA, etc. Examples of RNA include mRNA, shRNA, etc.

[0018] Lipid nanoparticles (LNPs) are lipid-based particles with a diameter of approximately 10 nm to 1,000 nm. They are used as non-viral drug delivery systems (DDS) for the delivery of nucleic acid drugs, etc. Lipid nanoparticles may encapsulate drugs. As described later in the Examples, the complex of this embodiment can be formed by mixing biocompatible polymer-modified polyphenols, metal ions, and lipid nanoparticles.

[0019] The inorganic / organic fine particles refer to inorganic fine particles, organic fine particles, composite fine particles thereof, etc. The diameter of the inorganic / organic fine particles is preferably about 10 nm to 1,000 nm. Examples of inorganic / organic fine particles include polystyrene particles, silica particles, and gold nanoparticles. As will be described later in the Examples, the composite of this embodiment can be formed by mixing a biocompatible polymer-modified polyphenol, a metal ion, and inorganic / organic fine particles.

[0020] A biocompatible polymer-modified polyphenol is a compound in which a biocompatible polymer and a polyphenol are bound together. In the composite of this embodiment, examples of the polyphenol include tannic acid, gallic acid, and derivatives thereof. Examples of derivatives of tannic acid or gallic acid include tannic acid or gallic acid in which one or more hydrogen atoms or hydroxyl groups have been substituted with a substituent. Examples of the substituent include a hydroxyl group, an amino group, a monovalent linear saturated hydrocarbon group having 1 to 10 carbon atoms, and a halogen atom. Examples of the monovalent linear saturated hydrocarbon group having 1 to 10 carbon atoms include a methyl group, an ethyl group, a propyl group, and a butyl group. Examples of the halogen atom include a fluorine atom and a chlorine atom.

[0021] A biocompatible polymer refers to a polymer that does not or is unlikely to cause significant harmful or adverse effects, such as strong inflammatory responses or injury, when administered to a living body. In the composite of this embodiment, biocompatible polymers include polyethylene glycol, acrylic resins, polyamino acids, polyvinylamines, polyallylamine, polynucleotides, polyacrylamides, polyethers, polyesters, polyurethanes, polysaccharides, and copolymers thereof. The biocompatible polymer may partially contain any group introduced during its synthesis. Examples of such groups include a portion of a polymerization initiator.

[0022] As will be described later in the Examples, functional molecules may be introduced into the biocompatible polymer. Examples of functional molecules include N-acetylgalactosamine (GalNAc), which is a hepatocyte targeting molecule, and cationic molecules that improve cellular uptake. Examples of cationic molecules include ethylenediamine-based molecules, more specifically, triethylenetriamine (TET), tetraethylenepentamine (TEP), spermine (S), and the like.

[0023] The weight-average molecular weight of the biocompatible polymer may be 1,000 to 50,000. The upper limit of the weight-average molecular weight of the biocompatible polymer may be 30,000, 20,000, or 10,000. The lower limit of the weight-average molecular weight of the biocompatible polymer may be 2,000, 3,000, 4,000, or 5,000. These upper and lower limits can be combined as appropriate.

[0024] Here, the weight-average molecular weight of a biocompatible polymer can be measured by size exclusion chromatography (SEC). Specifically, the biocompatible polymer is dissolved in a solvent and then passed through a column containing a packing material with many pores, along with a mobile phase solution. The polymers are separated by molecular weight in the column, and the resulting molecular weights are detected using a differential refractometer, ultraviolet-visible spectrophotometer, viscometer, light scattering detector, or the like. Dedicated SEC devices are widely available commercially, and measurements are generally made in terms of standard polyethylene glycol. The weight-average molecular weights used herein are measured in terms of standard polyethylene glycol.

[0025] The dispersity (Mw / Mn) of the biocompatible polymer is preferably 1.0 or more and less than 2.0, more preferably 1.0 to 1.5, and even more preferably 1.0 to 1.3. When the dispersity of the biocompatible polymer is within the above range, the conjugate of the present embodiment can exhibit high tumor accumulation.

[0026] The tumor accumulation is thought to be exerted by selective accumulation in tumors taking advantage of the increased vascular leakage of tumors, i.e., the enhanced permeability and retention effect (EPR effect), and it becomes possible to selectively deliver the complex of this embodiment to tumors.

[0027] When polyphenols are mixed with biomolecules, they spontaneously form complexes, which are thought to be formed through hydrophobic interactions and / or hydrogen bonds, and can be formed without chemical modification of the biomolecules.

[0028] When biocompatible polymer-modified polyphenols are mixed with biomolecules, they spontaneously form complexes, resulting in the biomolecule being attached to the biocompatible polymer via the polyphenol.

[0029] As described later in the Examples, the complex of this embodiment is formed spontaneously by mixing a biocompatible polymer-modified polyphenol, a metal ion, and a biomolecule in a buffer solution. The biocompatible polymer-modified polyphenol, the metal ion, and the biomolecule may be mixed simultaneously, or the three may be mixed in any order. In either case, the complex of this embodiment is formed spontaneously. When the three are mixed in order, it is preferable to mix the biomolecule, the biocompatible polymer-modified polyphenol, and the metal ion in that order. The buffer solution is not particularly limited, and for example, phosphate-buffered saline (PBS) or the like can be used.

[0030] The biocompatible polymer-modified polyphenol is preferably mixed so that the molar ratio of biomolecule to biocompatible polymer-modified polyphenol is 1:20 to 1:5,000, more preferably 1:25 to 1:3,000. The metal ion is preferably mixed so that the molar ratio of polyphenol to metal ion in the biocompatible polymer-modified polyphenol is 5:1 to 1:5, more preferably 3:1 to 1:3.3.

[0031] In the composite of this embodiment, the metal ion is not particularly limited as long as it can form a metal ion-polyphenol complex (MPN) having buffering capacity by crosslinking the polyphenol described above, and specific examples include iron (III) ions, cobalt (II) ions, zirconium (IV) ions, etc. The metal ion can be used by dissolving a metal salt that generates the above metal ion upon ionization in a solvent such as a buffer solution.

[0032] As will be described later in the Examples, metal ions such as iron (III) ions, cobalt (II) ions, and zirconium (IV) ions form pH-responsive complexes with polyphenols, thereby exhibiting buffering ability.

[0033] The complex of this embodiment is taken up into cells in a state encapsulated in a vesicle called an endosome. The endosomal membrane contains a proton pump called V-ATPase, which transports protons into the endosome until the pH inside the endosome reaches approximately 5-6. However, because the complex of this embodiment has buffering capacity, a decrease in the pH inside the endosome is suppressed, and a larger number of protons must flow into the endosome to lower the pH. Furthermore, anions also flow into the endosome to maintain the charge balance inside and outside the endosome, increasing the salt concentration and osmotic pressure inside the endosome. To resolve this high osmotic pressure, a large amount of water flows into the endosome, and the endosomal membrane cannot withstand the increased volume, causing it to collapse. As a result, it is believed that the complex of this embodiment can escape from the endosome (lysosome). This effect is called the proton sponge effect. In other words, the complex of this embodiment exhibits the proton sponge effect.

[0034] [Compound] In one embodiment, the present invention provides a biocompatible polymer-modified polyphenol. The biocompatible polymer-modified polyphenol of this embodiment is a novel compound. The biocompatible polymer and polyphenol are the same as those described above.

[0035] More specific examples of the compound of this embodiment include tannic acid-modified polyethylene glycol (PEG-TA) shown in the following formula (1), and PEG-Poly[(L-Lysine) ｎ / (L-Lysine-Gallic Acid) ｍ ] etc.

[0036] In formula (1) and formula (2), p represents the number of polyethylene glycol units. In formula (2), n represents the number of lysine residues, and m represents the number of gallic acid-modified lysine residues. p may be approximately 45 to 4,500. Furthermore, n may be approximately 10 to 200. Furthermore, m may be approximately 5 to 100. As will be described later in the Examples, by using the compounds represented by formula (1) and formula (2), a biomolecule can be taken up into the cytoplasm of a cell, and further escaped from an endosome and delivered to the cytoplasm.

[0037]

[0038]

[0039] As will be described later in the Examples, a part of the galloyl group structure of the tannic acid-modified polyethylene glycol may be a quinone structure. The chemical formula of the tannic acid-modified polyethylene glycol in which a part of the galloyl group structure is a quinone structure is shown in the following formula (3). In the following formula (3), n represents the number of polyethylene glycol units.

[0040]

[0041] As will be described later in the Examples, a functional molecule may be introduced into the compound of this embodiment. Examples of functional molecules include N-acetylgalactosamine (GalNAc), which is a hepatocyte targeting molecule, and cationic molecules that improve cellular uptake. Examples of cationic molecules include ethylenediamine-based molecules, more specifically, triethylenetriamine (TET), tetraethylenepentamine (TEP), spermine (S), and the like.

[0042] [Kit] In one embodiment, the present invention provides a kit for delivering a biomolecule to the cytoplasm by forming a complex with the biomolecule, comprising a biocompatible polymer-modified polyphenol and a metal ion, and administering the complex to a subject.

[0043] As will be described later in the Examples, the kit of this embodiment enables efficient delivery of biomolecules into the cytoplasm of target cells.

[0044] In the kit of this embodiment, the biocompatible polymer, polyphenol, metal ion, biomolecule, etc. are the same as those described above.

[0045] As described above, when a biocompatible polymer-modified polyphenol, a metal ion, and a biomolecule are mixed in a buffer solution, a complex is spontaneously formed. By administering this complex to a subject, the biomolecule can be efficiently delivered into the cytoplasm of the subject's cells.

[0046] The complex may be added to the culture medium of target cells in vitro, or may be administered to a target living body in vivo. The living body may be the same as those described above. Routes of administration to the living body include intravenous injection.

[0047] [Other Embodiments] In one embodiment, the present invention provides a method for delivering a biomolecule to the cytoplasm, comprising administering to a subject a complex of a biocompatible polymer-modified polyphenol, a metal ion, and the biomolecule. As described below in the Examples, the method of this embodiment can efficiently deliver a biomolecule to the cytoplasm of a cell of a subject.

[0048] In the method of this embodiment, the biocompatible polymer, polyphenol, metal ion, biomolecule, etc. are the same as those described above.

[0049] As described above, when a biocompatible polymer-modified polyphenol, a metal ion, and a biomolecule are mixed in a buffer solution, a complex is spontaneously formed. By administering this complex to a subject, the biomolecule can be efficiently delivered into the cytoplasm of the subject's cells.

[0050] The complex may be added to the culture medium of target cells in vitro, or may be administered to a target living body in vivo. The living body may be the same as those described above. Routes of administration to the living body include intravenous injection.

[0051] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0052] [Experimental Example 1] <PEG １０ｋ -Poly[L-Lysine ｎ / (L-Lysine-Gallic Acid) ｍ ]> (1.1 Overview) In the following schemes (1) and (2), １０ｋ -Poly[(L-Lysine) ｎ / (L-Lysine-Gallic Acid) ｍ ] (hereinafter referred to as “PEG-P[(Lys) ｎ / (Lys-GA) ｍ Here, n+m represents the degree of polymerization of Lys, and m represents the number of Gallic Acid (GA) introduced.

[0053]

[0054]

[0055] As shown in scheme (1), PEG １０ｋ -NH ２ PEG-P[Lys(TFA)] was synthesized by N-carboxyanhydride (NCA) polymerization using Lys(TFA)-NCA as the monomer. ｎ＋ｍ The TFA group in the side chain was deprotected under basic conditions, and PEG-PLys was synthesized. ｎ＋ｍ obtained.

[0056] Next, as shown in scheme (2), the carboxyl group of gallic acid was converted to PEG-PLys. ｎ＋ｍ and PEG-P[Lys ｎ / (Lys-GA) ｍ ] was obtained.

[0057] (1.2 Reagents) The reagents used are listed below. Unless otherwise specified, commercially available reagents and solvents were used as they were. α-Methoxy-ω-amino-poly(ethylene glycol) (PEG-NH ２ ) [Mn: 10k] (NOF Corp.), benzene (Nacalai Tesque), N-ε-trifluoroacetyl-L-lysine-N-carboxyanhydride (Lys(TFA)-NCA) (Chuo Kasei Co., Ltd.), and dimethyl sulfoxide (DMSO) (Fujifilm Wako Pure Chemical Industries, Ltd.) were distilled under an argon atmosphere and used. (b.p. 189°C) Diethyl ether (Kanto Chemical) Methanol (Kanto Chemical) 5 mol / L NaOH (Fujifilm Wako Pure Chemical Industries) Dimethyl sulfoxide (DMSO) (Nacalai Tesque) D-PBS(-) (Fujifilm Wako Pure Chemical Industries) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride n-hydrate (DMT-MM) (Fujifilm Wako Pure Chemical Industries) Gallic acid (Gallic Acid, GA) (Tokyo Chemical Industry Co., Ltd.) L(+)-ascorbic acid sodium salt (L(+)-sodium ascorbate) (Fujifilm Wako Pure Chemical Industries) D-sorbitol (Tokyo Chemical Industry Co., Ltd.) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Dojindo Laboratories) Sodium chloride (NaCl) (Fujifilm Wako Pure Chemical Industries) Cy5-NHS (Lumiprobe) 1-methyl-2-pyrrolidinone (Sigma-Aldrich) Lithium bromide (Sigma-Aldrich)

[0058] (1.3 Measuring equipment) Nuclear Magnetic Resonance (NMR) apparatus (BRUKER AVANCEIII400 (400 MHz, BRUKER BioSpin) Gel Permeation Chromatography (GPC) apparatus (Jasco International) Column: TSK-gel super AW3000 + super AW4000 (Tosoh), Superdex200 Increase 10 / 300 GL (GE Healthcare) Detector: RI-2031, UV-2030

[0059] (1.4 Synthesis method) 《PEG-P[Lys(TFA)]ｎ＋ｍ Synthesis of PEG-NH ２ 500 mg (0.050 mmol) of the product was weighed out and dissolved in 2.0 mL of benzene, followed by lyophilization. 348 mg (1.3 mmol, 26 equivalents) (n + m = 25) and 1,600 mg (6.0 mmol, 120 equivalents) (n + m = 105) of Lys(TFA)-NCA were weighed into a 100 mL two-necked recovery flask under an argon atmosphere. PEG-NH ２ 5 mL of DMSO was added to each of the Lys(TFA)-NCA solutions. 10 mL of DMSO was added to each of the Lys(TFA)-NCA solutions to dissolve the Lys(TFA)-NCA solution. ２ The resulting mixture was added to the solution and stirred at room temperature under an argon atmosphere for 72 hours. The reaction solution was then added dropwise to 300 mL of diethyl ether, and purified by reprecipitation. The resulting mixture was then dried under reduced pressure to obtain white solid PEG-PLys(TFA) in amounts of 745 mg (n + m = 25) and 1,750 mg (n + m = 105), with yields of 91% (n + m = 25) and 86% (n + m = 105).

[0060] Figure 1 shows the PEG-P[Lys(TFA)] ２５ of １ H-NMR spectrum (solvent: DMSO-d ６ ) is shown below. ２５ of １ The results of H-NMR analysis are shown below.

[0061] １ H-NMR spectrum of PEG-P[Lys(TFA)] ２５

[0062] Figure 2 shows the PEG-P[Lys(TFA)] １０５ of １ H-NMR spectrum (solvent: DMSO-d ６ ) is shown below. １０５ of １ The results of H-NMR analysis are shown below.

[0063] １ H-NMR spectrum of PEG-P[Lys(TFA)] １０５ ​​​１ H-NMR (DMSO-d ６ , 25°C): attribution as above １ H-NMR spectrum of PEG-P[Lys(TFA)] ２５ Same as above.

[0064] Figure 3 shows the PEG-P[Lys(TFA)] １０５ The GPC curves are shown in Table 1. The GPC curves were obtained using TSK-gel super AW3000 and super AW4000 columns, NMP (50 mM LiBr) as the eluent, and RI-2031 as the detector, at a flow rate of 0.30 mL / min and a measurement temperature of 40°C. Table 1 below also shows the GPC curves of PEG-P[Lys(TFA)] ２５ and PEG-PLys(TFA) １０５ The Mw / Mn of both polymers is shown.

[0065]

[0066] 《PEG-P[Lys(TFA)] ｎ＋ｍ Deprotection of PEG-P[Lys(TFA)] in a 50 mL recovery flask ２５ 500 mg and PEG-P[Lys(TFA)] １０５ 500 mg of each sample was weighed out, added to a mixture of 2 mL of 5 M NaOH and 8 mL of methanol, and stirred overnight at room temperature. The reaction solution was placed in a dialysis membrane (MWCO: 3.5 kDa) and dialyzed twice with 2 L of 0.1 M HCl and then with 2 L of pure water. The solution was lyophilized to obtain a white solid PEG-P[Lys] ｎ＋ｍ were obtained in amounts of 367 mg (n+m=25) and 331 mg (n+m=105), yields of 92% (n+m=25) and 90% (n+m=25).

[0067] Figure 4 shows the PEG-P[Lys] ２５ of １ H-NMR spectrum (solvent: D ２ O) is shown below. ２５ of １ The results of H-NMR analysis are shown below.

[0068] １ H-NMR spectrum of PEG-P[Lys]​２５

[0069] Figure 5 shows the PEG-P[Lys] １０５ of １ H-NMR spectrum (solvent: D ２ O) is shown below. １０５ of １ The results of H-NMR analysis are shown below.

[0070] １ H-NMR spectrum of PEG-P[Lys] １０５ １ H-NMR (D ２ 0, 25°C): attributions as above １ H-NMR spectrum of PEG-PLys ２５ Same as above.

[0071] PEG-P[Lys] of GA ｎ＋ｍ Bonding to PEG-PLys ｎ＋ｍ Gallic acid (GA) and sodium L(+)-ascorbate were weighed into a vial. The amount of GA added was PEG-P[Lys] ｎ＋ｍ The molar ratio of Lys was 100%, and the molar ratios were 25%, 50%, and 75%. Sodium L(+)-ascorbate was PEG-P[Lys] ｎ＋ｍ The amount was 30 times the chemical equivalent of GA. DMT-MM and GA were weighed out in separate screw tubes. D-PBS(-) was then added to dissolve the weighed-out reagents. While stirring the polymer solution in an ice bath, the DMT-MM solution was added all at once, and the mixture was stirred at 4°C for 6 hours to carry out the galloyl group introduction reaction of the polymer. After the reaction, diluted hydrochloric acid was added to lower the pH of the galloyl group-introduced polymer solution to 4. Next, ultrafiltration (MWCO: 3 kDa) was carried out 3 to 5 times at 4°C using ultrapure water to remove impurities. The purified polymer solution was transferred to a centrifuge tube and freeze-dried to remove the solvent, and a light brown solid PEG-P[Lys ｎ / (Lys-GA) ｍ The yields were calculated to be 60-80%.

[0072] ​​6 and 7 show the results of PEG-P[Lys ｎ / (Lys-GA) ｍ ]of １ H-NMR spectrum (solvent: DMSO-d ６ ) is shown below. ｎ / (Lys-GA) ｍ ]of １ The results of H-NMR analysis are shown below. １ From the H-NMR spectrum, PEG-P[Lys] ｎ＋ｍ The GA introduction rate for the Lys side chain was calculated.

[0073] １ H-NMR spectrum of PEG-P[PEG-P[Lys １９ / (Lys-GA) ６ ]

[0074] １ H-NMR spectrum of PEG-P[PEG-P[Lys ７２ / (Lys-GA) ３３ ] １ H-NMR (DMSO-d ６ , 25°C): attribution as above １ H-NMR spectrum of PEG-P[PEG-P[Lys １９ / (Lys-GA) ６ ]Same as.

[0075] Figure 8 shows the PEG-P[PEG-P[Lys １９ / (Lys-GA) ６ ] is shown in Figure 9. ７２ / (Lys-GA) ３３ The GPC curve was obtained using a Superdex 200 increase 10 / 300 GL column, 10 mM HEPES, 140 mM NaCl (pH 7.4) as the eluent, a UV-2030 detector, detection at a wavelength of 220 nm, a flow rate of 0.75 mL / min, and room temperature.

[0076] (1.5 Analysis) 《PEG-P[Lys(TFA)] ｎ＋ｍ ​​》From the GPC curve, PEG-P[Lys(TFA)] ２５ M ｗ / M ｎ was calculated to be 1.07. １０５ M ｗ / M ｎ was calculated to be 1.10. This result confirmed that these polymers had narrow molecular weight distributions.

[0077] PEG-PLys ｎ＋ｍ 》 １ The degree of polymerization of PLys was calculated to be DP=25 and DP=105 from the ratio of the integral values ​​of the peak derived from the initiator and the peak derived from Lys in the H-NMR spectrum.

[0078] 《PEG-P[PEG-P[Lys ｎ / (Lys-GA) ｍ ]]》 １ From the ratio of the integrals of the peaks derived from PLys and GA in the H-NMR spectrum, the number of GA introduced was calculated to be 6 (n = 25) and 33 (n = 105), and the number average molecular weights were Mn = 16,300 (n = 25) and Mn = 30,940 (n = 105). Furthermore, the GPC curve confirmed that the obtained polymer had a unimodal, narrow molecular weight distribution. Furthermore, PEG-P[PEG-P[Lys] with a Lys polymerization degree of 25 was ｎ / (Lys-GA) ｍ Regarding PEG-P [PEG-P[Lys ｎ / (Lys-GA) ｍ ]] was also successfully synthesized.

[0079] [Experimental Example 2] <Synthesis of PEG-TA> (2.1 Overview) The following schemes (3) and (4) show methods for synthesizing tannic acid-modified PEG (PEG-TA).

[0080]

[0081]

[0082] PEG-NH with a molecular weight of 10kJ ２ and PEG-NH with a molecular weight of 5kJ２ were reacted with tannic acid (TA) by Michael addition reaction, and then the ketone group in the side chain was reduced under acidic conditions. After dialysis, PEG-TA was obtained.

[0083] (2.2 Reagents) The reagents used are as follows: α-methoxy-ω-amino-poly(ethylene glycol) (PEG １０ｋ -NH ２ ) [Mn: 10k] (NOF Corp.) α-methoxy-ω-amino-poly(ethylene glycol) (PEG ５ｋ -NH ２ ) [Mn: 5k] (NOF Corp.), Tannic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), Sodium bicarbonate (NaHCO ３ ) (FUJIFILM Wako Pure Chemical Industries) Acetic acid (CH ３ COOH) (Nacalai Tesque), {2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid} (HEPES) (Nacalai Tesque), and sodium chloride (NaCl) (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0084] (2.3 Measuring Instruments) Nuclear Magnetic Resonance (NMR) apparatus (BRUKER AVANCEIII400 (400 MHz, BRUKER BioSpin) Gel Permeation Chromatography (GPC) apparatus (Jasco International) Column: Superdex75 Increase 10 / 300 GL (Cytiva) Detector: RI-2031, UV-2030 Nanospectrophotometer NanoDrop One (Thermo Fisher Scientific)

[0085] (2.4 Synthesis method) PEG １０ｋ -TA synthesis》《PEG of TA １０ｋ -NH ２ Binding to 52 mg (0.030 mmol) of TA was weighed out into a 200 mL beaker and added to 50 mM NaHCO ３ (pH 8.5) 80 mL. ３ PEG dissolved in 40 mL of (pH 8.5) １０ｋ-NH ２ 100 mg (0.010 mmol) of the reaction mixture was added dropwise to the reaction mixture, which was then stirred at room temperature for 5 hours. The reaction mixture was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed for 1 hour against 2 L of pure water that had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes.

[0086] <<Reduction of the Side Chain Ketone Group of TA>> The reaction solution was transferred to a 200 mL beaker, and 2 mL of acetic acid was added dropwise and stirred for 5 minutes. The reaction solution was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed twice with 2 L of pure water, which had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes. The resulting solution was freeze-dried to obtain a yellow solid PEG. １０ｋ -TA was obtained in a yield of 68 mg.

[0087] In FIG. １０ｋ -TA's １ The H-NMR spectrum of PEG is shown in FIG. １０ｋ 11 shows the GPC curve of α-TA. The GPC curve was acquired using a Superdex 75 Increase 10 / 300 GL column, 10 mM HEPES, 140 mM NaCl (pH 7.4) as the eluent, and a UV-2030 detector at a detection wavelength of 254 nm, a flow rate of 0.75 mL / min, and room temperature. The vertical axis of FIG. 11 represents signal intensity (relative value).

[0088] 《PEG ５ｋ -TA synthesis》《PEG of TA ５ｋ -NH ２ Binding to 52 mg (0.030 mmol) of TA was weighed out into a 200 mL beaker and added to 50 mM NaHCO ３ (pH 8.5) 80 mL. ３ PEG dissolved in 40 mL of (pH 8.5) ５ｋ -NH ２ 50 mg (0.010 mmol) of the reaction mixture was added dropwise to the reaction mixture, which was then stirred at room temperature for 5 hours. The reaction mixture was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed for 1 hour against 2 L of pure water that had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes.

[0089] <<Reduction of the Side Chain Ketone Group of TA>> The reaction solution was transferred to a 200 mL beaker, and 2 mL of acetic acid was added dropwise and stirred for 5 minutes. The reaction solution was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed twice with 2 L of pure water, which had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes. The resulting solution was freeze-dried to obtain a yellow solid PEG. ５ｋ -TA was obtained in a yield of 30 mg.

[0090] In FIG. ５ｋ -TA's １ The H-NMR spectrum of PEG is shown in FIG. ５ｋ 13 shows the GPC curve of α-TA. The GPC curve was acquired using a Superdex 75 Increase 10 / 300 GL column, 10 mM HEPES, 140 mM NaCl (pH 7.4) as the eluent, and a UV-2030 detector at a detection wavelength of 254 nm, a flow rate of 0.75 mL / min, and room temperature. The vertical axis of FIG. 13 represents signal intensity (relative value).

[0091] (2.5 Analysis) <<PEG １０ｋ -TA》 From the GPC curve, PEG １０ｋ It was confirmed that the -TA had a narrow molecular weight distribution. １ In the H-NMR spectrum, a peak derived from the initiator and a peak derived from the GA group were confirmed. Furthermore, from the absorbance at an absorption wavelength of 277 nm derived from the GA group, the TA introduction rate was calculated to be 92%.

[0092] 《PEG ５ｋ -TA》 From the GPC curve, PEG ５ｋ It was confirmed that the -TA had a narrow molecular weight distribution. １ In the H-NMR spectrum, a peak derived from the initiator and a peak derived from the GA group were confirmed. In addition, the TA introduction rate was calculated to be 81% from the absorbance at an absorption wavelength of 277 nm derived from the GA group. In the following examples, when PEG-TA is used, it means that PEG １０ｋ -TA was used.

[0093] [Experimental Example 3] <GA group and Fe ３＋(3.1 Overview) GA groups and metal ions crosslink to form metal ion-polyphenol complexes (MPN) with buffering capacity. Therefore, a spectrophotometer was used to confirm the complex formation between GA groups and iron ions and their pH responsiveness.

[0094] (3.2 Reagents) Gallic acid (GA) (Tokyo Chemical Industry Co., Ltd.) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries, Ltd.) D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) {2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid} (HEPES) (Nacalai Tesque, Inc.)

[0095] (3.3 Measuring equipment) JASCO V-650 spectrophotometer (Jasco)

[0096] (3.4 Confirmation of Complex Formation) <GA, FeCl ３ ・6H ２ Final concentration of O》 ・GA: 3mM ・FeCl ３ ・6H ２ O(Fe ３＋ ): 3 mM and 9 mM were dissolved in D-PBS(-).

[0097] GA and Fe ３＋ The two types of mixture ratio ([GA:Fe ３＋ ] = 1:1 and [GA:Fe ３＋ The mixture was mixed in a ratio of 1:3 (pH 7.0, pH ...

[0098] From the absorption spectrum, [GA:Fe ３＋ In the 1:3 solution, an increase in absorbance was observed around 550 nm, and the ３＋ [GA:Fe ３＋ It was confirmed that a complex was formed at a ratio of 1:3.

[0099] (3.5 Confirmation of pH responsiveness) <GA, FeCl ３ ・6H ２Final concentration of O》 GA: 3mM Fe ３＋ : 9 mM

[0100] GA and Fe ３＋ was dissolved in D-PBS(-) (pH 7.4) and D-PBS(-) adjusted to pH 4.0, and after incubation at room temperature for 2 hours, the absorption spectrum was measured using a spectrophotometer. Figure 15 is a graph showing the results of the absorption spectrum measurement.

[0101] From the absorption spectrum, the absorbance at around 550 nm of the pH 4.0 solution was lower than that of the pH 7.4 solution, indicating that GA and Fe ３＋ It was confirmed that the complex has pH responsiveness.

[0102] [Experimental Example 4] <Evaluation of physicochemical properties of protein metal-polyphenol network (MPN) complex> (4.1 Overview) When a protein MPN complex is formed by a protein, a polymer-modified polyphenol, and iron ions, the particle size increases. Therefore, particle size was measured by fluorescence spectroscopic correlation method using β-galactosidase (β-Gal) and an antibody as the protein. PEG-P[Lys ｎ / (Lys-GA) ｍ ] and PEG-TA were used. At the same time, complex formation was confirmed using transmission electron microscopy (TEM). Furthermore, the physical properties of the protein-MPN complex, such as pH response and surface zeta potential, were also evaluated. Furthermore, to evaluate stability in the blood environment, particle size changes in FBS solution were also measured.

[0103] (4.2 Reagents) Unless otherwise specified, commercially available reagents and solvents were used as they were. β-Galactosidase (β-Gal, Mw: 540 kDa) (Fujifilm Wako Pure Chemical Industries, Ltd.) Normal human IgG (antibody, Mw: 150 kDa) (Fujifilm Wako Pure Chemical Industries, Ltd.) Cy5-NHS (Lumiprobe) PEG-P[Lys ｎ / (Lys-GA) ｍ](m+n=25,105) PEG-TA (Mn=11,700) D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) 5 mol / L HCl (Fujifilm Wako Pure Chemical Industries, Ltd.) 5 mol / L NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium bicarbonate: Tokyo Chemical Industry Co., Ltd. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Dojindo Laboratories, Ltd.) Sodium chloride (NaCl) (Fujifilm Wako Pure Chemical Industries, Ltd.) Iron(III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) Fetal bovine serum (FBS) (Biosera)

[0104] (4.3 Measuring equipment) ・LSM710 (Carl Zeiss) ・Fluorescence spectrophotometer FP-8300 (Jasco) ・Zetasizer NanoZS (Malvern Panalytical) ・Nanospectrophotometer NanoDrop One (Thermo Fisher Scientific) ・JEM-1400 (JEOL)

[0105] (4.4 Introduction of fluorescent dye into protein) 20 mg of antibody and 15 mg of β-Gal were weighed into a vial and added to 50 mM NaHCO ３ The solution was dissolved in 10 mL of DMSO (pH 8.5). 0.7 mg of Cy5-NHS dissolved in DMSO was added thereto and stirred at room temperature for 2 hours. The reaction solution was added to an ultrafiltration tube (MWCO: 30 kDa) and centrifuged at 8000 x g for 20 minutes to remove unreacted Cy5-NHS. PBS(-) was used as the exchange solution. The final concentration was determined by measuring the absorbance at 280 nm, where protein-derived absorption is observed. Removal of unreacted fluorescent dye was confirmed by electrophoresis. As a result, Cy5-β-Gal (hereinafter referred to as β-Gal) and Cy5-IgG antibody (hereinafter referred to as antibody) were obtained.

[0106] (4.5 PEG-P[Lys ｎ / (Lys-GA) ｍ Evaluation of β-Gal-loaded MPN complex formation using β-Gal, PEG-P[Lys ｎ / (Lys-GA) ｍ ], FeCl３ ・6H ２ Final concentration of O》 ・β-Gal: 500 nM (prepared concentration: 2.0 μM) ・PEG-P[Lys １１ / (Lys-GA) １４ ]: 50 μM (prepared concentration: 200 μM) ・FeCl ３ ・6H ２ O(Fe ３＋ ): 220 μM (prepared concentration: 2.2 mM) Each was prepared by dissolving in D-PBS(-).

[0107] β-Gal solution, PEG-P[Lys １１ / (Lys-GA) １４ ] solution, Fe ３＋ The solution was mixed by pipetting. After that, PBS(-) was added to adjust the concentration, and the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 16.

[0108] β-Gal / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ The particle size of the complex was determined by the following formula: β-Gal, β-Gal / PEG-P[Lys １１ / (Lys-GA) １４ ] solution, the particle size was significantly increased compared to that of the β-Gal / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ The formation of a complex was demonstrated.

[0109] (4.6 PEG-P[Lys ｎ / (Lys-GA) ｍ Evaluation of antibody-loaded MPN complex formation using antibody, PEG-P[Lys ｎ / (Lys-GA) ｍ ], Fe ３＋ Final concentration of antibody: 200 nM PEG-P[Lys １１ / (Lys-GA) １４ ]: 20 to 600 μM (prepared concentration: 2,000 μM) ・Fe ３＋ : 90 to 2,800 μM (prepared concentration: 2.2 mM) Each was prepared by dissolving in D-PBS(-).

[0110] Antibody solution, PEG-P[Lys １１ / (Lys-GA) １４ ] solution, Fe ３＋ The solution was mixed by pipetting. １１ / (Lys-GA) １４ The molar ratio of PEG-P[Lys １１ / (Lys-GA) １４ The amount was 1 / 3 equivalent to the GA of 1000 mg / mL. Finally, PBS(-) was added to adjust the concentration, and the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 17.

[0111] Antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ The particle size of the complex was determined by the antibody, antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] solution, the particle size was significantly increased compared to that of the antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ The formation of a complex was suggested. １１ / (Lys-GA) １４ ] and Fe ３＋ It was observed that the particle size tended to increase with increasing amount of added.

[0112] (4.7 Evaluation of antibody-loaded MPN complex formation when the introduction rate of GA on the polymer is changed) ｎ / (Lys-GA) ｍ ], Fe ３＋ Final concentration of antibody: 200 nM PEG-P[Lys １９ / (Lys-GA) ６ ]: 20 μM (prepared concentration: 2,000 μM) ・PEG-P[Lys １１ / (Lys-GA) １４ ]: 20 μM (prepared concentration: 2,000 μM) ・PEG-P[Lys ９ / (Lys-GA) １６]: 20 μM (prepared concentration: 2,000 μM) ・Fe ３＋ : 25 to 110 μM (prepared concentration: 2.2 mM) Each was prepared by dissolving in D-PBS(-).

[0113] Antibody solution, each PEG-P[Lys ｎ / (Lys-GA) ｍ ] solution, Fe ３＋ The solution was mixed by pipetting. ｎ / (Lys-GA) ｍ The molar ratio of PEG-P[Lys ｎ / (Lys-GA) ｍ The amount was 1 / 3 equivalent to the GA of 1000 mg / mL. Finally, PBS(-) was added to adjust the concentration, and the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 18.

[0114] Antibody / PEG-P[Lys ｎ / (Lys-GA) ｍ ] / Fe ３＋ The particle size of the complex was determined by the GA introduction rate of PEG-P[Lys ｎ / (Lys-GA) ｍ ], the particle size was significantly increased compared to the particle size of the antibody solution. １１ / (Lys-GA) １４ The greatest increase in particle size of the MPN complex was observed when the following was used:

[0115] (4.8 Evaluation of antibody-loaded MPN complex formation when the degree of Lys polymerization on the polymer is changed) ｎ / (Lys-GA) ｍ ], Fe ３＋ Final concentration of antibody: 200 nM PEG-P[Lys １１ / (Lys-GA) １４ ]: 20 μM (prepared concentration: 2,000 μM) ・PEG-P[Lys ７２ / (Lys-GA) ３３ ]: 20 μM (prepared concentration: 2,000 μM) ・Fe ３＋: 90 to 200 μM (prepared concentration: 2.2 mM) Each was prepared by dissolving in D-PBS(-).

[0116] Antibody solution, each PEG-P[Lys ｎ / (Lys-GA) ｍ ] solution, Fe ３＋ The solution was mixed by pipetting. ｎ / (Lys-GA) ｍ The molar ratio of PEG-P[Lys ｎ / (Lys-GA) ｍ The amount was 1 / 3 equivalent to the GA of 1000 mg / mL. Finally, PBS(-) was added to adjust the concentration, and the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 19.

[0117] Antibody / PEG-P[Lys ｎ / (Lys-GA) ｍ ] / Fe ３＋ The particle size of the complex was determined for both PEG-P[Lys ｎ / (Lys-GA) ｍ ], the particle size was significantly increased compared to the particle size of the antibody solution. １１ / (Lys-GA) １４ ] was used, the PEG-P[Lys ７２ / (Lys-GA) ３３ The particle size of the MPN complex was found to be larger than that of the MPN complex obtained using the method described above.

[0118] (4.9 Evaluation of antibody-loaded MPN complex formation using TEM) <<Antibody, PEG-P[Lys ｎ / (Lys-GA) ｍ ], Fe ３＋ Final concentration of antibody: 6.6 μM PEG-P[Lys １１ / (Lys-GA) １４ ]: 660 μM (prepared concentration: 2,000 μM) ・PEG-P[Lys ７２ / (Lys-GA) ３３ ]: 660 μM (prepared concentration: 2,000 μM) ・Fe ３＋: 3.0 to 7.2 mM (prepared concentration: 20 mM) Each was prepared by dissolving in 1 mM HEPES buffer solution, pH 7.4.

[0119] Antibody solution, each PEG-P[Lys ｎ / (Lys-GA) ｍ ] solution, Fe ３＋ The solution was mixed by pipetting. ｎ / (Lys-GA) ｍ The molar ratio of PEG-P[Lys ｎ / (Lys-GA) ｍ The amount was 1 / 3 equivalent to the GA. Finally, 1 mM HEPS buffer solution, pH 7.4, was added to adjust the concentration. 5 μL of the sample solution was then dropped onto the copper grid, and the solution was removed after 1 minute. The grid was then stained with gadolinium acetate solution, washed with ultrapure water, and then air-dried for 1 day. TEM images of the sample-coated microgrid were taken using a JEM1400.

[0120] Figure 20 is a transmission electron microscope (TEM) image of the antibody. Figure 21 is a transmission electron microscope (TEM) image of the antibody / PEG-P[Lys １１ / (Lys-GA) １４ 22 shows a TEM image of antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ FIG. 23 shows a TEM image of antibody / PEG-P[Lys ７２ / (Lys-GA) ３３ ] / Fe ３＋ TEM image of the MPN composite.

[0121] Antibody / PEG-P[Lys ｎ / (Lys-GA) ｍ ] / Fe ３＋ In the TEM images of the complex, relatively large spherical particles were observed compared to the antibody alone, suggesting the formation of an antibody-loaded MPN complex.

[0122] (4.10 Evaluation of antibody-loaded MPN complex formation using PEG-TA) <<Antibody, PEG-TA, Fe ３＋Final concentration of: Antibody: 660 nM PEG-TA: 16 μM (prepared concentration: 80 μM) Fe ３＋ : 55 μM (prepared concentration: 2.2 mM) The antibody and PEG-TA were dissolved in D-PBS(-) and prepared. ３＋ was prepared by dissolving in ultrapure water.

[0123] Antibody solution, PEG-TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of PEG-TA / antibody was set to 25. The molar ratio of iron ions was set to 3.3 equivalents relative to the TA in PEG-TA. Finally, PBS(-) was added to adjust the concentration, and then particle size was measured by fluorescence spectroscopic correlation method using an LSM710. The particle size measurement results are shown in Figure 24. Furthermore, particle size was measured using a Zetasizer NanoZS. The particle size measurement results are shown in Figure 25.

[0124] Antibody / PEG-TA / Fe ３＋ The particle size of the MPN complex was significantly increased compared to that of the antibody alone and the antibody / PEG-TA. ３＋ The formation of MPN complexes was demonstrated.

[0125] (4.11 Zeta potential measurement of antibody-loaded MPN complex formation) <<Antibody, PEG-TA, Fe ３＋ Final concentration of: Antibody: 660 nM PEG-TA: 16 μM (prepared concentration: 80 μM) Fe ３＋ : 55 μM (prepared concentration: 2.2 mM) The antibody and PEG-TA were dissolved in 10 mM HEPES. ３＋ was prepared by dissolving in ultrapure water.

[0126] Antibody solution, PEG-TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of PEG-TA / antibody was set to 25. The molar ratio of iron ions was set to 3.3 equivalents relative to the TA of PEG-TA. Next, 10 mM HEPES was added to adjust the concentration, and the zeta potential was measured using a Zetasizer NanoZS. The measurement results of the zeta potential are shown in Figure 26.

[0127] The antibody alone showed a strong negative charge of -16 mV, but the antibody / PEG-TA / Fe ３＋ By forming the MPN complex, the zeta potential became −8 mV, the negative charge decreased, and the value became closer to 0.

[0128] (4.12 pH responsiveness evaluation of antibody-loaded MPN complex) <<Antibody, PEG-TA, Fe ３＋ Final concentration of: Antibody: 660 nM PEG-TA: 16 μM (prepared concentration: 80 μM) Fe ３＋ : 55 μM (prepared concentration: 2.2 mM) Each was prepared by dissolving in D-PBS(-) (pH 7.4), 50 mM HEPES buffer (pH 6.8), and 50 mM MES buffer (pH 6.0 and pH 5.5). ３＋ was prepared by dissolving in ultrapure water.

[0129] Antibody solution, PEG-TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of PEG-TA / antibody was set to 25. The molar ratio of iron ions relative to the TA in PEG-TA was set to 3.3 equivalents. Next, D-PBS(-) was added to adjust the concentration, and the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 27.

[0130] Antibody / PEG-TA / Fe at pH 6.8 ３＋ The particle size of the MPN complex is ３＋ The particle size of the antibody / PEG-TA / Fe complex at pH 5.5 and pH 6.0 was similar to that of the MPN complex. ３＋ Since the particle size of the MPN complex was equivalent to that of the antibody alone, antibody / PEG-TA / Fe ３＋ It was confirmed that the MPN complex is responsive to intracellular pH.

[0131] (4.13 Evaluation of complex formation in FBS) <<Antibody, PEG-TA, Fe ３＋ Final concentration of: Antibody: 660 nM PEG-TA: 16 μM (prepared concentration: 80 μM) Fe ３＋: 55 μM (prepared concentration: 2.2 mM) The antibody and PEG-TA were dissolved in D-PBS(-). ３＋ was dissolved in ultrapure water and then added to a mixed solution of FBS / D-PBS(-) (10 / 90, 30 / 70 (vol)) to achieve the above concentrations.

[0132] Antibody solution, PEG-TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of PEG-TA / antibody was set to 25. The molar ratio of iron ions relative to the TA in PEG-TA was set to 3.3 equivalents. Next, PBS(-) was added to adjust the concentration, and the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 28.

[0133] Antibody / PEG-TA / Fe ３＋ The particle size of the MPN complex did not change in FBS solutions of various concentrations, confirming that it was stable in FBS solutions.

[0134] [Experimental Example 5] <Evaluation of physicochemical properties of MPN complexes loaded with nucleic acid complexes (RNPs)> (5.1 Overview) When an MPN complex is formed by mixing Cas9-sgRNA RNP, PEG-TA, and metal ions, the particle size increases compared to RNP alone. Therefore, particle size measurements were performed using Cas9-GFP by fluorescence spectroscopic correlation. Zr, which has a different valence, was used as the metal ion. ４＋ , Fe ３＋ , Co ２＋ was used.

[0135] (5.2 Reagents) Cas9-GFP Protein (Sigma-Aldrich) sgRNA (Luciferase-2) (GenScript) PEG １０ｋ -TA (M ｎ = 11,700) ・PEG ５ｋ -TA (M ｎ = 6,700) Zirconium oxide (IV) (ZrO ２ ) (Zr ４＋ ) (FUJIFILM Wako Pure Chemical Industries, Ltd.) Cobalt (II) nitrate hexahydrate (Co(NO ３ ) ２ ・6H ２O) (Co ２＋ ) (Fujifilm Wako Pure Chemical Industries, Ltd.) D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) Iron(III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0136] (5.3 Measuring equipment) LSM710 (Carl Zeiss) JEM-1400 (JEOL)

[0137] (5.4 Zr ４＋ and Co ２＋ Evaluation of RNP-MPN complex formation using Cas9-sgRNA, PEG-TA, ZrO ２ , Co(NO ３ ) ２ ・6H ２ Final concentration of O》 Cas9-GFP: 0.2 μM sgRNA: 0.2 μM PEG １０ｋ -TA: 20μM PEG ５ｋ -TA: 20 μM ・ZrO ２ (Zr ４＋ ): 67μM ・Co(NO ３ ) ２ ・6H ２ O (Co ２＋ ): 22 μM Cas9-sgRNA, PEG-TA was dissolved in D-PBS (-) and prepared. ４＋ , Co ２＋ was prepared by dissolving in pure water.

[0138] Cas9-GFP and sgRNA were mixed and centrifuged twice at 12,000 × g for 5 minutes using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). １０ｋ -TA or PEG ５ｋ The mixture was left standing at room temperature for 15 minutes to prepare both RNP / PEG-TA solutions. ４＋ or Co ２＋ After adding the RNP / PEG-TA / Zr ４＋ Solution, both RNP / PEG-TA / Co ２＋was prepared. At this time, the molar ratio of PEG-TA / RNP was set to 25. The molar ratio of metal ions to TA in PEG-TA was set to 3.3 equivalents. Finally, the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figures 29 and 30.

[0139] RNP / PEG-TA / Zr ４＋ , RNP / PEG-TA / Co ２＋ The particle size of RNP / PEG-TA / Zr was significantly increased compared to that of RNP and RNP / PEG-TA, regardless of the molecular weight of PEG. ４＋ MPN complex, RNP / PEG-TA / Co ２＋ The formation of an MPN complex was suggested. On the other hand, the valence of the metal ion, Zr, ４＋ Co is better ２＋ The increase in particle size was greater than when using , suggesting the formation of a more stable complex.

[0140] (5.5 Fe ３＋ Evaluation of RNP-MPN complex formation using Cas9-sgRNA, PEG-TA, FeCl ３ ・6H ２ Final concentration of O》 Cas9-GFP: 0.2 μM sgRNA: 0.2 μM PEG １０ｋ -TA: 20 μM ・FeCl ３ ・6H ２ O(Fe ３＋ ): 67 μM Cas9-sgRNA, PEG-TA was dissolved in D-PBS(-). ３ ・6H ２ O was prepared by dissolving it in pure water.

[0141] Cas9-GFP and sgRNA were mixed and centrifuged twice at 12,000 × g for 5 minutes using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). １０ｋ -TA and PEG ５ｋ The mixture was then left to stand at room temperature for 15 minutes to prepare both RNP / PEG-TA solutions. ３＋ After adding the above and leaving it at room temperature for 15 minutes, both RNP / PEG １０ｋ-TA / Fe ３＋ An MPN complex solution was prepared. The molar ratio of PEG-TA / RNP was 100. The molar ratio of iron ions to TA in PEG-TA was 3.3 equivalents. Finally, particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figures 31 and 32.

[0142] Both RNP / PEG-TA / Fe ３＋ The particle size of RNP / PEG-TA / Fe was significantly increased compared to that of RNP and RNP / PEG-TA. ３＋ The formation of an MPN complex was suggested.

[0143] (5.6 Evaluation of RNP-loaded MPN complex formation using TEM) Cas9-sgRNA, PEG-TA, FeCl ３ ・6H ２ Final concentration of O》 Cas9-GFP: 0.2 μM sgRNA: 0.2 μM PEG １０ｋ -TA: 20 μM ・FeCl ３ ・6H ２ O(Fe ３＋ ): 67 μM Cas9-sgRNA, PEG-TA were dissolved in 1 mM HEPES buffer (pH 7.4). ３＋ was prepared by dissolving in pure water.

[0144] Cas9-GFP and sgRNA were mixed and centrifuged twice at 12,000 × g for 5 minutes using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). １０ｋ -TA and PEG ５ｋ The mixture was then left to stand at room temperature for 15 minutes to prepare both RNP / PEG-TA solutions. ３＋ After adding the above and leaving it at room temperature for 15 minutes, both RNP / PEG １０ｋ -TA / Fe ３＋An MPN complex solution was prepared. The molar ratio of PEG-TA / RNP was 100. The molar ratio of iron ions to TA in PEG-TA was 3.3 equivalents. Next, 5 μL of the sample solution was dropped onto a copper grid, and the solution was removed after 1 minute. The grid was then stained with gadolinium acetate solution. The grid was then washed with ultrapure water and air-dried for 1 day. TEM images were then taken using a transmission electron microscope.

[0145] Figure 33 shows a TEM image of RNP. Figure 34 shows a TEM image of RNP / PEG. １０ｋ 34 shows a TEM image of RNP / PEG-TA. １０ｋ Figure 35 shows the RNP / PEG １０ｋ -TA / Fe ３＋ In Figure 35, the arrowhead indicates RNP / PEG. １０ｋ -TA / Fe ３＋ The TEM image shows that the RNP / PEG complex is more soluble than the RNP alone. １０ｋ -TA / Fe ３＋ The complexes were observed as relatively large particles of approximately 70 to 100 nm, suggesting the formation of RNP-loaded MPN complexes.

[0146] [Experimental Example 6] <Evaluation of cytotoxicity of PEG-TA> (6.1 Overview) Synthesized PEG １０ｋ -TA and PEG １０ｋ -TA / Fe ３＋ The cytotoxicity of the compound was evaluated by CCK assay using Cell Counting Kit-8 (Dojindo Laboratories).

[0147] (6.2 Reagents) α-Methoxy-ω-amino-poly(ethylene glycol) (PEG １０ｋ -NH ２ ) [Mn:10k] (NOF) ・PEG １０ｋ -TA (M ｎ = 11,700) ・Iron (III) Chloride Hexahydrate (FeCl ３ ・6H ２ O) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋) (Fujifilm Wako Pure Chemical Industries), D-PBS (-) (Fujifilm Wako Pure Chemical Industries), Cell Counting Kit-8 (Dojindo Laboratories), Roswell Park Memorial Institute medium (RPMI) (Sigma-Aldrich), Fetal bovine serum (FBS) (Biosera), Trypsin-EDTA solution (Sigma-Aldrich), Penicillin / streptomycin (Sigma-Aldrich), HeLa-Luc cells (aneuploid epithelial cell line stably expressing luciferase) (American Type Culture Collection, ATCC)

[0148] (6.3 Measuring equipment) Multiplate reader (Spark) (Tecan)

[0149] (6.4 Cytotoxicity Evaluation) <PEG １０ｋ -NH ２ , PEG １０ｋ -TA, FeCl ３ ・6H ２ Final concentration of O》 PEG １０ｋ -NH ２ :5-250μM ・PEG １０ｋ -TA (Mn=11,700): 5 to 250 μM ・FeCl ３ ・6H ２ O(Fe ３＋ ): 16.5 to 825 μM Dissolved in D-PBS(-) to prepare. ３＋ was prepared by dissolving in pure water.

[0150] From the above, PEG １０ｋ -NH ２ Solution, PEG １０ｋ -TA solution, FeCl ３ ・6H ２ Further, each PEG solution was prepared. １０ｋ -TA solution, PEG １０ｋ -TA:Fe ３＋ Fe = 3:10 ３＋ PEG containing １０ｋ -TA / Fe ３＋ A solution was prepared.

[0151] <Cytotoxicity evaluation by CCK assay> 3.0 × 10 HeLa-Luc cells were cultured in a 96-well plate.３ The cells were seeded at 100 cells / plate and incubated at 37°C in 5% CO ２ The mixture was incubated under the same conditions for 24 hours. １０ｋ -NH ２ Solution, RNP / PEG １０ｋ -TA solution, PEG １０ｋ -TA / Fe ３＋ solution, FeCl ３ ・6H ２ 100 μL of O solution was added to each well and incubated for 72 hours. After washing with 150 μL / well of D-PBS(-), 110 μL / well of CCK measurement solution, a mixture of 10 μL of CCK and 100 μL of PBS(-), was added and incubated for 1 hour. After that, the absorbance at 460 nm was measured using a plate reader.

[0152] The cytotoxicity was then evaluated by normalizing the absorbance of the wells containing cells treated with each sample with the absorbance of the wells containing untreated cells. Figure 36 is a graph showing the results of measuring cell viability by CCK assay. As a result, PEG-TA alone was found to be cytotoxic, but Fe ３＋ It was confirmed that the addition of

[0153] Experimental Example 7 Evaluation of Antibody-MPN Complexes in Cultured Cells (7.1 Overview) The intracellular distribution of antibody-loaded MPN complexes was observed using a confocal microscope to confirm the intracellular uptake pathway. In addition, in this experiment, the antigen-recognition function of the loaded antibody was also evaluated by using an anti-nuclear pore complex protein antibody (anti-NPC antibody) that can target the cell nucleus.

[0154] (7.2 Reagents and cell lines) Cy5-labeled antibody (antibody) Alexa Fluor （Ｒ） 647 Anti-Nuclear Pore Complex Proteins Antibody (anti-NPC antibody) (Biolegend) PEG-P[Lys ｎ / (Lys-GA) ｍ ](m+n=25,105) ・PEG ５ｋ -TA (M ｎ = 11,700) ・PEG１０ｋ -TA (M ｎ =5,700) D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) Roswell Park Memorial Institute medium (RPMI) (Sigma-Aldrich) Fetal bovine serum (FBS) (Biosera) Trypsin-EDTA solution (Sigma-Aldrich) Penicillin / streptomycin (Sigma-Aldrich) 5 mol / L HCl (Fujifilm Wako Pure Chemical Industries, Ltd.) CT26 cells (mouse colon cancer cell line) (American Type Culture Collection, ATCC) 4T1-Luc cells (mouse breast cancer cell line) (American Type Culture Collection, ATCC) LysoTracker® red DND-99 (Thermo Fisher Scientific) Hoechst 33342 (Thermo Fisher Scientific) Paraformaldehyde (Nacalai Tesque) was used as a 4% paraformaldehyde / D-PBS(-) solution.

[0155] (7.3 Measurement equipment) Countess (Thermo Fisher Scientific) LSM710 (Carl Zeiss) Flow cytometer (Guava easyCyte 6-2L): (Merck Millipore, Ex / Em: 642 / 661 nm)

[0156] (7.4 Observation of intracellular distribution of antibody-loaded MPN complexes using a confocal microscope) <<Antibody, PEG-P[Lys ｎ / (Lys-GA) ｍ ], Fe ３＋ Final concentration of antibody: 3.3 μM PEG-P[Lys １１ / (Lys-GA) １４ ]: 330 μM (prepared concentration: 2,000 μM) ・Fe ３＋ : 1.5 mM (prepared concentration: 20 mM) Each was prepared by dissolving in PBS(-).

[0157] Antibody solution, each PEG-P[Lys １１ / (Lys-GA) １４ ] solution, Fe ３＋ The solution was mixed by pipetting.１１ / (Lys-GA) １４ The molar ratio of PEG-P[Lys １１ / (Lys-GA) １４ ] was used in an amount of 1 / 3 equivalent to GA.

[0158] <<Observation by confocal microscope>> 35mm ２ CT26 cells were cultured in a glass base dish at a density of 5.0 × 10 ４ The cells were seeded at 100 cells / dish and incubated at 37°C, 5% CO ２ The plates were pre-cultured for 24 hours under RT. 500 μL of the above antibody sample solution and 500 μL of RPMI medium were added to each dish and incubated for 48 hours. After washing with 1 mL of D-PBS(-), 1 mL of 100 nM LysoTracker red DND-99 / (D-PBS(-):RPMI = 1:9) solution was added and incubated for 30 minutes. After washing with 1 mL of D-PBS(-), the plates were incubated for 4 minutes with 4% paraformaldehyde / D-PBS(-). After washing with 1 mL of D-PBS(-), 1 mL of 5.0 μg / mL Hoechst / D-PBS(-) solution was added and incubated for 5 minutes. After washing twice with 1 mL of D-PBS(-), 2 mL of RPMI was added and the plates were observed under a confocal microscope. Figure 37 shows the microscopic images taken. FIG. 38 is a graph showing the results of calculating the coexistence efficiency of fluorescence derived from Lysotracker and fluorescence derived from Cy5 labeled antibody.

[0159] Antibody alone and antibody / PEG-P[Lys １１ / (Lys-GA) １４ In contrast, in the case of antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ In the MPN complex, the fluorescence from the antibody was observed to diffuse throughout the cytoplasm. The coexistence efficiency of Cy5 and Lysotracker fluorescence was also observed when the antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ The MPN conjugates consisted of antibody alone and antibody / PEG-P[Lys１１ / (Lys-GA) １４ This result was significantly reduced compared to the antibody / PEG-P[Lys １１ / (Lys-GA) １４ ] / Fe ３＋ We show that after being taken up into cells, MPN complexes can escape from lysosomes and translocate into the cytoplasm.

[0160] (7.5 Functional Evaluation of Anti-NPC Antibody-Loaded MPN Complexes Using Confocal Microscopy) ｎ / (Lys-GA) ｍ ], Fe ３＋ Final concentration of》 Anti-NPC antibody: 1.7 μM PEG-P[Lys １１ / (Lys-GA) １４ ]: 170 μM (prepared concentration: 2,000 μM) ・Fe ３＋ : 750 mM (prepared concentration: 20 mM) Each was prepared by dissolving in PBS(-).

[0161] Anti-NPC antibody solution, PEG-P[Lys １１ / (Lys-GA) １４ ] solution, Fe ３＋ The solution was mixed by pipetting. １１ / (Lys-GA) １４ The molar ratio of PEG-P[Lys １１ / (Lys-GA) １４ ] was used in an amount of 1 / 3 equivalent to GA.

[0162] <<Observation by confocal microscope>> 35mm ２ CT26 cells were cultured in a glass base dish at a density of 5.0 × 10 ４ The cells were seeded at 100 cells / dish and incubated at 37°C, 5% CO ２The plates were pre-cultured for 24 hours under RT. 100 μL of the antibody sample solution and 100 μL of RPMI medium were added to each dish and incubated for 48 hours. After washing with 1 mL of D-PBS(-), 1 mL of 100 nM LysoTracker® red DND-99 / (D-PBS(-):RPMI = 1:9) solution was added and incubated for 30 minutes. After washing with 1 mL of D-PBS(-), the plates were incubated for 4 minutes with 4% paraformaldehyde / D-PBS(-). After washing with 1 mL of D-PBS(-), 1 mL of 5.0 μg / mL Hoechst / D-PBS(-) solution was added and incubated for 5 minutes. After washing twice with 1 mL of D-PBS(-), 2 mL of RPMI was added and the plates were observed under a confocal microscope. Figure 39 shows the microscopic images taken.

[0163] When anti-NPC antibody was used alone, the fluorescence derived from the antibody was co-localized with lysosomes, but the co-localization of anti-NPC antibody with the nucleus was not observed. １１ / (Lys-GA) １４ ] / Fe ３＋ In the MPN complex, the anti-NPC antibody was observed to diffuse throughout the cytoplasm and coexist in the nucleus. １１ / (Lys-GA) １４ ] / Fe ３＋ We show that after cellular uptake, MPN complexes can escape from lysosomes, translocate into the cytoplasm, and target the nucleus.

[0164] (7.6 Evaluation of the amount of cellular uptake of antibody-loaded MPN complex) {Antibody, PEG-TA, Fe ３＋ Final concentration of antibody: 1.7 μM PEG １０ｋ -TA: 42.5 μM (prepared concentration: 200 μM) ・PEG ５ｋ -TA: 42.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 141 μM (prepared concentration: 20 mM) The antibody and each PEG-TA were dissolved in 10 mM HEPES. ３＋ was prepared by dissolving in ultrapure water.

[0165] Antibody solution, PEG-TA solution, Fe ３＋The solution was mixed by pipetting, with the molar ratio of PEG-TA / antibody being 25. The molar ratio of iron ions relative to the TA in PEG-TA was 3.3 equivalents.

[0166] Observation by confocal microscope: 2.5 × 10 4T1-Luc cells were placed in a 24-well plate. ４ The cells were seeded at 100 cells / plate and incubated at 37°C in 5% CO ２ The cells were pre-cultured for 24 hours under RT. 50 μL of the antibody sample solution and 150 μL of RPMI medium were added to each dish and incubated for 24 hours. The solution was then removed, the cells were washed twice with PBS(-), 150 μL of trypsin was added, and the cells were incubated at 37°C for 7 minutes. Then, 150 μL of RPMI medium was added and the cells were analyzed using a flow cytometer (FCM). Figure 40 is a graph showing the results of measuring the fluorescence of Cy5-labeled antibodies taken up by cells using a flow cytometer.

[0167] Antibody / PEG ５ｋ -TA and antibody / PEG １０ｋ The intracellular uptake of PEG-TA was reduced compared to the antibody alone. ５ｋ -TA / Fe ３＋ MPN complex, antibody / PEG １０ｋ -TA / Fe ３＋ The cellular uptake of the MPN complex was increased compared to the antibody alone, indicating that the formation of the MPN complex increases cellular uptake.

[0168] (7.7 Functional evaluation of anti-NPC antibody-loaded MPN complex using confocal microscope) Final concentrations of antibody and PEG-TA: Antibody: 1.7 μM PEG １０ｋ -TA: 42.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 141 μM (prepared concentration: 20 mM) The antibody and each PEG-TA were dissolved in 10 mM HEPES. ３＋ was prepared by dissolving in ultrapure water.

[0169] Antibody solution, PEG １０ｋ -TA solution, Fe ３＋The solution was mixed by pipetting, with the molar ratio of PEG-TA / antibody being 25. The molar ratio of iron ions relative to the TA in PEG-TA was 3.3 equivalents.

[0170] <<Observation by confocal microscope>> 35mm ２ 5.0 × 10 4T1-Luc cells were placed on a glass base dish. ４ The cells were seeded at 100 cells / dish and incubated at 37°C, 5% CO ２ The plates were pre-incubated for 24 hours under reduced pressure. 500 μL of the antibody sample solution and 500 μL of RPMI medium were added to each dish and incubated for 24 and 48 hours. After washing with 1 mL of D-PBS(-), 1 mL of a 100 nM LysoTracker® red DND-99 / (D-PBS(-):RPMI = 1:9) solution was added and incubated for 30 minutes. After washing with 1 mL of D-PBS(-), the plates were incubated for 4 minutes with a 4% paraformaldehyde / D-PBS(-) solution. After washing with 1 mL of D-PBS(-), 1 mL of a 5.0 μg / mL Hoechst / D-PBS(-) solution was added and incubated for 5 minutes. After washing twice with 1 mL of D-PBS(-), 2 mL of RPMI was added and the plates were incubated for 24 or 48 hours and observed by confocal microscopy. Figure 41 shows microscopic images taken after 24 hours of incubation. Figure 42 shows microscopic images taken after 48 hours of incubation. Figure 43 is a graph showing the results of measuring the colocalization efficiency of fluorescence derived from Lysotracker and fluorescence derived from Cy5 labeled antibody.

[0171] In the case of antibody alone and antibody / PEG-TA, the fluorescence from the antibody was observed to co-localize with lysosomes after both 24 and 48 hours of incubation. ３＋ In the MPN complex, the antibody-derived fluorescence was observed to diffuse throughout the cytoplasm. In particular, after 48 hours of incubation, the fluorescence was clearly visible throughout the cells. ３＋ The colocalization efficiency of Cy5 and Lysotracker-derived fluorescence in the MPN conjugate was significantly reduced compared to the antibody alone and antibody / PEG-TA.

[0172] (7.7 Functional evaluation of anti-NPC antibody-loaded MPN complex using confocal microscope) Final concentrations of antibody and PEG-TA: Anti-NPC antibody: 1.7 μM PEG １０ｋ -TA: 42.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 141 μM (prepared concentration: 20 mM) The antibody and each PEG-TA were dissolved in 10 mM HEPES. ３＋ was prepared by dissolving in ultrapure water.

[0173] Anti-NPC antibody solution, PEG １０ｋ -TA solution, Fe ３＋ The solution was mixed by pipetting, with the molar ratio of PEG-TA / antibody being 25. The molar ratio of iron ions relative to the TA in PEG-TA was 3.3 equivalents.

[0174] <<Observation by confocal microscope>> 35mm ２ 5.0 × 10 4T1 cells were placed on a glass-based dish. ４ The cells were seeded at 100 cells / dish and incubated at 37°C, 5% CO ２ The plates were pre-cultured for 24 hours under RT. 100 μL of the above antibody sample solution and 100 μL of RPMI medium were added to each dish and incubated for 48 hours. After washing with 1 mL of D-PBS(-), 1 mL of 100 nM LysoTracker® red DND-99 / (D-PBS(-):RPMI = 1:9) solution was added and incubated for 30 minutes. After washing with 1 mL of D-PBS(-), the plates were incubated for 4 minutes with 4% paraformaldehyde / D-PBS(-). After washing with 1 mL of D-PBS(-), 1 mL of 5.0 μg / mL Hoechst / D-PBS(-) solution was added and incubated for 5 minutes. After washing twice with 1 mL of D-PBS(-), 2 mL of RPMI was added and the plates were observed under a confocal microscope. Figure 44 shows the microscopic images taken.

[0175] When anti-NPC antibody was used alone, the antibody-derived fluorescence was co-localized with lysosomes, and the anti-NPC antibody was not observed to be co-localized with the nucleus. １０ｋ -TA / Fe ３＋In the MPN complex, the anti-NPC antibody was observed to be co-localized with the nucleus. １０ｋ -TA / Fe ３＋ We show that the MPN complex can translocate to the cytoplasm and target the nucleus.

[0176] [Experimental Example 8] <Functional evaluation of RNP-loaded MPN complex on cultured cells> (8.1 Overview) A complex using Luc gene-editing RNP was introduced into HeLa-Luc cells, and toxicity was evaluated by CCK assay and knockout efficiency was measured based on luciferase luminescence intensity.

[0177] (8.2 Reagents and Cell Lines) Cas9 Nuclease Protein NLS (Cas9) (Nippon Gene) Luciferase knockout gene knockout sgRNA (sgRNA) (GenScript) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ -TA (Mn=11,700) ・PEG ５ｋ -TA (Mn=6,700) D-PBS(-) (Fujifilm Wako Pure Chemical Industries) Luciferase Assay System (Promega) Cell culture lysis 5x reagent (Promega) Cell Counting Kit-8 (Dojindo Laboratories) Roswell Park Memorial Institute medium (RPMI) (Sigma-Aldrich) Fetal bovine serum (FBS) (Biosera) Trypsin-EDTA solution (Sigma-Aldrich) Penicillin / streptomycin (Sigma-Aldrich) HeLa-Luc cells (aneuploid epithelial cell line stably expressing luciferase) (American Type Culture Collection, ATCC) ProDeliverIn (OZ Biosciences)

[0178] (8.3 Measuring equipment) Glomax 96 microplate luminometer (Promega) Multiplate reader (Spark) (Tecan)

[0179] (8.4 Cytotoxicity Evaluation) Cas9-sgRNA, PEG １０ｋ -TA FeCl ３ ・6H ２ Final concentration of O» Cas9: 90 nM sgRNA: 90 nM PEG １０ｋ -TA (Mn=11,700): 9 μM ・Fe ３＋ :30μM Cas9-sgRNA, PEG １０ｋ -TA was prepared by dissolving it in D-PBS(-). ３＋ was prepared by dissolving in pure water.

[0180] Cas9 and sgRNA were mixed and centrifuged twice at 12,000 × g for 5 minutes using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). １０ｋ -TA was added, and the mixture was left standing at room temperature for 15 minutes. １０ｋ A Fe-TA solution was prepared. ３＋ After adding the above and leaving it at room temperature for 15 minutes, RNP / PEG １０ｋ -TA / Fe ３＋ An MPN complex solution was prepared. As a positive control, ProDeliverIn, a cationic reagent, was mixed at a ratio of ProDeliverIn / RNP = 1 μg / 1 μL to prepare an RNP / ProDeliverIn solution.

[0181] <Cytotoxicity evaluation by CCK assay> 3.0 × 10 HeLa-Luc cells were cultured in a 96-well plate. ３ The cells were seeded at 100 cells / plate and incubated at 37°C in 5% CO ２ The RNP solution, RNP / PEG, and １０ｋ -TA solution, RNP / PEG １０ｋ -TA / Fe ３＋The MPN complex solution and RNP / ProDeliverIn solution were added to each well at 100 μL / well and incubated for 72 hours. After washing with 150 μL / well of D-PBS(-), 110 μL / well of CCK measurement solution, a mixture of 10 μL of CCK and 100 μL of PBS(-), was added and incubated for 1 hour. The absorbance at 460 nm was then measured using a plate reader to evaluate cytotoxicity. Figure 45 is a graph showing the results of measuring cell viability by CCK assay.

[0182] RNP / PEG １０ｋ -TA / Fe ３＋ In the group to which the MPN complex was added, there was no significant decrease in cell viability compared to NT (untreated cells), and no significant cytotoxicity was confirmed.

[0183] (8.5 Evaluation of Knockout Efficiency) After the cytotoxicity evaluation, the medium was collected and washed with D-PBS(-). Then, 50 μL of Cell Culture Lysis was added to each well and incubated for 15 minutes. Then, 20 μL of the lysate was transferred to a dish for fluorescence measurement, luciferin was added, and the luciferase-induced luminescence intensity was measured using a plate reader to evaluate the knockout efficiency of the luciferase gene. Figure 46 is a graph showing the results of measuring the luciferase-induced luminescence intensity.

[0184] RNP / PEG １０ｋ -TA / Fe ３＋ In cells to which the MPN complex solution was added, the cells were treated with RNP alone, RNP / PEG, and １０ｋ A significant decrease in luciferase luminescence intensity was observed compared to cells administered with -TA. This result confirmed that the knockout efficiency was improved by conjugating RNP to MPN. １０ｋ -TA / Fe ３＋ The MPN complex exhibited gene knockout efficiency equivalent to that achieved with the commercially available product ProDeliverIn.

[0185] [Experimental Example 9] <Effects on subcutaneous tumor mouse model (pharmacokinetics, intratumor kinetics, antitumor effect)> (9.1 Overview) Antibody / PEG-TA / Fe in 4T1-Luc (mouse colon cancer cell) subcutaneous tumor mouse model ３＋ The functions of the complex were evaluated, specifically, the pharmacokinetics, intratumoral dynamics, and antitumor effect when loaded with a therapeutic antibody.

[0186] (9.2 Reagents, Cells, and Animals) Cy5-labeled antibody (antibody) Alexa Fluor （Ｒ） 647 anti-Nuclear Pore Complex Proteins Antibody (anti-NPC antibody) (Biolegend) Mouse Anti-S100A4 Recombinant Antibody (TAB-0136CL, hereafter referred to as anti-S100A4 antibody) (Creative Biolabs) PEG １０ｋ -TA (Mn=11,701) ・PEG ５ｋ -TA (Mn = 6,701) Tannic acid (Mw: 1,701) (Fujifilm Wako Pure Chemical Industries) 4T1-Luc cells (mouse breast cancer cell line) (American Type Culture Collection, ATCC) BALB / c mice (Charles River)

[0187] (9.3 Equipment and facilities) ・Countess (Thermo Fisher Scientific) ・Multi-bead shocker (Yasui Kikai) ・Multi-plate reader (Spark) (Tecan) ・In vivo imaging system (PerkinElmer) ・Cryostat (Leica Biosystems) ・Fluorescence microscope (BZX-700) (Keyence)

[0188] (9.4 Pharmacokinetics of antibody-loaded MPN complex) Antibody / PEG-TA / Fe ３＋ To evaluate the blood retention and tumor accumulation of the MPN complex, the antibody sample was intravenously injected into a mouse model with a 4T1-Luc subcutaneous tumor, and the antibody content in the blood and tumor after a certain period of time was measured by fluorescence intensity measurement. １０ｋ -TA / Fe ３＋ For comparison, the MPN complex was prepared using antibody alone and antibody / PEG. １０ｋ-TA, antibody / TA / Fe ３＋ The MPN complex was evaluated.

[0189] <<Final concentrations of antibody and PEG-TA>> Antibody: 3.3 μM PEG １０ｋ -TA: 82.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 272 μM (preparation concentration: 20 mM) antibody and PEG １０ｋ -TA was prepared by dissolving it in 10 mM HEPES. ３＋ was prepared by dissolving in ultrapure water.

[0190] Antibody solution, PEG １０ｋ -TA solution, Fe ３＋ The solution was mixed by pipetting. １０ｋ The molar ratio of TA / antibody was 25. The molar ratio of iron ions was 3.3 equivalents relative to the TA of PEG-TA.

[0191] <<Preparation of a 4T1-Luc Subcutaneous Tumor Mouse Model>> 4T1-Luc cell suspension (4.0 × 10 ７ 100 μL of the solution (cells / mL) was subcutaneously injected into BALB / c mice.

[0192] <Evaluation of pharmacokinetics> Tumor size is approximately 200 mm ３ 100 μL of the prepared sample solution was administered via the tail vein to model mice that had reached 100 μL. 48 hours after sample administration, the mice were dissected, and blood and various organs were collected. Five times the weight of passive lysis buffer was added, and the organs were homogenized using a multi-bead shocker. Blood was also collected from the tail vein 2 and 24 hours later. Subsequently, the mice were centrifuged at 10,000 × g for 5 minutes at 4°C, and 50 μL of the supernatant was transferred to a 96-well plate. The fluorescence intensity of the antibody was measured using a multiplate reader to evaluate pharmacokinetics. Figure 47 is a graph showing the results of organ accumulation evaluation. Figure 48 is a graph showing the results of blood retention evaluation.

[0193] Antibody / PEG １０ｋ -TA / Fe ３＋ The pharmacokinetics of the MPN conjugate showed behavior similar to that of the antibody alone, which has high blood retention and tumor accumulation. １０ｋ-TA / Fe ３＋ The MPN complex showed a tendency to have improved tumor accumulation compared to the antibody alone. ３＋ The blood retention and tumor accumulation of the MPN complex were evaluated using antibody / PEG-TA / Fe ３＋ The values ​​were lower than those of the MPN complex. These results indicate that PEG is effective in improving blood retention and tumor accumulation.

[0194] (9.5 Intratumoral Behavior of Anti-NPC Antibody-MPN Conjugate) To evaluate the intratumoral behavior of the antibody-MPN conjugate, an anti-NPC antibody / PEG-TA / Fe ３＋ The MPN complex sample was intravenously injected into a 4T1-Luc subcutaneous tumor mouse model, and after a certain period of time, the tumor was excised and sectioned to observe the intratumoral behavior of the antibody. ３＋ The MPN complexes were compared using antibody alone, antibody / PEG-TA, and antibody / TA / Fe. ３＋ The MPN complex was evaluated.

[0195] 《Antibody, PEG １０ｋ -TA, TA, Fe ３＋ Final concentration of: Anti-NPC antibody: 1.7 μM PEG １０ｋ -TA: 41.7 μM (prepared concentration: 200 μM) ・Fe ３＋ : 138 μM (prepared concentration: 20 mM) TA: 41.7 μM (prepared concentration: 200 μM) Antibody and PEG １０ｋ -TA, TA was prepared by dissolving in PBS(-), and Fe ３＋ was prepared by dissolving in ultrapure water.

[0196] Antibody solution, PEG １０ｋ -TA solution and TA solution, Fe ３＋ The solution was mixed by pipetting. １０ｋ The molar ratio of TA / antibody and TA / antibody was 25. The molar ratio of iron ions was 3.3 equivalents relative to the TA of PEG-TA.

[0197] <<Preparation of a 4T1-Luc Subcutaneous Tumor Mouse Model>> 4T1-Luc cell suspension (4.0 × 10 ７100 μL of the solution (cells / mL) was subcutaneously injected into BALB / c mice.

[0198] <Evaluation of pharmacokinetics> Tumor size is approximately 200 mm ３ 100 μL of the above sample solution was administered via the tail vein to model mice that had reached 100 μL. In vivo fluorescence imaging (IVIS) measurements were performed 48 hours after sample administration. Figure 49 shows images illustrating the results of in vivo fluorescence imaging. Immediately after in vivo fluorescence imaging, the mice were dissected and the tumors were removed. The tumors were cut into 6 μm sections using a cryostat and fixed with 4% paraformaldehyde. Subsequently, the tumors were stained with Hoechst and observed under a fluorescence microscope. Figure 50 shows a fluorescence microscope image of the sections. In Figure 50, the arrowheads indicate the areas in the nuclei where antibody-derived fluorescence was detected. Figure 51 is a graph showing the antibody-derived fluorescence intensity observed in the nuclei.

[0199] As shown in Figure 49, it was confirmed that all anti-NPC antibody samples accumulated in tumors. ３＋ The MPN complex had the lowest fluorescence intensity, which was consistent with the quantitative results of tumor accumulation shown in Figure 47. Figures 50 and 51 show that the anti-NPC antibody / PEG complex １０ｋ -TA / Fe ３＋ The MPN complex showed higher antibody fluorescence intensity in the nucleus than the other samples, indicating that the antibody was sufficiently transported to the cytoplasm and nucleus in vivo. ３＋ The MPN complex had lower tumor accumulation than the anti-NPC antibody alone and the anti-NPC antibody / PEG-TA, but had a higher colocalization efficiency with the nucleus. ３＋ It was shown that is crucial for translocation to the cytoplasm.

[0200] (9.6 Antitumor Effect of MPN Complexes Loaded with Therapeutic Antibodies) To demonstrate the function of the antibody-MPN complex, the antitumor effect of an MPN complex loaded with an anti-S100A4 antibody that activates the tumor suppressor gene (p53) was evaluated. ３＋The MPN complex sample was intravenously injected into a 4T1-Luc subcutaneous tumor mouse model, and the tumor size was measured. In this experiment, antibody / PEG-TA / Fe(2+) complexes were prepared using PEG-TA with PEG molecular weights of 10k and 5k. ３＋ In addition to the MPN complex, anti-S100A4 antibody alone and anti-S100A4 antibody / PEG were used as controls. １０ｋ -TA, control antibody / PEG １０ｋ -TA, anti-S100A4 antibody / TA / Fe ３＋ The MPN complex was evaluated.

[0201] 《Antibody, PEG-TA, TA, Fe ３＋ Final concentration of Anti-S100A4 antibody: 3.3 μM PEG １０ｋ -TA: 82.5 μM (prepared concentration: 200 μM) ・PEG ５ｋ -TA: 82.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 272 μM (prepared concentration: 20 mM) TA: 82.5 μM (prepared concentration: 200 μM) Antibody and PEG-TA, TA was prepared by dissolving in PBS (-), Fe ３＋ was prepared by dissolving in ultrapure water.

[0202] Antibody solution, both PEG-TA solutions and TA solution, Fe ３＋ The solution was mixed by pipetting, with the molar ratios of PEG-TA / antibody and TA / antibody being 25. The molar ratio of iron ions relative to the TA in PEG-TA was 3.3 equivalents.

[0203] <<Preparation of a 4T1-Luc Subcutaneous Tumor Mouse Model>> 4T1-Luc cell suspension (5.0 × 10 ７ 100 μL of the solution (cells / mL) was subcutaneously injected into BALB / c mice.

[0204] <Evaluation of pharmacokinetics> The tumor size in the model mouse was approximately 30 mm ３The time when the tumor size reached 100 μL was defined as day 0, and on days 0, 4, and 8, 100 μL of the above sample solution (anti-S100A4 antibody: 50 μg / mouse) was administered via the tail vein. For comparison, a group administered with D-PBS(-) was also prepared. The tumor size was measured with a caliper every two days until day 16 to evaluate the tumor size. At the same time, the mice were weighed and any adverse events were also confirmed. Figure 52 shows the change in tumor size over time, and Figure 53 shows the change in body weight over time. The tumor size was calculated using the following formula (F1): V=(W ２ × L) / 2 (V: volume, W: minor axis, L: major axis) ... (F1)

[0205] As shown in Figure 52, both anti-S100A4 antibody / PEG-TA / Fe ３＋ The tumor size in the MPN complex-administered group was shown to be significantly suppressed compared to the tumor sizes in the other sample and PBS(-)-administered groups. Furthermore, the MPN complex loaded with the control antibody showed a tumor growth curve similar to that of the PBS(-)-administered group, confirming that the anti-S100A4 antibody exerted its anti-tumor effect by functioning within the tumor. Furthermore, as shown in Figure 53, all sample-administered groups showed similar changes in body weight.

[0206] [Experimental Example 10] <Effect on subcutaneous tumor mouse model (evaluation of knockout efficiency)> (10.1 Overview) RNP / PEG in 4T1-Luc subcutaneous tumor mouse model １０ｋ -TA / Fe ３＋ The knockout efficiency of the MPN complex was evaluated.

[0207] (10.2 Reagents, Cells, and Animals) Cas9 Nuclease Protein NLS (Cas9) (Nippon Gene) sgRNA (Luciferase-2) (GenScript) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ-TA (Mn=11,700) 4T1-Luc cells (a mouse breast cancer cell line stably expressing luciferase) (American Type Culture Collection, ATCC) BALB / c mice (Charles River) Passive lysis 5x buffer (Promega) Micro BCA ＴＭ Protein Assay kit (Thermo Fisher Scientific)

[0208] (10.3 Measuring equipment) Glomax 96 microplate luminometer (Promega), multiplate reader (Spark) (Tecan), multi-beads shocker (Yasui Kikai)

[0209] (10.4 RNP / PEG １０ｋ -TA / Fe ３＋ Confirmation of in vivo function of MPN complex) RNP / PEG １０ｋ -TA / Fe ３＋ To evaluate the gene knockout efficiency of the MPN complex, RNP / PEG １０ｋ -TA / Fe ３＋ The MPN complex was intravenously injected into a 4T1-Luc subcutaneous tumor mouse model, and the luciferase luminescence intensity of the tumor was measured after a certain period of time had passed.

[0210] 《Cas9-sgRNA, PEG １０ｋ -TA, Fe ３＋ Final concentration of Cas9 Nuclease protein NLS: 1.0 μM sgRNA: 1.0 μM PEG １０ｋ -TA (Mn=11,700): 100 μM ・Fe ３＋ :333μM Cas9-sgRNA, PEG １０ｋ -TA was prepared by dissolving it in D-PBS(-). ３＋ was prepared by dissolving in pure water.

[0211] Cas9 and sgRNA were mixed and centrifuged twice at 12,000 × g for 5 minutes using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). １０ｋ -TA was added, and the mixture was left standing at room temperature for 15 minutes.１０ｋ A Fe-TA solution was prepared. ３＋ After adding the above and leaving it at room temperature for 15 minutes, RNP / PEG １０ｋ -TA / Fe ３＋ An MPN complex solution was prepared.

[0212] <<Preparation of a 4T1-Luc Subcutaneous Tumor Mouse Model>> 4T1-Luc cell suspension (1.0 × 10 ６ 100 μL of the solution (cells / mL) was subcutaneously injected into BALB / c mice.

[0213] <<Evaluation of Knockout Efficiency>> 200 μL of the above-mentioned prepared solution and D-PBS(-) were administered via the tail vein to a 4T1-Luc subcutaneous tumor mouse model. Eight days after sample administration, the tumor was collected, and 3 times the weight of Passive Lysis Buffer was added. The tumor was then pulverized using a multi-bead shocker at 1,500 rpm for 30 seconds, 8 times. The suspension was then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected. The supernatant was then diluted 50-fold with PBS(-), and the BCA assay standard reagent and sample were mixed at a sample:standard reagent ratio of 1:15, followed by incubation at 37°C for 20 minutes. The absorbance at 562 nm was measured using a plate reader, and the protein amount of each sample was calculated from the calibration curve. Furthermore, 10 μL of the supernatant was transferred to a fluorescence measurement plate, 100 μL of luciferin was added, and the luminescence intensity of luciferase was measured using a plate reader to evaluate the knockout efficiency of the luciferase gene. Figure 54 is a graph showing the measurement results of the luminescence intensity of luciferase.

[0214] RNP / PEG １０ｋ -TA / Fe ３＋ The MPN complex-administered mice showed a decrease in luciferase luminescence intensity compared to the PBS(-)-administered mice, confirming that the luciferase gene had been knocked out.

[0215] [Summary] In order to improve the activity of physiologically active proteins, we have constructed a protein delivery system driven by the complex formation between polyphenol-introduced polymers and metal ions.３＋ The MPN complex promoted antibody cytoplasmic translocation and enhanced the antibody-derived antitumor effect. Furthermore, the RNP-loaded MPN complex loaded with Cas9 / sgRNA RNP enhanced gene knockout efficiency. Furthermore, this RNP-loaded MPN complex was also successful in in vivo gene knockout in tumors.

[0216] [Experimental Example 11] <Evaluation of physical properties of PEG-TA> (11.1 Overview) PEG ５ｋ -TA (M ｎ = 6,700) and PEG １０ｋ -TA (M ｎ The structure of the compound (M = 11,700) was evaluated. UV absorption due to the structure was confirmed using a JASCO V-650 spectrophotometer, and the presence of specific functional groups was confirmed by FT-IR measurement.

[0217] (11.2 Reagents) Unless otherwise specified, commercially available reagents and solvents were used as is. ５ｋ -TA (M ｎ =6,700) ・PEG １０ｋ -TA (M ｎ = 11,700) D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.) Tannic acid (Mw: 1,701) (Fujifilm Wako Pure Chemical Industries, Ltd.) TA (quinone) α-methoxy-ω-amino-poly(ethylene glycol) (PEG-NH ２ ) [Mn: 5k] (NOF Corp.) α-Methoxy-ω-amino-poly(ethylene glycol) (PEG-NH ２ ) [Mn: 10k] (NOF Corp.) Sodium bicarbonate (Tokyo Chemical Industry Co., Ltd.)

[0218] (11.3 Measuring equipment) JASCO V-650 spectrophotometer (JASCO) FT-IR: JASCO

[0219] (11.4 Preparation of Oxidized TA) TA Concentration: TA: 0.26 mg / mL

[0220] TA was dissolved in 50 mM sodium bicarbonate buffer (pH 8.5) in a beaker and stirred for 24 hours. The reaction solution was then transferred to a dialysis membrane (MWCO: 3500 Da) and dialyzed for 24 hours against 2 L of purified water, which had been subjected to 10 minutes of ultrasonication and 5 minutes of Ar bubbling. The dialyzed reaction solution was then passed through a 0.45 μm filter and lyophilized to obtain TA (quinone).

[0221] (11.5 TA, TA (quinone), PEG ５ｋ -TA and PEG １０ｋ -UV spectrum measurement of TA) 《TA, TA (quinone), PEG ５ｋ -TA, PEG １０ｋ - Final concentration of TA》 TA: 0.073 mg / mL TA (quinone): 0.073 mg / mL PEG ５ｋ -TA: 0.29mg / mL ・PEG １０ｋ -TA: 0.5 mg / mL Each was prepared by dissolving in D-PBS(-).

[0222] TA solution, TA (quinone) solution, PEG solution were measured using a JASCO V-650 spectrophotometer. ５ｋ -TA solution, PEG １０ｋ The UV absorption of each of the -TA solutions was measured, and the results are shown in Figure 55.

[0223] PEG ５ｋ -TA solution and PEG １０ｋ The PEG-TA solution exhibited similar UV absorption to that of TA, ５ｋ -TA and PEG １０ｋ It was shown that the TA structure is contained in -TA. In addition, UV absorption at wavelengths of 360 nm or more was observed in TA (quinone) solution, PEG, and ５ｋ -TA solution and PEG １０ｋ -TA solution, so PEG ５ｋ -TA and PEG １０ｋ It was suggested that part of the galloyl group structure of -TA was converted to a quinone structure. The chemical formula of tannic acid-modified polyethylene glycol, in which part of the galloyl group structure was converted to a quinone structure, is shown in formula (3) below. In formula (3), n represents the number of polyethylene glycol units.

[0224]

[0225] (11.6 PEG-NH ２ (Mn: 10k) + TA and PEG-NH ２ Preparation of (Mn: 5k) + TA) «TA, PEG-NH ２ (Mn: 10k), PEG-NH ２ Final concentration of (Mn: 5k)》 TA: 0.17 mg / mL PEG-NH ２ (Mn:10k) :1mg / mL ・PEG-NH ２ (Mn: 5k): 0.5 mg / mL Each was prepared by dissolving in ultrapure water.

[0226] TA to PEG １０ｋ -NH ２ (Mn: 10k) or PEG ５ｋ -NH ２ (Mn: 5k) was dissolved in the same solution. ２ The molar ratio of each was 1:1. The reaction solution was then freeze-dried to obtain PEG. １０ｋ -NH ２ (Mn: 10k) + TA and PEG ５ｋ -NH ２ (Mn: 5k) + TA was obtained.

[0227] (11.7 FT-IR Measurement) Using FT-IR, TA, MeO-PEG-NH ２ (Mn: 10k and 5k), PEG １０ｋ -TA, PEG ５ｋ -TA, MeO-PEG-NH ２ (Mn: 10k and 5k) + TA were measured. The measurement results are shown in Figures 56 and 57.

[0228] PEG １０ｋ -TA and PEG ５ｋ -TA (1,200 cm －１ , 1,600-1,800cm －１ , 3,100-3,600cm －１ ), PEG (2,800-3,000cm －１ ) and PEG １０ｋ -TA and PEG５ｋ -TA is PEG-NH ２ It was suggested that both the and TA structures exist.

[0229] [Experimental Example 12] <Synthesis of PEG-TA Introduced with a Hepatocyte Targeting Molecule> (12.1. Overview) In order to impart functionality to PEG-TA, PEG-TA was synthesized by introduc- ing a hepatocyte targeting molecule (N-acetylgalactosamine). As shown in the following scheme (5), α-GalNAc-PEG3-Thiol was converted to ACA-PEG-NH ２ After the introduction of TA,

[0230]

[0231] (12.2. Reagents) ACA-PEG １０ｋ -NH ２ (HE010005-10K, Mn: 10K, Biochempeg Scientific Inc.) Tannic acid (Mw: 1701, Fujifilm Wako Pure Chemical Industries) D-PBS(-) (Fujifilm Wako Pure Chemical Industries) α-GalNAc-PEG3-Thiol: Sussex Research Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) (Sigma Aldrich) Acetic acid (CH ３ COOH) (Nacalai Tesque) Sodium bicarbonate (NaHCO ３ ) (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0232] (12.3. Measurement equipment) Nuclear Magnetic Resonance (NMR) device (BRUKER AVANCEIII400 (400 MHz, BRUKER BioSpin) Nanospectrophotometer NanoDrop One (Thermo Fisher Scientific)

[0233] (12.4. Synthesis of GalNac-introduced PEG-TA) GalNac-PEG １０ｋ Synthesis of GalNac-ACA-PEG １０ｋ -NH ２ Binding to α-GalNAc-PEG in a 30 mL beaker ３-Thiol 20mg (0.05mmol), ACA-PEG １０ｋ -NH ２ 200 mg (0.025 mmol) of the reaction mixture and 15 mg (0.05 mmol) of LAP were weighed out and irradiated with 380 nm ultraviolet light at 20 W for 5 hours. The reaction solution was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed three times in water. The resulting solution was freeze-dried to obtain a white solid, GalNac-PEG. １０ｋ -NH ２ The yield was 200 mg.

[0234] GalNac-PEG of TA １０ｋ -NH ２ Binding to 52 mg (0.030 mmol) of TA was weighed out into a 200 mL beaker and added to 50 mM NaHCO ３ (pH 8.5) 80 mL. ３ GalNac-PEG dissolved in 40 mL of (pH 8.5) １０ｋ -NH ２ 100 mg (0.010 mmol) of the reaction mixture was added dropwise to the reaction mixture, which was then stirred at room temperature for 5 hours. The reaction mixture was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed for 1 hour against 2 L of pure water that had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes.

[0235] <<Reduction of the Side Chain Ketone Group of TA>> The reaction solution was transferred to a 200 mL beaker, and 2 mL of acetic acid was added dropwise and stirred for 5 minutes. The reaction solution was then transferred to a dialysis membrane (MWCO: 6-8 kDa) and dialyzed twice with 2 L of pure water, which had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes. The resulting solution was freeze-dried to obtain a yellow solid PEG. １０ｋ The yield of D-TA was 68 mg. ２ GalNac-PEG dissolved in O １０ｋ , and Figure 59 shows GalNac-PEG dissolved in d-DMSO. １０ｋ -TA's １ The H-NMR spectrum is shown.

[0236] (Analysis) GalNac ACA-PEG １０ｋ -NH ２ Bonding to １In the H-NMR spectrum, a peak (3.3-4.0 ppm) derived from the initiator and a peak (2.0 ppm) derived from GalNac were confirmed. The GalNac introduction rate was calculated to be 90%.

[0237] GalNac-PEG of TA １０ｋ -NH ２ Bonding to GalNac-PEG-NH ２ of １ A peak derived from TA was confirmed in the H-NMR spectrum, confirming quantitative introduction of TA.

[0238] [Experimental Example 13] <Synthesis of PEG-TA with Cationic Molecules> (13.1. Overview) In order to impart functionality to PEG-TA, PEG-TAs were synthesized incorporating ethylenediamine molecules such as triethylenetriamine (TET), tetraethylenepentamine (TEP), and spermine (S). As shown in the following schemes (6) to (11), MeO-PEG １０ｋ After introducing an ethylenediamine-based molecule to the —COOH, TA was bound.

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] 13.2. Reagents α-hydroxyl-ω-carboxyl-poly(ethylene glycol) (MeO-PEG １０ｋ-COOH, Mn: 10k, NOF Corp.) Tannic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) N,N'-bis(2-aminoethyl)-1,2-ethanediamine (TET, Tokyo Chemical Industry Co., Ltd.) N(2-aminoethyl)-N-(2-((2-aminoethyl)amino)ethyl-1,2-ethanediamine (TEP, Tokyo Chemical Industry Co., Ltd.) N,N'-bis(3-aminopropyl)butane-1,4-diamine (Spermine, Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium bicarbonate (NaHCO ３ , Fujifilm Wako Pure Chemical Industries) Acetic acid (CH ３ COOH) (Nacalai Tesque), {2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid} (HEPES, Nacalai Tesque), Sodium chloride (NaCl, Fujifilm Wako Pure Chemical Industries, Ltd.), Sodium hydroxide (NaOH, Nacalai Tesque),

[0246] (13.3. Measuring equipment) Nuclear magnetic resonance (NMR) apparatus (BRUKER AVANCEIII400 (400 MHz, BRUKER BioSpin) Gel permeation chromatography (GPC) apparatus (Jasco International) Column: Superdex200 Increase 10 / 300 GL (Cytiva) Column: Superdex75 Increase 10 / 300 GL (Cytiva) Detector: RI-2031, UV-2030 JASCO V-650 spectrophotometer (Jasco)

[0247] (13.4. Polymer Synthesis) PEG １０ｋ Synthesis of -TET-TA] [PEG of TET １０ｋ Bonding to -COOH] 29 mg (0.2 mmol) of TET was weighed out into a 200 mL beaker and dissolved in 10 mM NaHCO ３ (pH 8.5) 80 mL. ３ PEG dissolved in 40 mL of (pH 8.5) １０ｋ100 mg (0.010 mmol) of —COOH was added dropwise, and 1.5 mmol of DMT-MM was added, followed by stirring at room temperature for 5 hours. Thereafter, dialysis was carried out against 0.5 mL of NaOH and 2 L of distilled water, and then against distilled water alone. ２ Dissolved in O １ The H-NMR spectrum is shown in Figure 60.

[0248] [PEG of TA １０ｋ Binding to TET] 10 mg (0.006 mmol) of TA was weighed out in a 200 mL beaker and added to 50 mM NaHCO ３ (pH 8.5) 60 mL. ３ PEG dissolved in 40 mL of (pH 8.5) １０ｋ 30 mg (0.003 mmol) of -TET was added dropwise and stirred at room temperature for 5 hours. The reaction solution was then transferred to a dialysis membrane (MWCO = 6-8 kDa) and dialyzed for 1 hour against 2 L of pure water that had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes.

[0249] [Reduction of the side chain ketone group of TA] The reaction solution was transferred to a 200 mL beaker, and 5 mL of a 10-fold diluted solution of 0.5 mL of acetic acid was added dropwise and stirred for 5 minutes. The reaction solution was then transferred to a dialysis membrane (MWCO = 6-8 kDa) and dialyzed twice with 2 L of pure water, which had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes. The resulting solution was freeze-dried to obtain a yellow solid PEG. １０ｋ The yield of 30 mg of PEG-TET was 30 mg. ＴＭ 200 Increase 10 / 300 GL, eluent: 10 mM HEPES, 140 mM NaCl (pH 7.4), flow rate: 0.75 mL / min, detector: UV-2030, detection wavelength: 254 nm, measurement temperature: room temperature) is shown in FIG.

[0250] <<Synthesis of PEG-TEP-TA>> [PEG of TEP １０ｋ Bonding to -COOH] 38 mg (0.2 mmol) of TEP was weighed out into a 200 mL beaker and added to 10 mM NaHCO ３ (pH 8.5) 80 mL. ３ PEG dissolved in 40 mL of (pH 8.5) １０ｋ100 mg (0.010 mmol) of —COOH was added dropwise, and 1.5 mmol of DMT-MM was added, followed by stirring at room temperature for 5 hours. Thereafter, dialysis was carried out against 0.5 mL of NaOH and 2 L of distilled water, and then against distilled water alone. ２ Dissolved in O １ The H-NMR spectrum is shown in Figure 62.

[0251] [TEP PEG １０ｋ -Binding to TEP] 10 mg (0.006 mmol) of TA was weighed out into a 200 mL beaker and added to 50 mM NaHCO ３ (pH 8.5) 60 mL. ３ PEG dissolved in 40 mL of (pH 8.5) １０ｋ 30 mg (0.003 mmol) of -TEP was added dropwise, and the mixture was stirred at room temperature for 5 hours. Thereafter, the reaction solution was transferred to a dialysis membrane (MWCO = 6 to 8 kDa) and dialyzed for 1 hour against 2 L of pure water that had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes.

[0252] [Reduction of the side chain ketone group of TA] The reaction solution was transferred to a 200 mL beaker, and 5 mL of a 10-fold diluted solution of 0.5 mL of acetic acid was added dropwise and stirred for 5 minutes. The reaction solution was then transferred to a dialysis membrane (MWCO = 6-8 kDa) and dialyzed twice with 2 L of pure water, which had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes. The resulting solution was freeze-dried to obtain a yellow solid PEG. １０ｋ -TEP was obtained in a yield of 30 mg. GPC curve (column: Superdex ＴＭ 200 Increase 10 / 300 GL, eluent: 10 mM HEPES, 140 mM NaCl (pH 7.4), flow rate: 0.75 mL / min, detector: UV-2030, detection wavelength: 254 nm, measurement temperature: room temperature) is shown in FIG.

[0253] <<Synthesis of PEG-S-TA>> [PEG of Spermine １０ｋ Bonding to —COOH] 40 mg (0.2 mmol) of spermine was weighed out in a 200 mL beaker and dissolved in 10 mM NaHCO ３ (pH 8.5) 80 mL. ３ PEG dissolved in 40 mL of (pH 8.5)１０ｋ 100 mg (0.010 mmol) of —COOH was added dropwise, and 1.5 mmol of DMT-MM was added, followed by stirring at room temperature for 5 hours. Thereafter, dialysis was carried out against 0.5 mL of NaOH and 2 L of distilled water, and then against distilled water alone. ２ Dissolved in O １ The H-NMR spectrum is shown in Figure 63.

[0254] [PEG of TA １０ｋ -Binding to spermine] 10 mg (0.006 mmol) of TA was weighed out in a 200 mL beaker and added to 50 mM NaHCO ３ (pH 8.5) 60 mL. ３ PEG dissolved in 40 mL of (pH 8.5) １０ｋ 30 mg (0.003 mmol) of -TET was added dropwise and stirred at room temperature for 5 hours. The reaction solution was then transferred to a dialysis membrane (MWCO = 6-8 kDa) and dialyzed for 1 hour against 2 L of pure water that had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes.

[0255] [Reduction of the side chain ketone group of TA] The reaction solution was transferred to a 200 mL beaker, and 5 mL of a 10-fold diluted solution of 0.5 mL of acetic acid was added dropwise and stirred for 5 minutes. The reaction solution was then transferred to a dialysis membrane (MWCO = 6-8 kDa) and dialyzed twice with 2 L of pure water, which had been subjected to ultrasonic irradiation for 10 minutes and Ar bubbling for 5 minutes. The resulting solution was freeze-dried to obtain a yellow solid PEG. １０ｋ The yield of 30 mg of PEG-TET was 30 mg. ＴＭ 75 Increase 10 / 300 GL, eluent: 10 mM HEPES, 140 mM NaCl (pH 7.4), flow rate: 0.75 mL / min, detector: UV-2030, detection wavelength: 280 nm, measurement temperature: room temperature) are shown in Figure 64.

[0256] (13.5. Analysis) 《PEG １０ｋ -TET-TA》 From the GPC curve, PEG １０ｋ It was confirmed that PEG-TET-TA has a narrow molecular weight distribution. １０ｋ -TET's １In the H-NMR spectrum, peaks derived from PEG and TET were observed, confirming quantitative introduction. Furthermore, the introduction rate of TA was quantitatively confirmed from the absorbance at the absorption wavelength of 290 nm derived from the GA group.

[0257] 《PEG １０ｋ -TEP-TA》 From the GPC curve, PEG １０ｋ -TEP-TA was confirmed to have a narrow molecular weight distribution. １０ｋ -TEP's １ In the H-NMR spectrum, peaks derived from PEG and TEP were observed, confirming quantitative introduction. Furthermore, the introduction rate of TA was quantitatively confirmed from the absorbance at the absorption wavelength of 290 nm derived from the GA group.

[0258] 《PEG １０ｋ -Spermine-TA》 From the GPC curve, PEG １０ｋ -Spermine-TA was confirmed to have a narrow molecular weight distribution. １０ｋ -Spermine's １ In the H-NMR spectrum, a peak derived from PEG and a peak derived from spermine (near 2.3 ppm) were observed, confirming quantitative introduction. Furthermore, the introduction rate of TA was quantitatively confirmed from the absorbance at an absorption wavelength of 290 nm derived from the GA group.

[0259] [Experimental Example 14] <Confirmation of complex formation between synthetic polymer and metal ion> (14.1. Overview) GA groups and metal ions crosslink to form metal ion-polyphenol complexes (MPN) with buffering capacity. Therefore, complex formation between each polymer and iron ions was confirmed using a spectrophotometer.

[0260] (14.2. Reagents) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ -TA (Mn=11,701) ・PEG １０ｋ -TET-TA (Mn=11,847) ・PEG １０ｋ -TEP-TA (Mn=11,890) ・PEG １０ｋ-S-TA (Mn=11,903) Tannic acid (Fujifilm Wako Pure Chemical Industries) D-PBS (-) (Fujifilm Wako Pure Chemical Industries)

[0261] (14.3. Measuring equipment) ・JASCO V-650 spectrophotometer (Jasco)

[0262] (4.4. Confirmation of Complex Formation) <<Polymer, FeCl ３ ・6H ２ Final concentration of O》 PEG １０ｋ -TA: 0.04mM PEG １０ｋ -TET-TA: 0.04mM PEG １０ｋ -TEP-TA: 0.04mM PEG １０ｋ -S-TA: 0.04mM ・FeCl ３ ・6H ２ O(Fe ３＋ ): 0.12 mM

[0263] PEG １０ｋ -TA, PEG １０ｋ -TET-TA, PEG １０ｋ -TEP-TA, PEG １０ｋ -S-TA is D-PBS(-), Fe ３＋ was prepared by dissolving in ultrapure water.

[0264] Polymer solution and Fe ３＋ The above concentrations were mixed to prepare a solution. After incubation at room temperature for 15 minutes, the absorption spectrum was measured using a spectrophotometer. Figure 65 is a graph showing the results of the absorption spectrum measurement.

[0265] (14.5. Analysis) From the absorption spectrum in Figure 65, a peak is observed around 550 nm in all samples, and various polymers and Fe ３＋ It was confirmed that a complex was formed.

[0266] [Experimental Example 15] <Evaluation of the formation of MPN complexes loaded with GalNac-modified β-Gal> (15.1. Overview) MPN complexes were formed using not only proteins but also inorganic / organic particles, nucleic acids, and lipid nanoparticles, and the change in particle size was measured. The particle size was measured by dynamic light scattering (DLS) or fluorescence spectroscopy correlation spectroscopy (FCS).

[0267] (15.2. Reagents) D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) β-D-galactosidase (β-Gal, Mw: 540 kDa, Fujifilm Wako Pure Chemical Industries, Ltd.) PEG １０ｋ -TA ・GalNac-PEG １０ｋ -TA

[0268] (15.3. Measuring equipment) ・LSM710 (Carl Zeiss Co., Ltd.) ・Zetasizer Ultra (Malvern Co., Ltd.)

[0269] (15.4. Evaluation of GalNac-modified β-Gal-loaded MPN complex formation) <<Introduction of Alexa 647 into β-Gal complex>> β-Gal: 10 mg Alexa Flour 647-NHS: 0.12 mg

[0270] 10 mg of β-Gal was weighed into a 20 mL vial and diluted with 50 mM NaHCO ３ The Alexa Flour 647-NHS was dissolved in 10 mL of DMSO (pH 8.0). 0.12 mg of Alexa Flour 647-NHS dissolved in DMSO was added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was subjected to ultrafiltration (MWCO: 10 kDa) twice using PBS, and then unreacted Alexa Flour 647-NHS was removed using a PD-10 column (solvent: PBS). Next, ultrafiltration (MWCO: 10 kDa) was again performed twice using PBS, and Alexa Flour 647-modified β-Gal (Alexa 647-β-Gal) was recovered in solution. The β-Gal concentration was then calculated from the absorbance at 280 nm, which is the protein-derived absorption wavelength.

[0271] 《Alexa647-β-Gal, PEG-TA, GalNac-PEG-TA, Fe ３＋ Final concentration》 ・Alexa647-β-Gal (β-Gal): 370 nM ・PEG-TA: 92.5 μM ・GalNac-PEG １０ｋ -TA: 92.5 μM ・Fe ３＋ : 305μM (prepared concentration: 6.1mM)

[0272] Alexa647-β-Gal, PEG-TA, and GalNac-PEG-TA were prepared by dissolving them in PBS(-). ３＋was prepared by dissolving in ultrapure water.

[0273] Alexa647-β-Gal solution, PEG-TA solution, GalNac-PEG １０ｋ -TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of polymer / β-Gal was set to 250. The molar ratio of iron ions was set to 3.3 equivalents relative to the TA of the polymer. Finally, PBS(-) was added to adjust the concentration, and then the particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. The particle size measurement results are shown in Figure 66.

[0274] β-Gal / PEG １０ｋ -TA / Fe ３＋ MPN complex and β-Gal / GalNac-PEG-TA / Fe ３＋ The particle size of the MPN complexes was significantly increased compared to that of β-Gal alone, indicating the formation of β-Gal MPN complexes.

[0275] [Experimental Example 16] <Confirmation of the formation of MPN complexes loaded with various molecules> (16.1. Overview) MPN complexes loaded with not only proteins but also inorganic / organic particles, nucleic acids, and lipid nanoparticles were formed, and changes in particle size were measured. Particle size was measured using dynamic light scattering (DLS) or fluorescence spectroscopy correlation spectroscopy (FCS).

[0276] (16.2. Reagents) Unless otherwise specified, commercially available reagents and solvents were used as is. D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.) β-D-galactosidase (β-Gal, Mw: 540 kDa, Fujifilm Wako Pure Chemical Industries, Ltd.) LipidLaunch ＴＭ　 SORT LNP Exploration Kit (Cayman Chemical) Iron(III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) D-PBS (-) (Fujifilm Wako Pure Chemical Industries) Sodium acetate (Fujifilm Wako Pure Chemical Industries) Alexa Fluor 647-TUG1 (8058.7 g / mol) (GeneDesign. Inc.) PEG ５ｋ -TA (M ｎ=6,700) ・PEG １０ｋ -TA (M ｎ = 11,700) ・GalNac-PEG １０ｋ -TA (M ｎ = 12,400) ・PS particles-COOH (50 nm) (Abvigen) ・PEG-P[Lys １１ / (Lys-GA) １４ Alexa Fluor 647-TUG1 (TUG1, 8058.7 g / mol) (Gene Design, Inc.) Zirconium (IV) chloride (ZrCl ４ ) (Zr ４＋ ) (Fujifilm Wako Pure Chemical Industries) ・UltraPure ＴＭ DNase / RNase-Free Distilled Water (Thermo Fisher Scientific)

[0277] (16.3 Measuring equipment) Zetasizer Ultra (red) (Malvern Panalytical) LSM710 (Carl Zeiss)

[0278] (16.4 Evaluation of physicochemical properties of LNP-MPN complexes) LNP, PEG ５ｋ -TA (M ｎ When an MPN complex is formed by mixing metal ions, the particle size increases compared to LNP alone. Therefore, particle size measurements were performed using a Zetasizer Ultra (red). The metal ions used were Fe, ３＋ was used.

[0279] [LNP formation] LipidLaunch was used according to the kit instructions. ＴＭ Each solution in the SORT LNP Exploration Kit was dispensed into a 1.5 mL tube to a total volume of 100 μL. Then, 12.5 μL of Alexa Fluor647-TUG1 (2 mg / mL) was added to another 1.5 mL tube, and 285.7 μL of 50 mM sodium acetate (pH 4.0) was added thereto. LipidLaunch was added to this solution. ＴＭ100 μL of SORT LNP Exploration Kit solution was added and stirred by pipetting for 15 seconds. This solution was allowed to stand for 10 minutes, after which the reaction solution was added to an ultrafiltration tube (MWCO: 30 kDa) and centrifuged twice at 2,300 × rpm for 20 minutes to remove unreacted lipids, etc. Finally, the solution was diluted to 500 μL using D-PBS(-) to obtain LNPs incorporating Alexa Fluor 647-TUG1.

[0280] [Confirmation of LNP-MPN complex formation] <LNP, PEG ５ｋ -TA, FeCl ３ ・6H ２ Final concentration of LNP: 20-fold dilution of the solution at the time of preparation PEG ５ｋ -TA: 11.2 μM (prepared concentration: 224 μM) ・FeCl ３ ・6H ２ O: 12.9 μM (prepared concentration: 370 μM)

[0281] LNP and PEG ５ｋ -TA is D-PBS(-), FeCl ３ ・6H ２ O was prepared by dissolving it in ultrapure water.

[0282] LNP solution, PEG ５ｋ -TA solution, FeCl ３ ・6H ２ The O solution was mixed by vortexing. ５ｋ -TA and FeCl ３ ・6H ２ The molar ratio of LNP solution to PEG was 1:1.2. ５ｋ A mixture of only the -TA solution was also prepared. After adjusting the concentration by adding D-PBS(-) to these solutions and the LNP solution, the particle size was measured using a Zetasizer Ultra (red). The particle size measurement results are shown in Figure 67.

[0283] LNP / PEG ５ｋ -TA / Fe ３＋ The particle size of the complex is LNP, LNP / PEG. ５ｋ -Since LNP / PEG significantly increased compared to TA, ５ｋ-TA / Fe ３＋ The formation of a complex was demonstrated.

[0284] [PEG ５ｋ -TA and FeCl ３ ・6H ２ Evaluation of LNP-MPN complex formation when the ratio of LNP, PEG, ５ｋ -TA, FeCl ３ ・6H ２ Final concentration of LNP: 20-fold dilution of the solution at the time of preparation PEG ５ｋ -TA: 11.2-22.4 μM (prepared concentration: 224 μM) ・FeCl ３ ・6H ２ O: 12.9-33.6 μM (prepared concentration: 370 μM)

[0285] LNP and PEG ５ｋ -TA is D-PBS(-), FeCl ３ ・6H ２ O was prepared by dissolving it in ultrapure water.

[0286] LNP solution, PEG ５ｋ -TA solution, FeCl ３ ・6H ２ The O solution was mixed by vortexing. ５ The final concentration of k-TA was 11.2 μM, and PEG ５ｋ -TA and FeCl ３ ・6H ２ The molar ratio of PEG to PEG was 1:1.2 and 1:3. ５ The final concentration of k-TA was 22.4 μM, and PEG ５ｋ -TA and FeCl ３ ・6H ２ A solution with a molar ratio of 1:1.2 was also prepared. D-PBS(-) was added to each of these solutions to adjust the concentration, and then the particle size was measured using a Zetasizer Ultra (red). The particle size measurement results are shown in Figure 68.

[0287] Each LNP / PEG ５ｋ -TA / Fe ３＋ The particle size of the complexes was significantly increased compared to LNP. ５ｋ -TA / Fe ３＋The formation of a complex was suggested. ５ｋ -TA and FeCl ３ ・6H ２ As the amount of O increased, the particle size increased.

[0288] (16.5. Evaluation of physicochemical properties of PS particle-MPN complex) When an MPN complex is formed by mixing PS particles, metal ions, and PEG-TA, the particle size increases compared to the PS particles alone. Therefore, particle size was measured using Zetasizer Ultra (red). Fe was used as the metal ion. ３＋ was used.

[0289] [Confirmation of PS particle-MPN complex formation] <<PS particles, PEG ５ｋ -TA, PEG １０ｋ -TA, FeCl ３ ・6H ２ Final concentration of O》 PS particles: 0.86 mg / mL PEG ５ｋ -TA: 60 μM (prepared concentration: 880 μM) ・PEG １０ｋ -TA: 60 μM (prepared concentration: 880 μM) ・FeCl ３ ・6H ２ O: 76 to 180 μM (prepared concentration: 370 μM) Each was prepared by dissolving in ultrapure water.

[0290] PS particle solution, PEG ５ｋ -TA solution, FeCl ３ ・6H ２ O solution, or PS particle solution, PEG １０ｋ -TA solution, FeCl ３ ・6H ２ The PEG-TA and FeCl 0 solutions were mixed by vortexing. ３ ・6H ２ The molar ratios of PS particles to O were 1:1.2 and 1:3. Solutions of PS particles alone were also prepared, and ultrapure water was added to each of these solutions to adjust the concentration. The particle diameters were measured using a Zetasizer Ultra (red). Figure 69 shows the results of the particle diameter measurements.

[0291] Each PS / PEG ５ｋ -TA / Fe３＋ Conjugates and PS / PEG １０ｋ -TA / Fe ３＋ The particle size of the complexes was significantly increased compared to that of PS particles. ５ｋ -TA / Fe ３＋ Conjugates and PS / PEG １０ｋ -TA / Fe ３＋ The formation of a complex was suggested. ３ ・6H ２ As the amount of O increased, the particle size increased.

[0292] (16.6. Evaluation of Physicochemical Properties of Nucleic Acid-MPN Complexes) Nucleic acid and metal ions were mixed, and PEG-TA or PEG-P[Lys １１ / (Lys-GA) １４ When an MPN complex is formed by mixing the metal ions Zr and ZrO, the particle size increases compared to the case of nucleic acid alone. ４＋ was used.

[0293] [Confirmation of nucleic acid MPN complex formation] <TUG1, PEG １０ｋ -TA, PEG-P[Lys １１ / (Lys-GA) １４ ], ZrCl ４ Final concentration of》 TUG1: 3.36 μM PEG １０ｋ -TA: 672 μM (prepared concentration: 940 μM) ・PEG-P[Lys １１ / (Lys-GA) １４ ]: 672 μM (prepared concentration: 940 μM) ・ZrCl ４ : 307-613 μM (prepared concentration: 2146 μM)

[0294] All of these are UltraPure ＴＭ It was prepared by dissolving in DNase / RNase-free distilled water.

[0295] TUG1 solution, PEG １０ｋ -TA solution, ZrCl ４ solution, or TUG1 solution, PEG-P[Lys １１ / (Lys-GA) １４] Solution, ZrCl ４ The solutions were mixed by vortexing. １０ｋ -TA and ZrCl ４ , PEG-P[Lys １１ / (Lys-GA) １４ ]) and ZrCl ４ The molar ratios were 1:0, 1:0.45, and 1:0.9, respectively. Solutions of TUG1 alone were also prepared, and D-PBS(-) was added to each of these solutions to adjust the concentration, and the particle diameters were measured using an LSM710. Figures 70 and 71 show the results of particle diameter measurements.

[0296] Each TUG1 / PEG １０ｋ -TA / Zr ４＋ Conjugate and TUG1 / PEG-P[Lys １１ / (Lys-GA) １４ ] / Zr ４＋ The particle size of each complex was significantly increased compared to that of TUG1. １０ｋ -TA / Zr ４＋ Conjugates and TUG1 / PEG-P[Lys １１ / (Lys-GA) １４ ] / Zr ４＋ The formation of a complex was suggested. １０ｋ The particle size of the TUG1 / PEG-P[Lys １１ / (Lys-GA) １４ ] complex, the particle size was significantly increased. １１ / (Lys-GA) １４ It was suggested that only the complex was formed. ４ As the amount of HCl increased, the particle size increased.

[0297] Experimental Example 7: Evaluation of physicochemical properties of MPN complexes loaded with nucleic acid complexes (RNPs) (17.1. Overview) The formation of MPN complexes loaded with Cas9-sgRNA RNPs was confirmed using newly synthesized PEG-ethylenediamine-TA. Specifically, the increase in particle size was measured using fluorescence spectroscopic correlation spectroscopy.

[0298] (17.2. Reagents) Cas9-GFP Protein (Sigma-Aldrich) sgRNA (Luciferase-2) (GenScript) D-PBS (-) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ -TET-TA (Mn=11,847) ・PEG １０ｋ -TEP-TA (Mn=11,890) ・PEG １０ｋ -S-TA (Mn=11,903) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0299] (17.3. Measuring equipment) LSM710 (Carl Zeiss)

[0300] (17.4. Evaluation of RNP-MPN Complex Formation) Cas9-sgRNA, Polymer, FeCl ３ ・6H ２ Final concentration of O》 ・Cas9-GFP: 0.2 μM ・sgRNA: 0.2 μM ・Polymer (PEG-TET-TA, PEG-TEP-TA, PEG-S-TA): 20 μM ・FeCl ３ ・6H ２ O(Fe ３＋ ): 67 μM

[0301] Cas9-sgRNA and polymer were dissolved in D-PBS(-). ３ ・6H ２ O was prepared by dissolving it in pure water.

[0302] Cas9-GFP and sgRNA were mixed and centrifuged twice for 5 minutes at 12,000 × g using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). Then, polymers (PEG-TET-TA, PEG-TEP-TA, PEG-S-TA) were added and the mixture was left at room temperature for 15 minutes to prepare both RNP / polymer solutions. Furthermore, Fe ３＋ After adding the above and leaving it at room temperature for 15 minutes, both RNP / polymer / Fe ３＋An MPN complex solution was prepared. The polymer / RNP molar ratio was 100. The molar ratio of iron ions was 3.3 equivalents relative to the polymer-containing TA. Finally, particle size was measured by fluorescence spectroscopic correlation spectroscopy using an LSM710. Figures 72 and 73 show the particle size measurement results.

[0303] (17.5. Analysis) From Figures 72 and 73, both RNP / polymer / Fe ３＋ The particle size of RNP / polymer / Fe was significantly increased compared to that of RNP and RNP / PEG-TA. ３＋ The formation of an MPN complex was suggested.

[0304] [Experimental Example 18] <Functional Evaluation of Antibody-Loaded MPN Complex> (18.1. Overview) The formation and functionality of the antibody-MPN complex were further confirmed. Specifically, electron microscopic image observation, buffering effect, and therapeutic effect and mechanism in an orthotopic breast cancer transplant model were confirmed.

[0305] (18.2. Reagents) Anti-S100A4 antibody (Creative Biolabs) Anti-NPC antibody (Biolegend) PEG-TA TA: Wako Pure Chemical Industries p53 (D2H9O) Rabbit mAb #32532 (Anti-p53 antibody, Cell Signaling) HRP-modified Anti-Rabbit mAb (Cell Signaling) TUNEL Assay Kit-HRP-DAB (ab206386, Abcam) Fuji DryChem Slide (GOT, GPT) (Fujifilm Wako Pure Chemical) Roswell Park Memorial Institute medium (RPMI, Sigma Aldrich Co., LLC.), fetal bovine serum (FBS, Biosera Inc.), trypsin-EDTA solution (Trp, Sigma Life Science Co., Ltd.), penicillin / streptomycin (PS, Sigma Life Science Co., Ltd.), PBST (Fujifilm Wako Pure Chemical Industries, Ltd.), TMB solution (Fujifilm Wako Pure Chemical Industries, Ltd.), Ab206386-TUNEL Assay Kit-HRP-DAB (Abcam), 5M hydrochloric acid (Nacalai Tesque)

[0306] (18.3. Measuring equipment) ・JEM-1400 (JEOL) ・LSM710 (Carl Zeiss Co., Ltd.) ・Fuji DryChem NX500 (Fujifilm Wako Pure Chemical Industries) ・pH meter (Horiba) ・All-in-one fluorescence microscope BZ-X800 (Keyence) ・Cryostat (Leica)

[0307] (18.4. Electron microscope image observation of antibody-loaded MPN complex) <<Antibody, PEG-TA, Fe ３＋ Final concentrations of: Anti-NPC antibody: 660 nM PEG-TA: 16 μM (prepared concentration: 80 μM) Fe ３＋ : 55μM (prepared concentration: 2.2mM)

[0308] Each was dissolved in 1 mM HEPES buffer (pH 7.4). ３＋ was prepared by dissolving in ultrapure water.

[0309] Antibody solution, both PEG-TA solutions and TA solution, Fe ３＋ The solution was mixed by pipetting. The molar ratio of PEG-TA / antibody and TA / antibody was 25. The molar ratio of iron ions to TA in PEG-TA was 3.3 equivalents. 5 μL of the sample solution was then dropped onto the copper grid, and the solution was removed after 1 minute. The grid was then stained with gadolinium acetate solution, washed with ultrapure water, and then air-dried for 1 day. TEM images of the sample-coated microgrid were taken using a JEM1400. Figure 74 shows a TEM image of the antibody sample. The scale bar indicates 100 nm.

[0310] Antibody / PEG １０ｋ -TA / Fe ３＋ MPN conjugate or antibody / PEG ５ｋ -TA / Fe ３＋ TEM images of the MPN complex showed spherical particles of approximately 30 nm. ３＋ TEM images of the MPN complexes showed aggregated particles. １０ｋ -TA / Fe ３＋ MPN complex, antibody / PEG ５ｋ -TA / Fe ３＋ The formation of the MPN complex was confirmed.

[0311] (18.5. Measurement of buffer effect of antibody-loaded MPN complex) <<Antibody, PEG １０ｋ -TA, TA, Fe ３＋ Final concentration of Anti-NPC antibody: 660 nM PEG １０ｋ -TA: 16 μM (prepared concentration: 80 μM) ・TA: 16 μM (prepared concentration: 80 μM) ・Fe ３＋ : 55μM (prepared concentration: 2.2mM)

[0312] Each was prepared by dissolving in D-PBS(-) (pH 7.4). ３＋ was prepared by dissolving in ultrapure water.

[0313] Antibody solution, PEG １０ｋ -TA solution, Fe ３＋ solution, antibody solution, TA solution, Fe３＋ The solution was mixed by pipetting. １０ｋ The molar ratios of TA / antibody and TA / antibody were 25. The molar ratio of iron ions was 3.3 equivalents relative to the TA in PEG-TA. 5 μL of 0.1 N hydrochloric acid was added dropwise to the sample solution, and the pH change was measured. The measurement was stopped when the pH became 3 or less and stabilized. The results are shown in Figure 75.

[0314] Antibody / TA / Fe ３＋ MPN conjugates and antibody / PEG １０ｋ -TA / Fe ３＋ It was confirmed that the MPN complex suppressed the decrease in pH when HCl was added. １０ｋ -TA / Fe ３＋ It was shown that the MPN complex can significantly suppress the decrease in pH.

[0315] (18.6. Evaluation of blood stability of antibody-loaded MPN complex) {Antibody, PEG-TA, TA, Fe ３＋ Final concentration of Anti-NPC antibody: 3.3 μM PEG １０ｋ -TA: 82.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 272μM (prepared concentration: 20mM) ・TA: 82.5μM (prepared concentration: 200μM)

[0316] Antibodies and PEG １０ｋ -TA, TA was prepared by dissolving in PBS(-), and Fe ３＋ was prepared by dissolving in ultrapure water.

[0317] <<Evaluation of Stability in Blood>> Balb / C mice were administered 200 μL of the above prepared solution via the tail vein. Two and 48 hours after sample administration, the mice were dissected and blood was collected. The blood was centrifuged at 5,000 × g for 10 minutes at 20°C, diluted 10-fold with D-PBS(-), and the particle size of the sample was measured using FCS. The results are shown in Figure 76.

[0318] Antibody / TA / Fe ３＋ When the MPN complex was administered into the blood, the particle size increased by more than 200%. ３＋This was thought to be due to the interaction of the MPN complex with blood components. ３＋ No change in particle size of the MPN complex was observed, demonstrating that this MPN complex is stable even in the fluid environment of blood.

[0319] (18.7. Toxicity Evaluation of Antibody-Loaded MPN Complexes) <<Antibody, PEG １０ｋ -TA, TA, Fe ３＋ Final concentration of Anti-S100A4 antibody: 3.3 μM PEG １０ｋ -TA: 82.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 272μM (prepared concentration: 20mM) ・TA: 82.5μM (prepared concentration: 200μM)

[0320] Antibodies and PEG １０ｋ -TA, TA was prepared by dissolving in PBS(-), and Fe ３＋ was prepared by dissolving in ultrapure water.

[0321] Toxicity Evaluation: Model mice were administered 200 μL of the above-mentioned prepared solution into the tail vein three times every four days. Four days after the third administration, the mice were dissected and blood was collected. The blood was centrifuged at 5,000 × g for 10 minutes at 20°C to collect serum. 10 μL of the serum was dropped onto a Fuji DriChem slide (GOT, GPT) corresponding to each blood marker and measured using the DriChem slide. The results are shown in Figure 77.

[0322] Antibody / TA / Fe ３＋ Administration of the MPN complex resulted in increased GOT and GPT levels and liver toxicity. ３＋ Administration of the MPN complex did not result in an increase in GOT or GPT. ３＋ The MPN complex was confirmed to be non-toxic.

[0323] (18.8 Therapeutic Effect of Therapeutic Intracellular Antibody-Loaded MPN Complexes on Orthotopic Breast Cancer Models) <<Antibody, PEG-TA, TA, Fe ３＋ Final concentration of Anti-S100A4 antibody: 3.3 μM PEG １０ｋ-TA: 82.5 μM (prepared concentration: 200 μM) ・PEG ５ｋ -TA: 82.5 μM (prepared concentration: 200 μM) ・Fe ３＋ : 272μM (prepared concentration: 20mM) ・TA: 82.5μM (prepared concentration: 200μM)

[0324] The antibody, PEG-TA, and TA were dissolved in PBS(-) and prepared. ３＋ was prepared by dissolving in ultrapure water.

[0325] Antibody solution, PEG-TA solution, Fe ３＋ solution, antibody solution, TA solution, Fe ３＋ The solution was mixed by pipetting. １０ｋ The molar ratio of TA / antibody and TA / antibody was 25. The molar ratio of iron ions was 3.3 equivalents relative to the TA of PEG-TA.

[0326] <<Preparation of a 4T1-Luc Subcutaneous Tumor Mouse Model>> 4T1-Luc cell suspension (5.0 × 10 ７ 50 μL of the solution (cells / mL) was injected into the mammary gland of BALB / c mice.

[0327] <Evaluation of antitumor effect> The tumor size of the model mouse was approximately 20 mm ３ The time when the tumor size reached 100 μL was defined as day 0, and on days 0, 4, and 8, 100 μL of the above sample solution (anti-S100A4 antibody: 50 μg / mouse) was administered to the tail vein. For comparison, a group administered with D-PBS(-) was also prepared. Tumor size was measured with a caliper every two days until day 16 to evaluate tumor size. At the same time, the mice were weighed and adverse events were also confirmed. Figure 78 shows the change in tumor size over time, and Figure 79 shows the change in body weight over time. Tumor size was calculated using the following formula (F1): V=(W ２ × L) / 2 (V: volume, W: minor axis, L: major axis) ... (F1)

[0328] As shown in Figure 78, anti-S100A4 antibody / PEG-TA / Fe ３＋ The tumor size in the MPN complex administration group was shown to be significantly suppressed compared to the tumor sizes in the other sample and PBS(-) administration groups. Furthermore, as shown in Figure 79, all sample administration groups showed similar changes in body weight.

[0329] (18.9. Confirmation of the mechanism of anticancer activity by immunostaining) <Immunostaining observation> Paraffin blocks were prepared from the tumor prepared in 18.8 above. 10 μm-thick sections were prepared from the paraffin blocks using a cryostat and deparaffinized using xylene and alcohol. Endogenous peroxidase was removed by incubation in 30% hydrogen peroxide for 10 minutes. Subsequently, the blocks were washed with PBST solution and 10% normal goat serum solution, and then incubated overnight at 4°C with anti-p53 antibody diluted 50-fold with PBST. Subsequently, the blocks were washed with PBST and incubated at room temperature for 30 minutes with HRP-labeled anti-rabbit antibody diluted 1,000-fold with PBST. Finally, HRP was developed with TMB, and the tissue was stained with hematoxylin and observed. The resulting images are shown in Figure 80.

[0330] Anti-S100A4 antibody / PEG-TA / Fe ３＋ In the images of the tumor samples administered with the MPN complex, brown spots derived from p53 protein were observed, confirming that the expression of p53 protein had increased.

[0331] (18.10. Confirmation of the mechanism of anticancer action by TUNEL assay) <<TUNEL assay>> Paraffin blocks were prepared from the tumors prepared in 18.8 above. 10 μm-thick sections of the paraffin blocks were prepared using a cryostat and deparaffinized using xylene and alcohol. Then, according to the TUNEL assay kit protocol, the section samples were treated with proteinase K, inactivated endogenous peroxidase, subjected to terminal deoxynucleotidyl transferase reaction, labeling reaction, DAB staining, and counterstaining. Then, the sections were observed using an all-in-one fluorescence microscope. The results are shown in Figure 81.

[0332] Anti-S100A4 antibody / PEG-TA / Fe ３＋ In the image of the tumor sample administered with the MPN complex, brown spots derived from apoptotic cells were observed. ３＋It was confirmed that the MPN complex induced apoptosis in cancer cells.

[0333] [Experimental Example 19] <Evaluation of blood retention of GalNac-modified β-Gal-loaded MPN complex> (19.1. Overview) β-Gal ternary complex (β-Gal / PEG-TA / Fe ３＋ ) was evaluated for blood retention.

[0334] (19.2. Reagents) Unless otherwise specified, commercially available reagents were used as is. β-D-galactosidase (β-Gal, Mw: 540 kDa, Fujifilm Wako Pure Chemical Industries, Ltd.) PEG-TA (Mn=11700) Alexa Fluor 647-β-Gal D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) BALB / c mice (Charles River Japan Inc.) Passive Lysis Buffer (Promega)

[0335] (9.3. Measuring equipment) Multiplate reader (Spark, Tecan Group Ltd.)

[0336] (9.4. Evaluation of Pharmacokinetics) Alexa Fluor 647-β-Gal solution, PEG １０ｋ -TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of polymer / β-Gal was set to 250. The molar ratio of iron ions was set to 3.3 equivalents relative to the TA of the polymer. Finally, PBS(-) was added to adjust the concentration.

[0337] 200 μL of the above-mentioned prepared solution was administered to the model mice via the tail vein. Blood was collected 1 hour and 2 hours after sample administration and centrifuged at 5,000 × g for 10 minutes at 20°C. 100 μL of plasma components were collected, and 200 μL of passive lysis buffer was added. The fluorescence intensity (Ex / Em: 640 nm / 680 nm) of the solution was then measured using a TECAN to evaluate blood retention. The results are shown in Figure 82.

[0338] The blood retention of the Alexa647-β-Gal-loaded MPN complex was improved by 4 times after 1 hour and 6 times after 2 hours compared to Alexa647-β-Gal.

[0339] [Experimental Example 20] <Evaluation of target function of GalNac-modified β-Gal-loaded MPN complex> (20.1. Overview) Functionality of the β-Gal ternary complex was evaluated. Specifically, the cellular uptake of the GalNac-loaded MPN complex was measured in the presence and absence of an inhibitor.

[0340] (10.2. Reagents) Unless otherwise specified, commercially available reagents were used as is. Alexa Fluor 647-β-Gal, PEG-TA (Mn=11,700), tannic acid (Mw: 1,701) (Fujifilm Wako Pure Chemical Industries, Ltd.), D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.), Dulbecco's modified Eagle's medium (DMEM, Sigma Aldrich Co., LLC), fetal bovine serum (FBS, Biosera Inc.), trypsin-EDTA solution (Trp, Sigma life science Co., Ltd.). Penicillin / streptomycin (PS, Sigma Life Science Co., Ltd.) Passive Lysis Buffer (Promega) N-acetylgalactosamine (GalNac, Fujifilm Wako Pure Chemical Industries) Human liver cancer cells (HepG2 cells, cells overexpressing ASGPR (asialoglycoprotein receptor)) (American Type Culture Collection, ATCC)

[0341] (10.3. Measuring equipment) Flow cytometer (Guava easyCyte 6-2L, Merck Millipore, Ex / Em: 642 / 661 nm)

[0342] (10.4. Cellular uptake efficiency of β-Gal-loaded MPN complex) Alexa647-β-Gal, PEG-TA, GalNac-PEG-TA, Fe ３＋ , Final concentration of GalNac》 ・Alexa647-β-Gal (β-Gal): 460 nM ・PEG-TA: 115 μM ・GalNac-PEG-TA: 115 μM ・Fe ３＋: 380μM (prepared concentration: 6.1mM) ・GalNac: 10mM (prepared concentration: 20mM)

[0343] Alexa647-β-Gal, PEG-TA, GalNac-PEG-TA, and GalNac were dissolved in PBS(-) and prepared. ３＋ was prepared by dissolving in ultrapure water.

[0344] Alexa647-β-Gal solution, PEG-TA solution or GalNac-PEG-TA solution, Fe ３＋ The solution was mixed by pipetting. At this time, the molar ratio of polymer / β-Gal was set to 250. The molar ratio of iron ions was set to 3.3 equivalents relative to the TA of the polymer. Finally, PBS(-) was added to adjust the concentration.

[0345] [Evaluation of the amount of β-Gal ternary complex uptake in cells] FBS and PS were added to DMEM at 10 wt % and 2 wt %, respectively, to prepare a cell culture medium. HepG2 cells were suspended in the cell culture medium, and 1.25 × 10 ５ A cell suspension was prepared at a concentration of 5.0 × 10 cells / mL. 400 μL of this cell suspension was seeded into a 24-well plate (5.0 × 10 cells / well). ４ The cells were incubated at 37°C for 24 hours. After removing the medium and washing once with PBS, 200 μL of each prepared Alexa647-β-Gal-loaded MPN complex solution, 200 μL of the inhibitor GalNAc solution, and PBS solution were added, followed by incubation at 37°C for 24 hours. After incubation for the specified time, the solution was removed, the cells were washed twice with PBS, and 150 μL of Trp was added and incubated at 37°C for 7 minutes. Next, 150 μL of 10% FBS-PBS was added, and the Alexa647-derived fluorescence (Ex / Em: 642 / 664 nm) was measured using a flow cytometer (FCM) to evaluate the cellular uptake of each sample. The amount of Alexa647-β-Gal uptake without the addition of inhibitors was set to 100%. The results are shown in Figure 83.

[0346] As a result, Alexa647-β-Gal / PEG-TA / Fe ３＋The amount of Alexa647-β-Gal / GalNac-PEG-TA / Fe uptake into cells was not reduced even in the presence of GalNac (inhibitor). ３＋ The amount of uptake into cells was reduced in the presence of GalNac (an inhibitor).

[0347] [Experimental Example 20] <RNP / PEG-TA / Fe ３＋ Pharmacokinetic evaluation of the complex > (21.1. Overview) RNP / PEG-TA / Fe in a tumor mouse model subcutaneously implanted with 4T1-Luc (mouse colon cancer cells) ３＋ The pharmacokinetics of the complex was evaluated.

[0348] (21.2. Reagents, Cells, and Animals) Label IT Nucleic Acid Labeling Kit, Cy5 (Milas) Cas9 Nuclease protein NLS (Cas9) (Nippon Gene) sgRNA (Luciferase-2) (GenScript) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ -TA (Mn=11,701) ・PEG ５ｋ -TA (Mn=6,701) 4T1-Luc cells (a mouse breast cancer cell line stably expressing luciferase) (American Type Culture Collection, ATCC) BALB / c mice (Charles River)

[0349] (21.3. Equipment and facilities) Countess (Thermo Fisher Scientific) Multi-beads shocker (Yasui Kikai) Multi-plate reader (Spark) (Tecan)

[0350] (21.4. RNP / PEG-TA / Fe ３＋ Pharmacokinetics of the complex) RNP / PEG-TA / Fe ３＋The blood retention and organ accumulation of the MPN complex were evaluated. Cy5-labeled RNP samples were prepared and intravenously injected into a 4T1-Luc subcutaneous tumor mouse model, and the content in the blood and each organ was measured by fluorescence intensity measurement after a certain period of time. In this experiment, RNP / PEG-TA / Fe ３＋ RNP alone was used as a comparison for the MPN complex.

[0351] <<Preparation of Cy5-Labeled RNP>> Labeling IT Reagent and RNA were mixed at a ratio of Labeling IT Reagent:RNA = 0.25:1 (v:w) and incubated at 37 ° C. for 1 hour. Then, purification was performed using a G50 column to prepare Cy5-labeled sgRNA.

[0352] Cas9 and Cy5-labeled sgRNA were mixed and centrifuged twice at 12,000 × g for 5 minutes using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution).

[0353] <<Final concentrations of RNP and PEG-TA>> Cas9 Nuclease protein NLS: 1.0 μM sgRNA: 1.0 μM PEG １０ｋ -TA (Mn=11,701): 100μM PEG ５ｋ -TA (Mn=6,701): 100μM ・Fe ３＋ : 333 μM

[0354] Cas9-sgRNA and PEG-TA were dissolved in D-PBS(-). ３＋ was prepared by dissolving in pure water.

[0355] PEG-TA was added to the RNP and allowed to stand at room temperature for 15 minutes. ３＋ was added, and the mixture was left standing at room temperature for 15 minutes. ３＋ An MPN complex solution was prepared with a PEG-TA / RNP molar ratio of 100. The molar ratio of iron ions relative to the TA in PEG-TA was 3.3 equivalents.

[0356] <<Preparation of a 4T1-Luc Subcutaneous Tumor Mouse Model>> 4T1-Luc cell suspension (5.0 × 10 ７ 100 μL of the solution (cells / mL) was subcutaneously injected into BALB / c mice.

[0357] Evaluation of Pharmacokinetics: 100 μL of the prepared sample solution was administered via the tail vein to 4T1-Luc tumor-bearing model mice in which tumors were visually visible. Six hours after sample administration, the mice were dissected, and blood and various organs were collected. Three times the weight of passive lysis buffer was added, followed by homogenization using a multi-bead shocker. Blood was also collected from the tail vein 2 and 24 hours later. Subsequently, the mixture was centrifuged at 10,000 × g for 5 minutes at 4°C, and 50 μL of the supernatant was transferred to a 96-well plate. The fluorescence intensity of RNP was measured using a multiplate reader to evaluate pharmacokinetics. Figure 84 is a graph showing the results of organ accumulation evaluation. Figure 85 is a graph showing the results of tumor accumulation evaluation. Figure 86 is a graph showing the results of blood retention evaluation.

[0358] (11.5. Analysis) RNP / PEG-TA / Fe ３＋ The pharmacokinetics of the MPN complex showed improved blood retention, organ accumulation, and tumor accumulation compared to RNP alone.

[0359] Experimental Example 22: Functional evaluation of RNP-loaded MPN complexes in cultured cells (22.1. Overview) MPN complexes using Luc gene-editing RNPs were introduced into HeLa-Luc cells, and toxicity was evaluated using a CCK assay. Furthermore, knockout efficiency was measured based on luciferase luminescence intensity.

[0360] (22.2. Reagents and Cell Lines) Cas9 Nuclease Protein NLS (Cas9) (Nippon Gene) Luciferase knockout gene knockout sgRNA (sgRNA) (GenScript) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ -TA (Mn=11,701) ・PEG１０ｋ -TET-TA (Mn=11,847) ・PEG １０ｋ -TEP-TA (Mn=11,890) ・PEG １０ｋ -S-TA (Mn=11,903) Luciferase Assay System (Promega) Cell Counting Kit-8 (Dojindo Laboratories) Roswell Park Memorial Institute medium (RPMI, Sigma-Aldrich) Fetal bovine serum (FBS, Biosera) Trypsin-EDTA solution (Sigma-Aldrich) Penicillin / streptomycin (Sigma-Aldrich) HeLa-Luc cells (aneuploid epithelial-like cell line stably expressing luciferase) (American Type Culture Collection, ATCC) ProDeliverIn (OZ Biosciences)

[0361] (12.2. Measuring equipment) Glomax 96 microplate luminometer (Promega) Multiplate reader (Spark) (Tecan)

[0362] (22.3. Knockout Efficiency Evaluation) <RNP, Polymer, FeCl ３ ・6H ２ Final concentration of O» Cas9: 90 nM sgRNA: 90 nM Polymer (PEG １０ｋ -TA, PEG １０ｋ -TET-TA, PEG １０ｋ -TEP-TA, PEG １０ｋ -Spermine-TA): 9μM ・Fe ３＋ : 30 μM

[0363] Cas9-sgRNA and polymer were dissolved in D-PBS(-). ３＋ was prepared by dissolving in pure water.

[0364] Cas9 and sgRNA were mixed and centrifuged twice for 5 minutes at 12,000 × g using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-sgRNA solution (RNP solution). Polymer was then added and the mixture was left to stand at room temperature for 15 minutes to prepare an RNP / polymer solution.３＋ After adding the above, the mixture was left standing at room temperature for 15 minutes, and then the RNP / polymer / Fe ３＋ An MPN complex solution was prepared. As a positive control, ProDeliverIn, a cationic reagent, was mixed at a ratio of ProDeliverIn / RNP = 1 μg / 1 μL to prepare an RNP / ProDeliverIn solution.

[0365] <<Measurement of cell viability by CCK assay>> 2.0 × 10 HeLa-Luc cells were cultured in a 96-well plate. ３ The cells were seeded at 100 cells / plate and incubated at 37°C in 5% CO ２ The RNP solution, RNP / PEG, and １０ｋ -TA / Fe ３＋ MPN complex solution, RNP / PEG-TET-TA / Fe ３＋ MPN complex solution, RNP / PEG-TEP-TA / Fe ３＋ MPN complex solution, RNP / PEG-S-TA / Fe ３＋ 100 μL / well of the MPN complex solution and RNP / ProDeliverIn solution were added to each well and incubated for 72 hours. After washing with 150 μL / well of D-PBS(-), 110 μL / well of a CCK measurement solution prepared by mixing 10 μL of CCK and 100 μL of PBS(-) was added and incubated for 1 hour. The absorbance at 460 nm was then measured using a plate reader to confirm cell viability.

[0366] <<Evaluation of Knockout Efficiency>> After confirming cell viability, the medium was collected and washed with D-PBS(-). 50 μL of Cell Culture Lysis was added to each well and incubated for 15 minutes. Then, 20 μL of the lysate was transferred to a fluorescence measurement dish, luciferin was added, and the luciferase-induced luminescence intensity was measured using a plate reader. The luciferase gene knockout efficiency was evaluated by normalizing the measured values ​​by cell viability. Figure 87 is a graph showing the results of measuring luciferase-induced luminescence intensity.

[0367] (22.4. Analysis) RNP / Polymer / Fe ３＋In cells treated with the MPN complex solution, a significant decrease in luciferase luminescence intensity was observed compared to untreated cells or cells treated with RNP alone. Furthermore, conjugating ethylenediamine molecules to PEG-TA improved the gene knockout effect.

[0368] [Experimental Example 23] <Pharmacokinetics of RNP-loaded MPN complex formation and in vivo genome editing evaluation> (23.1. Overview) RNP ternary complex (RNP / PEG-TA / Fe ３＋ Specifically, the RNP sample was administered to a genetically engineered mouse (Ai9 td Tomato mouse) that emits red fluorescence upon genome editing, and the fluorescence intensity of each organ was measured.

[0369] (23.2. Reagents) Cas9 Nuclease Protein NLS (Cas9, Nippon Gene) sgRNA (Ai9_L) (GenScript Japan) sgRNA (Ai9_R) (GenScript Japan) Iron (III) chloride hexahydrate (FeCl ３ ・6H ２ O)(Fe ３＋ ) (Fujifilm Wako Pure Chemical Industries) PEG １０ｋ -TA (Mn=11,701) ・PEG １０ｋ -S-TA (Mn=11,903) ・GalNac-PEG-TA (M ｎ = 12,400) D-PBS (-) (Fujifilm Wako Pure Chemical Industries) LipidLaunch ＴＭ SORT LNP Exploration Kit (Cayman Chemical) and Ai9 td-Tomato mice (Jackson Laboratory).

[0370] (23.3. Measuring equipment) Multiplate reader (Spark) (Tecan) In vivo imaging system (PerkinElmer)

[0371] (23.4. Genome Editing Efficiency Measurement) To evaluate the gene knockout efficiency of the RNP MPN complex, we used RNP / polymer / Fe ３＋The MPN complex was intravenously injected into a 4T1-Luc subcutaneous tumor mouse model, and the luciferase luminescence intensity of the tumor was measured after a certain period of time. ３＋ RNP alone and LNP-encapsulated RNP were used as comparison subjects for the MPN complex.

[0372] Cas9-sgRNA, polymer, Fe ３＋ Final concentrations of Cas9: 2 μM sgRNA: 2 μM (Ai9_L 1 μM + Ai9_R 1 μM) Polymer (PEG １０ｋ -TA, PEG-S-TA): 200 μM ・Fe ３＋ : 660 μM Cas9-sgRNA, polymer was dissolved in D-PBS(-). ３＋ was prepared by dissolving in pure water.

[0373] Cas9 and sgRNA were mixed and centrifuged twice for 5 minutes at 12,000 × g using an ultrafiltration membrane (MWCO: 10 kDa) to prepare a Cas9-gRNA solution (RNP solution). Then, the respective polymer solutions were mixed and left to stand at room temperature for 15 minutes to prepare an RNP / polymer solution. Furthermore, Fe ３＋ was added, and the mixture was allowed to stand at room temperature for 15 minutes. ３＋ An MPN complex solution was prepared.

[0374] <<Final concentrations of Cas9-sgRNA and LNP>> Cas9: 288 nM sgRNA: 288 nM LNP: 800 μM Cas9-sgRNA was prepared by dissolving it in D-PBS(-).

[0375] Follow the instructions in the kit to use LipidLaunch. ＴＭ Each solution in the SORT LNP Exploration Kit was dispensed into a 5 mL tube to a total volume of 1 mL. Then, the mixture was mixed with 20 mM MES buffer (pH 6.0) 50 mM NaCl at 0.09 mL / min, RNP at 0.09 mL / min, and LNP at 0.02 mL / min. After that, the mixture was centrifuged twice at 3,500 × g for 15 minutes. After removing the EtOH, the mixture was centrifuged five times at 3,500 × g for 15 minutes to concentrate the LNP to the desired amount.

[0376] Evaluation of gene editing efficiency and hematotoxicity: Ai9 td-Tomato mice were administered 200 μL of the above-mentioned prepared solution and D-PBS(-) via the tail vein. Eight days after sample administration, various organs were collected and measured by in vivo fluorescence imaging (IVIS). Genome editing efficiencies in the liver, muscle, and brain, calculated based on fluorescence intensity, are shown in Figures 88, 89, and 90, respectively.

[0377] By targeting liver parenchymal cells, the GalNac-loaded RNP MPN complex demonstrated higher genome editing efficiency in the liver compared to the RNP MPN complex, LNP, and RNP alone. Furthermore, the RNP MPN complex prepared using PEG-S-TA demonstrated higher genome editing efficiency in muscle and brain compared to the RNP MPN complex, LNP, and RNP alone. Furthermore, the RNP MPN complex demonstrated higher genome editing efficiency than RNP alone in various organs. These results suggest that the RNP MPN complex may be a useful tool for genome editing in various organs in vivo.

[0378] [Summary] This drug delivery system, composed of polyphenol-modified polymers and metal ions, was able to improve the blood retention, blood stability, and cytoplasmic transport of physiologically active proteins. Furthermore, it was confirmed that this drug delivery system can encapsulate not only proteins but also any molecule to form MPN complexes. Furthermore, it was shown that use of this drug delivery system can improve the in vivo pharmacokinetics and therapeutic efficacy of any drug. Furthermore, it was shown that the activity of any drug can be improved by introducing tissue-targeting molecules or cationic molecules into the polyphenol-modified polymer.

[0379] The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited to each embodiment, but is limited only by the scope of the claims.

[0380] According to the present invention, a technique for efficiently delivering a biomolecule into the cytoplasm can be provided.

Claims

1. Complexes of biocompatible polymer-modified polyphenols, metal ions, and biomolecules.

2. The complex according to claim 1, wherein the polyphenol is at least one selected from the group consisting of tannic acid, gallic acid, and derivatives thereof.

3. The composite of claim 1 or 2, wherein the biocompatible polymer comprises at least one selected from the group consisting of polyethylene glycol, acrylic resin, polyamino acid, polyvinylamine, polyallylamine, polynucleotide, polyacrylamide, polyether, polyester, polyurethane, polysaccharide, and copolymers thereof.

4. The composite according to claim 1 or 2, wherein the weight-average molecular weight of the biocompatible polymer is 1,000 to 50,000.

5. The complex according to claim 1 or 2, wherein the metal ion is an iron (III) ion, a cobalt (II) ion, or a zirconium (IV) ion.

6. The conjugate according to claim 1 or 2, wherein the biomolecule is an antibody or an antigen-binding fragment thereof, a physiologically active protein, a nucleic acid, a lipid nanoparticle, or an inorganic / organic microparticle.

7. Biocompatible polymer-modified polyphenols.

8. A kit for delivering a biomolecule to the cytoplasm by forming a complex with the biomolecule and administering the complex to a subject, the kit comprising a biocompatible polymer-modified polyphenol and a metal ion.

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