Surface-modified nano-diamond

Abstract

Provided is a surface-modified nanodiamond which is capable of selectively accumulating a sufficient amount of boron atoms in cancer tissues and cancer cells, and which has high killing properties against cancer cells. The surface-modified nanodiamond according to the present disclosure has nanodiamond particles, and a group containing a boron cluster and a group having a cancer cell recognition ability as surface-modifying groups for the nanodiamond particles.

Description

Technical Field

[0001] The present disclosure relates to a surface-modified nanodiamond. The present disclosure claims priority to Japanese Patent Application No. 2022-083332 filed in Japan on May 20, 2022, the contents of which are incorporated herein by reference. Background Art

[0002] Boron neutron capture therapy (BNCT) is a method of treating neutrons by 10 B) alpha rays released by nuclear reactions with neutrons ( 4 He nuclei) and lithium nuclei ( 7 Li nuclei) to destroy (kill) cancer cells. The alpha rays and 7 The movable distance of Li nuclei is about several micrometers, which is almost the same as the size of cells. Therefore, as long as sufficient amounts of boron atoms (boron compounds) can be selectively accumulated in cancer tissues and cancer cells, cancer cells can be selectively killed.

[0003] Currently, para-borylphenylalanine (BPA) is used clinically as a boron agent for BNCT (Patent Document 1). It is known that BPA enters cancer cells via the L-amino acid transporter (LAT-1) expressed on the surface of cancer cells, but since the cell membrane permeation (intracellular entry and exit) of BPA based on LAT-1 is reversible and it is a low molecular compound and metabolized (excreted) rapidly, in order to maintain the BPA concentration in cancer cells during neutron irradiation, it is necessary to increase the BPA concentration outside the cells. That is, in BNCT, high concentrations of BPA need to be continuously administered.

[0004] In order to overcome the above-mentioned problems of BPA, drugs (nanomedicines) that can selectively transport boron atoms into cancer cells and maintain the boron atoms in a nanoparticle matrix have been studied, and various boron-containing nanoparticles have been studied so far (Non-Patent Document 1).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2009-51766

[0008] Non-patent literature

[0009] Non-patent literature 1: M. Nishikawa et al., Bull. Chem. Soc. Jpn. 2021, 94, 2302-2312. Summary of the invention

[0010] Problems to be solved by the invention

[0011] However, the drug described in Non-Patent Document 1 shows a certain degree of remarkable effect as a drug in boron neutron capture therapy (BNCT), but a higher effect is required from the viewpoint of the killing activity against cancer cells.

[0012] Therefore, an object of the present invention is to provide a surface-modified nanodiamond that can selectively and sufficiently accumulate boron atoms in cancer tissues and cancer cells and has high toxicity to cancer cells.

[0013] Solutions for solving problems

[0014] The inventors of the present disclosure have conducted in-depth research to achieve the above-mentioned purpose, and found that the following surface-modified nanodiamond can solve the above-mentioned problem, wherein the surface-modified nanodiamond has: nanodiamond particles and a group containing boron clusters and a group having cancer cell recognition ability as a surface modification group of the nanodiamond particles. The invention of the present disclosure is completed based on these insights.

[0015] That is, the present disclosure relates to a surface-modified nanodiamond having: a nanodiamond particle; and a group including a boron cluster and a group having a cancer cell recognition ability as a surface modification group of the nanodiamond particle.

[0016] The surface-modified nanoparticles of the present disclosure preferably further have a group containing a hydrophilic polymer chain as a surface-modifying group.

[0017] The hydrophilic polymer chain in the group comprising a hydrophilic polymer chain is preferably a polyglycerol chain.

[0018] The groups comprising boron clusters are preferably bonded to the nanodiamond particles via hydrophilic polymer chains.

[0019] The group having cancer cell recognition ability is preferably bonded to the nanodiamond particle via a hydrophilic polymer chain.

[0020] Preferably, the group having cancer cell recognition ability is bonded to a hydrophilic polymer chain via an amide bond, an ester bond or a thioester bond, and the hydrophilic polymer chain is bonded to the nano-diamond particle.

[0021] It is preferred that the group having a cancer cell recognition ability is a group derived from a molecule having a cancer cell recognition ability, and the molecule having a cancer cell recognition ability is boronic acid, amino acid, peptide, protein, folic acid or porphyrin.

[0022] The boron atoms in the group containing boron clusters are preferably condensed to 10 B.

[0023] It should be noted that the present disclosure also describes a composition for boron neutron capture therapy comprising the surface-modified nanodiamond.

[0024] Effects of the Invention

[0025] The surface-modified nanodiamond disclosed in the present invention can selectively and sufficiently accumulate boron atoms in cancer tissues and cancer cells, and has a high degree of lethality to cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It represents the DND-PG of Example 1 13 C-NMR measurement results.

[0027] Figure 2 is the DND-PG-COOH of Example 1 1 A graph showing the results of H-NMR measurement.

[0028] Figure 3 is the DND-PG-COOH of Example 1 13 C-NMR measurement results.

[0029] Figure 4 This is a diagram showing the FT-IR spectrum of DND-PG(OTs)-COOH of Example 1.

[0030] Figure 5 is the DND-PG(BSH)-COOH of Example 1 1 Figure 1 H-NMR spectrum.

[0031] Figure 6 This is a diagram showing the FT-IR spectrum of DND-PG(BSH)-COOH of Example 1.

[0032] Figure 7 is the DND-PG(BSH)-PBA of Example 1 1 Figure 1 H-NMR spectrum.

[0033] Figure 8 FT-IR spectra of (a) DND-PG(OTs)-COOH and (b) DND-PG(N 3 )-COOH FT-IR spectrum, (c)DND-PG( 10 FT-IR spectra of (BSH)-COOH, (d)DND-PG( 10 FT-IR spectra of (BSH)-PBA, (e)DND-PG( 10 Figure 3 shows the FT-IR spectrum of BSH)-c(RGDyK).

[0034] Fig. 9 is the DND-PG ( 10 (a) in BSH)-COOH 1 H-NMR spectrum, (b) 13 C-NMR spectrum, (c) 10 Figure 1. B-NMR spectrum.

[0035] Fig.10 It is the embodiment 2 10 In BSH(Pgy), (a) 1 H-NMR spectrum, (b) 10 Figure 1. B-NMR spectrum.

[0036] Fig.11 is (a) DND-PG ( 10 BSH)-PBA 1 H-NMR spectrum, (b) DND-PG ( 10 BSH)-c(RGDyK) 1 Figure 1 H-NMR spectrum.

[0037] Fig.12 This is a graph showing the results of a cytotoxicity test (cell survival rate in B16 mouse melanoma cells) using the nanoparticles of Example 2. (n=6, Student's t-test using blank (PBS): * p<0.5, ** p<0.01, *** p<0.001)

[0038] Fig.13 This is a graph showing the cell survival rate relative to the irradiation dose in a colony formation assay when B16 cells exposed to the nanoparticles of Example 2 were irradiated with thermal neutrons.

[0039] Fig.14 This is a graph showing the cell survival rate relative to the irradiation dose in a colony formation assay of B16 cells exposed to the nanoparticles of Example 2 when subjected to thermal neutron irradiation. (Significance of gradient difference * p<0.5, **p<0.01, ***p<0.001)

[0040] Fig.15 It represents a) control, b) DND-PG ( 10 BSH)-PBA and c)DND-PG ( 10 Figure 1 is a TEM image of the introduction of BSH)-c(RGDyK) into cells.

[0041] Fig.16The figures show TEM images of the cell introduction experiments of d) DND-PG-COOH, e) DND-PG-PBA and f) DND-PG-c(RGDyK) in Example 2. DETAILED DESCRIPTION

[0042] [Surface modified nanodiamond]

[0043] The surface modified nanodiamond (hereinafter sometimes referred to as "ND") disclosed herein has nanodiamond particles and a group containing a boron cluster and a group having cancer cell recognition ability as a surface modification group for surface modification of the nanodiamond particles. The surface modified nanoparticles disclosed herein preferably further have a group containing a hydrophilic polymer chain as a surface modification group. The surface modified ND disclosed herein may have only one of the surface modification groups, or may have two or more of the surface modification groups.

[0044] The group comprising boron clusters and the group having cancer cell recognition ability may be present in the same surface modification group or in different surface modification groups. In addition, in the case where the surface modified nanodiamond disclosed herein has a group comprising a hydrophilic polymer chain as the surface modification group, the group comprising boron clusters, the group having cancer cell recognition ability and the group comprising a hydrophilic polymer chain may be present in the same surface modification group or in different surface modification groups. That is, the surface modification group may simultaneously comprise two or three of the group comprising boron clusters, the group having cancer cell recognition ability and the group comprising a hydrophilic polymer chain, or may consist of only any one of them.

[0045] The surface modified ND disclosed in the present invention has a tendency to improve the lethality to cancer cells by having a group containing a boron cluster. It is believed that the reason is that when cancer cells are killed by α particles and lithium nuclei produced by the nuclear reaction of neutrons and boron, the surface modified ND disclosed in the present invention has a group containing a boron cluster, so the concentration of boron atoms in the surface modified ND becomes high, and the result is that the lethality to cancer cells is improved. In addition, the group containing a boron cluster can have the following properties: non-specifically accumulate the surface modified ND disclosed in the present invention in its interior and surroundings to cancer tissues and cancer cells. As such a boron cluster, the carborane described later can be exemplified. However, the group containing a boron cluster and the group having the ability to recognize cancer cells are independent groups. In addition, the group having the ability to recognize cancer cells has the following properties: selectively recognize cancer tissues and / or cancer cells from multiple cells, preferably specifically recognize specific types of cancer cells, accumulate the surface modified ND disclosed in the present invention in its interior and surroundings, and further improve the lethality to cancer cells derived from boron atoms. It should be noted that the surface modified ND of the present invention has a tendency to be more lethal to cancer cells compared to the surface modified ND having only a group containing a boron atom and having the ability to recognize cancer cells (e.g., a group derived from an aromatic boronic acid such as phenylboronic acid). It is believed that the reason is that, as described above, the surface modified ND of the present invention independently has a group containing a boron cluster and a group having the ability to recognize cancer cells, thereby improving the lethality to cancer cells through their synergistic effect. In addition, in the case where the surface modified ND of the present invention has a group containing a hydrophilic polymer chain, there is a tendency that the stability under physiological conditions tends to be more excellent. Moreover, there is a tendency that the movement of the surface modified ND to cancer tissue / cancer cells becomes easy.

[0046] The surface modification groups in the surface modified ND disclosed herein may simultaneously include: a group containing a boron cluster and a group containing a hydrophilic polymer chain. For example, the group containing a boron cluster may be bonded to the ND particle via a hydrophilic polymer chain. By making the surface modification groups adopt the above-mentioned scheme, there is a tendency that the killing activity of the surface modified ND disclosed herein to cancer cells is further improved, and the stability under physiological conditions is more excellent.

[0047] The surface modification group in the surface modified ND disclosed in the present invention may simultaneously have: a group having the ability to recognize cancer cells and a group containing a hydrophilic polymer chain. For example, the group having the ability to recognize cancer cells may be bonded to the ND particle via a hydrophilic polymer chain. In addition, the group having the ability to recognize cancer cells may be bonded to the hydrophilic polymer chain via an amide bond, an ester bond, or a thioester bond, and the hydrophilic polymer chain is bonded to the ND particle. By making the surface modification group adopt the above-mentioned scheme, there is a tendency that a sufficient amount of boron atoms can be selectively accumulated in cancer tissues and cancer cells, and the stability under physiological conditions is more excellent.

[0048] The surface modification groups in the surface modified ND of the present disclosure may simultaneously include: a group containing a boron cluster, a group having cancer cell recognition ability, and a group containing a hydrophilic polymer chain. For example, the group containing a boron cluster and the group having cancer cell recognition ability may be bonded to the ND particle via the same hydrophilic polymer chain.

[0049] The ND particles constituting the surface-modified ND disclosed in the present invention preferably contain primary particles of nanodiamond. In addition, secondary particles formed by the agglomeration (agglomeration) of a plurality of the primary particles may also be included. Moreover, the surface-modified ND disclosed in the present invention may also have one or more substituents other than the group containing boron clusters, the group having cancer cell recognition ability, and the group containing hydrophilic polymer chains on the surface of the ND particles. As such substituents, for example, amino, hydroxyl, and carboxyl groups can be listed.

[0050] The ND particles in the surface modified nanodiamond disclosed in the present invention are not particularly limited, for example, detonation ND, high temperature and high pressure ND, chemical vapor deposition ND (ND formed by crushing a diamond film using the CVD method), etc. can be used. Among them, detonation ND is preferred from the perspective of having better dispersibility in the dispersion medium and a single-digit nanometer particle size of the primary particles. In the ND particles, nitrogen-defect centers (NV centers), silicon-defect centers (SiV centers), etc. having defects with fluorescent properties and magnetic properties may also exist in their diamond crystal structure.

[0051] [Groups containing boron clusters]

[0052] In the surface-modified ND disclosed herein, a group containing a boron cluster can be formed by introducing a boron cluster into the ND particle. The ND particle can be an ND particle having a group containing a hydrophilic polymer chain as a surface modification group. In this case, the group containing a boron cluster can be directly bonded to the surface of the ND particle, or can be bonded to the ND particle via a hydrophilic polymer chain.

[0053] (Boron Clusters)

[0054] The boron cluster is a cluster having a boron atom in the molecule. There is no particular limitation on the boron cluster, and examples thereof include carborane, dodecaborate, 10 H 10 The boron ion cluster shown has the molecular formula M(C 2 B 9 H 10 ) 2 The sandwich ion cluster shown (M is a transition element). The carborane may be a molecular formula of C 2 B 9 H 11The nested carborane (ortho, meta, para) represented by CB 11 H 12 The closed carborane shown. The dodecaborate (BH) is a 12 H 12 The ion cluster shown. The molecular formula is M(C 2 B 9 H 10 ) 2 The transition element M of the sandwich ion cluster shown may be Fe, Co, Ni, Mo, etc. These boron clusters may have substituents such as thiol, hydroxyl, amino, carboxyl, etc.

[0055] The boron atoms in the boron cluster can be 10 B. 11 B etc., but the boron atom is preferably concentrated to 10 B.

[0056] In the surface modified ND disclosed in the present invention, when the boron cluster is introduced into the ND particle or the ND particle having a group containing a hydrophilic polymer chain as a surface modification group, the boron cluster can be bonded to the ND particle or the hydrophilic polymer chain as the surface modification group via a single bond or a linking group. As the linking group, for example, alkenyl, thioether bond, sulfonium bond, sulfoxide group, secondary amine group, tertiary amine group, quaternary amine group (quaternary ammonium cation group), amide bond, ether bond, ester bond, thioester bond, heterocycle (for example, 1,2,3-triazole ring), carbonyl group, acetal bond and groups formed by bonding of two or more thereof. Among them, the linking group is preferably a group containing a thioether bond, a group containing a 1,2,3-triazole ring, or a group containing a thioether bond and a 1,2,3-triazole ring.

[0057] In the surface-modified ND of the present disclosure, in the CH 2 When introducing a boron cluster into an ND particle in which at least one of the OH groups is substituted with a carboxyl group, from the viewpoint of reactivity, the boron cluster is preferably introduced into the ND particle, for example, (1) by a reaction of a thiol group, a hydroxyl group, an amino group or the like substituent possessed by the boron cluster with a hydroxyl group of a polyglycerol chain, or (2) by a reaction (i.e., a click reaction, etc.) of an alkynyl group possessed by the boron cluster with a polyglycerol chain in which a part of the hydroxyl groups are substituted with an azide group. In the case of (1), in the surface-modified ND disclosed herein, the boron cluster is bonded to the polyglycerol chain via a thioether bond, an ether bond, etc. as a connecting group. In the case of (2), in the surface-modified ND disclosed herein, the boron cluster is bonded to the polyglycerol chain via a 1,2,3-triazole ring (preferably a thioether bond and a 1,2,3-triazole ring).

[0058] [Groups with cancer cell recognition ability]

[0059] In the surface-modified ND disclosed in the present invention, a group having the ability to recognize cancer cells can be formed by introducing a molecule having the ability to recognize cancer cells into the ND particles. The ND particles can be ND particles having a group containing a hydrophilic polymer chain as a surface modification group. In this case, the group having the ability to recognize cancer cells can be directly bonded to the surface of the ND particles, or can be bonded to the ND particles via a hydrophilic polymer chain.

[0060] (Molecules with cancer cell recognition ability)

[0061] Examples of molecules having cancer cell recognition capabilities include boric acid, amino acids, peptides, proteins, folic acid, and porphyrin. For example, by introducing boric acid into ND particles, a group derived from boric acid can be formed. In addition, by introducing peptides and proteins into ND particles, a group derived from peptides and a group derived from proteins can be formed.

[0062] The boric acid is not particularly limited, and examples thereof include compounds represented by the following formula (A).

[0063] R a2 -B(-R a1 ) 2 (A)

[0064] In formula (A), R a1 represents a hydroxyl group which is optionally protected by a protecting group. a2 represents a hydrogen atom or a monovalent organic group.

[0065] As the R a1 The hydroxyl group in the hydroxyl group may have a protecting group, for example, methyl, ethyl, tert-butyl, etc. 1-4 Alkyl; a group that forms an acetal bond with the oxygen atom constituting the hydroxyl group (for example, methoxymethyl, etc. 1-4 Alkyl-OC 1-4 a group that forms an ester bond with the oxygen atom constituting the hydroxyl group (for example, an acetyl group, a benzoyl group), and the like.

[0066] As R a2 The monovalent organic group in the formula (a) may include: a substituted or unsubstituted hydrocarbon group (a monovalent hydrocarbon group, particularly a monovalent aliphatic or aromatic hydrocarbon group); a substituted or unsubstituted heterocyclic group (a monovalent heterocyclic group); a group formed by bonding two or more of the monovalent hydrocarbon group and / or the monovalent heterocyclic group, etc. From the viewpoint of ease of manufacture and stability under physiological conditions, the monovalent organic group is preferably a substituted or unsubstituted monovalent aromatic hydrocarbon group or a group containing an aromatic heterocycle (i.e., a group that is the heterocyclic group and has aromaticity).

[0067] Examples of the aromatic hydrocarbon group include aromatic hydrocarbon groups having 6 to 18 carbon atoms (preferably 6 to 14). Examples of the aromatic heterocyclic ring-containing group include aromatic heterocyclic ring-containing groups having 4 to 18 carbon atoms (preferably 6 to 14) and having 1 to 4 at least one hetero atom selected from nitrogen atoms, oxygen atoms, and sulfur atoms.

[0068] The above-mentioned boric acid is not particularly limited, and examples thereof include: preferably aromatic boric acid, more preferably phenylboric acid such as 3-aminophenylboric acid, 4-aminophenylboric acid, and 4-aminomethylphenylboric acid, and pyridineboric acid such as (2-aminopyridin-4-yl)boric acid, (5-aminopyridin-3-yl)boric acid, (2-aminopyridin-5-yl)boric acid, and (4-aminopyridin-2-yl)boric acid.

[0069] The amino acid is not particularly limited, and examples thereof include leucine, valine, isoleucine, phenylalanine, tyrosine, lysine, arginine, histidine, and derivatives thereof. These amino acids can be introduced into ND particles via the amino group or carboxyl group in the molecule (e.g., the COOH group of DND-PG-COOH).

[0070] The peptide is not particularly limited, and examples thereof include peptides having an RGD sequence. The RGD sequence refers to Arg-Gly-Asp (arginine-glycine-aspartic acid). Such RGD sequences are not particularly limited, and examples thereof include: GRGDNP (Gly-Arg-Gly-Asp-Asn-Pro), Cyclo (-Arg-Gly-Asp-D-Phe-Cys), Cyclo [-Arg-Gly-Asp-D-Phe-Lys (Cys)], Cyclo (-Arg-Gly-Asp-D-Tyr-Lys), Cyclo (-Arg-Gly-Asp-D-Phe-Lys). Among them, Cyclo (-Arg-Gly-Asp-D-Tyr-Lys) is preferred. When these peptides are introduced into ND particles, peptides having Lys as a free amino group are preferred from the perspective of being able to form an amide bond through their ε-amino group. Furthermore, the terminal COOH of the peptide may be introduced into the ND particle (particularly the COOH group of DND-PG-COOH) via or without a spacer.

[0071] The protein is not particularly limited, and for example, IgG antibodies targeting cancer cells (particularly receptors and sugar chains on the surface of cancer cells) are preferred, and specific examples include anti-EGFR antibodies (anti-epidermal growth factor receptor antibodies, "Erbitux" (cetuximab)) and the like.

[0072] In the surface modified ND disclosed in the present invention, when introducing a molecule having the ability to recognize cancer cells into an ND particle or an ND particle having a group containing a hydrophilic polymer chain as a surface modification group, the molecule having the ability to recognize cancer cells can be introduced directly or via a substituent possessed by the molecule. That is, the molecule having the ability to recognize cancer cells can be combined with the ND particle or the hydrophilic polymer chain as a surface modification group via a single bond or a connecting group, and the connecting group is a group derived from a substituent possessed by the molecule having the ability to recognize cancer cells, preferably, for example, a thioether bond, a sulfonium bond, a sulfoxide group, a secondary amine group, a tertiary amine group, a quaternary amine group (quaternary ammonium cation group), an amide bond, an ether bond, an ester bond, a thioester bond, a carbonyl group or an acetal bond. Among them, an amide bond is more preferred.

[0073] In the surface-modified ND of the present disclosure, in the CH 2 When a molecule having the ability to recognize cancer cells is introduced into the ND particles in which at least one of the OH groups replaces the carboxyl group, the molecule having the ability to recognize cancer cells is preferably introduced via a substituent such as a thiol group, a hydroxyl group, an amino group, etc. from the viewpoint of reactivity. In this case, in the surface-modified ND of the present invention, the group having the ability to recognize cancer cells is bonded to the polyglycerol chain via an amide bond, an ester bond, or a thioester bond, and the hydrophilic polymer chain is bonded to the nanodiamond particle.

[0074] [Group containing a hydrophilic polymer chain]

[0075] Examples of the hydrophilic polymer chain include polymer chains containing structural units derived from monomers having hydrophilic groups, such as polyether chains (polyethylene oxide, polypropylene oxide, copolymers thereof, etc.), polyglycerol chains (C 3 H 6 O(CH 2 CH(OH)CH 2 Among them, from the viewpoint of stability under physiological conditions, a polyglycerol chain is preferred. That is, the group comprising a hydrophilic polymer chain is preferably a polyglycerol chain.

[0076] The polyglycerol chain is preferably a polyglycerol chain represented by the following formula (1), for example.

[0077] -(X 1 C 3 H 5 )-(OC 3 H 5 )p-(X 2 R 1 )q(1)

[0078] [In formula (1), p represents an integer greater than or equal to 1, and q represents an integer satisfying q=p+2. X 1represents a divalent group, [X 1 C 3 H 5 ] from X 1 The bond arm extending to the left is bonded to the ND particle. 2 R 1 ] indicates the end of the polyglycerol chain, X 2 represents a single bond or a divalent group, R 1 represents a hydrogen atom or a monovalent organic group]

[0079] In the formula (1), [X 1 C 3 H 5 ] by [-X 1 -CH 2 -C(-)H-CH 2 -] indicates that [X 1 C 3 H 5 ] 1 Bonded to ND particles, two Cs bonded to [OC 3 H 5 ] or [X 2 R 1 ] 2 Bonding. 1 C 3 H 5 ] 1 It should be noted that one or more R 1 is a monovalent organic group, R 1 It is preferred that the one or more monovalent organic groups in the group have the group containing a boron cluster or the group having cancer cell recognition ability.

[0080] As X 1 The divalent group in the formula (A) includes, for example, amino (-NR a -), amide bond (-NR a -C(=O)-), ether bond (-O-), ester bond (-OC(=O)-), thioester bond (-S-(=O)-), phosphinate (-PH(=O)O-), phosphonate (-P(-OH)(=O)O-), phosphate (-OP(=O)(OH)-O-), thioether bond (-S-), carbonyl (-C(=O)-), carbamate bond (-R a N-C(=O)-O-), imide bond (-C(=O)-NR a -C(=O)-), thiocarbonyl (-C(=S)-), siloxane bond (-Si-O-), sulfate ester group (-OS(=O)-), 2 -O-), sulfonyl (-S(=O) 2-O-), sulfone (-S(=O) 2 -), sulfoxide (-S(=O)-), groups formed by bonding two or more of them, etc. In the asymmetric divalent group, the direction of the divalent group relative to the ND particle side and the R side is not particularly limited. In addition, the R a represents a hydrogen atom or a monovalent organic group. As the divalent group, among them, -NR a -, -O-, -C(=O)O-, -NR a- C(=O)-, -PH(=O)O-, -S-, more preferably -NR a -、-O-、-S-、-NR a- C(═O)—, or —C(═O)O—.

[0081] As R 1 The monovalent organic group in, for example, can be listed as: substituted or unsubstituted hydrocarbon group (monovalent hydrocarbon group, especially monovalent aliphatic or aromatic hydrocarbon group); substituted or unsubstituted heterocyclic group (monovalent heterocyclic group); a group formed by bonding two or more of the monovalent hydrocarbon group and / or the monovalent heterocyclic group; the group containing boron clusters, the group having cancer cell recognition ability, etc. The bonded group can be directly bonded or bonded via a connecting group. As the connecting group, for example, it can be listed as: amino, ether bond, ester bond, phosphinic acid group, thioether bond, carbonyl group, organic group substituted amide group, organic group substituted carbamate bond, organic group substituted imide bond, thiocarbonyl group, siloxane bond, a group formed by bonding two or more of them, etc.

[0082] In the formula (1), [OC 3 H 5 ] is derived from [-O-CH 2 -C(-)H-CH 2 -], and the structure of glycerol shown in [X 1 C 3 H 5 ] together to form a polyglycerol chain. p represents [OC 3 H 5 ] is an integer greater than 1, preferably 3 to 2000, more preferably 5 to 500, and further preferably 10 to 200. p is optionally the same or different in the plurality of groups containing polyglycerol chains.

[0083] In the formula (1), [X 2 R 1 ] indicates the end of the polyglycerol chain, and [X 1 C 3 H 5 ] or [OC 3 H5 ] in C bonding.

[0084] The [X 2 R 1 ] 2 represents a single bond or a divalent group. Examples of the divalent group include the above-mentioned [X 1 C 3 H 5 ] 1 The divalent groups shown and described by way of example. 2 R a With R 1 are optionally bonded to form a ring. 2 Preferably -NR a -、-NR a C(=O)-, -O-, -C(=O)O-, -OC(=O)-, -SC(=O)-, -C(=O)-, -PH(=O)O-, -OP(=O)(OH)-O-, -S-, -OS(=O) 2 -O-, -OS(=O) 2 -, more preferably -NR a -、-O-、-NR a C(=O)-, -OC(=O)-, -SC(=O)-, -C(=O)-. Said[X 1 C 3 H 5 ] 1 With the [X 2 R 1 ] 2 Optionally the same or different. 2 R 1 ] are optionally the same or different. In addition, the [X 2 R 1 ] 2 The plurality of groups containing a polyglycerol chain may be the same or different. q represents an integer greater than or equal to 3, and its value depends on the value of p, satisfying q=p+2. q may be the same or different in the plurality of groups containing a polyglycerol chain.

[0085] In the R 1 When a plurality of R in the formula (1) is a monovalent organic group, 1 As the monovalent organic group, the following can be cited as R aThe monovalent organic group is exemplified and described as a group. Among them, substituted or unsubstituted hydrocarbon groups (monovalent hydrocarbon groups), substituted or unsubstituted heterocyclic groups (monovalent heterocyclic groups), and groups formed by bonding two or more of them can be listed. In addition, the monovalent organic group can also have an ionic form.

[0086] The bonded groups may be directly bonded or bonded via a connecting group. The hydrocarbon group in the substituted or unsubstituted hydrocarbon group is preferably an alkyl group, more preferably an alkyl group having 1 to 18 carbon atoms, further preferably an alkyl group having 1 to 6 carbon atoms, and particularly preferably an ethyl group, a propyl group, a butyl group or a hexyl group.

[0087] As [X 2 R 1 ] Specific examples include: OH, NH 2 , CH 3 , alkoxy, acyl, mono- or di-alkylamino, mono- or di-alkenylamino, alkylamide, alkenylamide, quaternary ammonium substituted alkoxy, chloroalkoxy, polyalkylene oxide, cyclic imide, carboxyl substituted alkylamino, carboxyl substituted alkyloxy; as the group containing the boron cluster, and having a carbonyl group, an amide bond, an ester bond, a thioester bond, a thioether bond, an ether bond, a heterocycle (such as a 1,2,3-triazole ring), and a group formed by bonding two or more of them; as the group having the ability to recognize cancer cells, and having a carbonyl group, an amide bond, an ester bond, or a thioester bond. Two or more of the [X 2 R 1 ]Optionally via R 1 Among them, a cyclic imide group and a carboxyl-substituted alkylamino group are preferred from the viewpoint of better water dispersibility of the surface-modified ND.

[0088] The number average degree of polymerization of glycerol in the polyglycerol chain is 3 to 2000, more preferably 5 to 500, and further preferably 10 to 200. If the number average degree of polymerization is large, the mutual repulsion between NDs can fully function, and the dispersibility of the nanodiamond particles can be further improved. If the number average degree of polymerization is below 5000, the polyglycerol chains between nanodiamonds can be inhibited from entangled with each other, and the dispersibility of ND in water can be further improved. It should be noted that the number average degree of polymerization is defined by the number of glycidyl units constituting the polyglycerol chain in the group bonded to the surface functional group of a raw nanodiamond, and the number of surface functional groups of the raw nanodiamond can be determined by measuring the elemental analysis value of the raw nanodiamond or the acid value, or by combining the two.

[0089] The polyglycerol chain may be a polyglycerol chain in which a part or all of the hydroxyl groups in the polyglycerol chain are substituted with amino groups (hereinafter referred to as a "nitrogen-substituted polyglycerol chain"). Examples of the nitrogen-substituted polyglycerol chain include X in at least a part of the polyglycerol chain represented by the formula (1): 2 It is a polyglycerol chain with amino groups.

[0090] The amino groups in the nitrogen-substituted polyglycerol chains are intended to improve the dispersibility of the surface-modified ND in blood (to prevent aggregation in blood), and at least a portion of the amino groups may be protected by a compound having an acidic functional group. Examples of such polyglycerol chains include polyglycerol chains represented by formula (1) wherein at least a portion of X 2 For amino (-NR a -) and at least part of R a A polyglycerol chain having a protective group. Examples of the acidic functional group include a carboxyl group, a sulfonyl group, and the like. Specifically, compounds containing the acidic functional group include: dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, and adipic acid; acid anhydrides such as oxalic anhydride, succinic anhydride, maleic anhydride, phthalic anhydride, and benzoic anhydride; and compounds having a sulfonyl group such as sulfuric acid. Among them, acid anhydrides are preferred from the viewpoint that the dispersibility of the surface-modified ND in blood is further improved by forming an amide bond and having a carboxyl group at the end. Examples of other protective groups include: alkyl groups such as methyl, ethyl, and propyl; and cycloalkyl groups such as benzyl and phenyl.

[0091] As the surface modification group of the surface modified ND, it is preferred to have a group derived from boric acid as the group having cancer cell recognition ability. In this case, the group derived from boric acid is preferably bonded to the ND particle via the hydrophilic polymer chain. In addition, the boron atom derived from boric acid is more preferably bonded to the hydrophilic polymer chain via an aromatic ring. The boron atom may be directly bonded to the aromatic ring or bonded via other connecting groups, but is preferably directly bonded, and is particularly preferably bonded to the carbon atom constituting the aromatic ring. The aromatic ring may have only one or more.

[0092] Examples of the aromatic ring include aromatic hydrocarbon rings and aromatic heterocyclic rings. Examples of the hydrocarbon ring include C 2+ rings such as a benzene ring and a naphthalene ring. 6-14 Hydrocarbon rings (especially C 6-10 Examples of the aromatic heterocyclic ring include a ring having a carbon atom and at least one hetero atom (for example, an oxygen atom, a sulfur atom, a nitrogen atom, etc.) among atoms constituting the ring, and a condensed ring thereof.

[0093] The aromatic ring is preferably a 4- to 10-membered ring, and more preferably a 6-membered ring. Examples of the 6-membered aromatic ring include a benzene ring and a pyridine ring.

[0094] The aromatic ring may or may not have a substituent. Examples of the substituent include: halogen atoms such as fluorine atoms, chlorine atoms, bromine atoms, iodine atoms; hydroxyl groups; thiol groups; carboxyl groups; amino groups; oxo groups, etc. Among them, as the substituent, it is preferred to have an electron-withdrawing group such as a fluorine atom. The electron-withdrawing group is preferably present in the meta position relative to the bonding position of the boron atom in the aromatic ring. It should be noted that the substituent corresponds to R in the formula (2) described later. 3 .

[0095] The aromatic ring is preferably bonded to the hydrophilic polymer chain via a divalent hydrocarbon group, a thioether bond, a sulfonium bond, a sulfoxide bond, a secondary amine group, a tertiary amine group, a quaternary amine group (quaternary ammonium cation group), an amide bond, an ether bond, an ester bond, a thioester bond, a carbonyl group, an acetal bond, or a group formed by connecting multiple thereof.

[0096] Examples of the divalent hydrocarbon group include a methylene group, an ethylene group, a propylene group, and a trimethylene group. The divalent hydrocarbon group preferably has 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms, from the viewpoint of improving the dispersibility of the surface-modified ND.

[0097] The embodiment in which the boron atom is directly bonded to the carbon atom constituting the aromatic ring and the aromatic ring is bonded to the hydrophilic polymer chain via a linking group bonded to the carbon atom constituting the aromatic ring will be described using a group represented by the following formula (2).

[0098]

[0099] In formula (2), Ar represents an aromatic ring, R 3 is a substituent optionally possessed by the aromatic ring, and is preferably an electron withdrawing group. m is an integer greater than 0. 4 is a group containing a boron atom derived from boric acid, and the boron atom is directly bonded to the carbon atom constituting the aromatic ring. n is an integer greater than 1. Y is a divalent hydrocarbon group, a thioether bond, a sulfonium bond, a sulfoxide group, a secondary amine group, a tertiary amine group, a quaternary amine group (quaternary ammonium cation group), an amide bond, an ether bond, an ester bond, a thioester bond, a carbonyl group, an acetal bond, or a group formed by connecting a plurality of them. The bond arm extending to the left from Y is bonded to a hydrophilic polymer chain (preferably a polyglycerol chain).

[0100] In R 4 A hydrogen atom, a hydroxyl group, an amino group, a sulfo group, a monovalent organic group, or other substituents may be bonded to the boron atom. The embodiment including the boron atom is not limited, and an embodiment having a group represented by the following formula (3) can be cited.

[0101] -B(-R 5 ) 2 (3)

[0102] In formula (3), R 5 Each of the two R represents a hydrogen atom, a hydroxyl group, an amino group, a sulfonic group, or a monovalent organic group. 5 Optionally, they are bonded to each other to form a ring (including a hydrocarbon ring). 5 When the two carbon atoms are bonded to each other to form a ring, the ring is optionally formed via an oxygen atom. In addition, the carbon atoms constituting the ring optionally form a double bond with an adjacent carbon atom. It should be noted that the number of carbon atoms constituting the ring is not particularly limited, for example, preferably 4 to 12, more preferably 4 to 10, and further preferably 4 to 6.

[0103] Examples of the monovalent organic group include hydrocarbon groups, among which an alkyl group is preferred, an alkyl group having 1 to 6 carbon atoms is more preferred, an alkyl group having 1 to 3 carbon atoms is further preferred, and a methyl group is particularly preferred.

[0104] Specific examples of the group represented by formula (3) include boric acid (-B(OH) 2 ), borane (-BH 2 ) etc. Among them, boric acid is preferred.

[0105] The content of boron atoms derived from boron clusters in the surface modified ND disclosed herein is not particularly limited, for example, preferably 1.0 mass % or more, more preferably 5.0 mass % or more, and further preferably 10 mass % or more. By making the content of boron atoms within the range, there is a tendency to selectively and sufficiently accumulate boron atoms in cancer tissues and cancer cells. The upper limit of the content of the boron atoms is not particularly limited, for example, 40.0 mass %.

[0106] In the surface-modified ND disclosed in the present invention, when the group having cancer cell recognition ability is a group derived from boronic acid, the content of the boron atom derived from the group derived from boronic acid is not particularly limited, and is, for example, 0.1 to 2.0% by mass, more preferably 0.2 to 1.5% by mass, and further preferably 0.3 to 1.0% by mass. When the content of the boron atom is within the above range, there is a tendency that the cancer cell recognition ability is excellent.

[0107] The boron atom may be 10 B. 11 B and any other isotope, without particular limitation. The boron atom is preferably concentrated to 10 B. Among them, it is particularly preferred that the boron atoms derived from the boron clusters are concentrated to 10 B.

[0108] The mass ratio of ND to the surface modification group in the surface modified ND disclosed herein [ND / surface modification group disclosed herein] is not particularly limited, and is preferably 0.1 to 2.0, more preferably 0.2 to 1.5, and further preferably 0.2 to 1.0. In particular, if the mass ratio is 0.2 or more, it is not easy to damage the characteristics as a nanodiamond material, and if the mass ratio is 1.5 or less (further less than 1.0), the modification degree of the surface modification group becomes sufficient, and the dispersibility under physiological conditions is more excellent. The mass ratio is based on the weight reduction rate measured by thermogravimetric analysis of the sample before the introduction of the boron substituent, and is obtained by the ratio of the weight of the remaining ND to the following value, which is obtained by adding the weight of the boron substituent introduced therein to the weight of the surface modification group containing the polyglycerol chain before the introduction of the boron substituent.

[0109] The volume average particle size (MV) of the surface modified ND disclosed in the present invention is, for example, 200 nm or less, preferably 100 nm or less, and more preferably 60 nm or less. The lower limit of the average particle size (MV) of the surface modified ND is, for example, 5 nm. The average particle size of the surface modified ND can be measured by dynamic light scattering.

[0110] The surface-modified NDs disclosed herein can be used for boron neutron capture therapy of tumor diseases.

[0111] Examples of the “tumor disease” in this specification include malignant melanoma, renal cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, hepatocellular carcinoma, biliary tract cancer, gastric cancer, ovarian cancer, esophageal cancer, urothelial carcinoma, colon cancer, bone cancer, skin cancer (e.g., malignant skin cancer), head and neck cancer, rectal cancer, anal cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid cancer, bladder cancer, malignant pleural mesothelioma, brain tumors (e.g., malignant brain tumors), and central nervous system tumors.

[0112] Boron neutron capture therapy using the surface-modified ND is performed, for example, in the following manner: a pharmaceutical agent comprising the surface-modified ND is administered to a mammal (e.g., a human) suffering from a tumor disease through any appropriate administration route in a manner such that the compound accumulates in the target site. The surface-modified ND is preferably selectively accumulated in the tumor. The preparation comprising the compound can be administered all at once or sequentially. The preparation can be administered repeatedly as needed.

[0113] After the surface-modified ND reaches the tumor, the site is irradiated with an effective amount of low-energy neutron beams such as thermal neutron beams or epithermal neutron beams. The site can be irradiated through the skin, or the site can be fully or partially exposed before irradiation. The administration of the surface-modified ND and the subsequent irradiation with thermal neutron beams or epithermal neutron beams can be repeated as needed.

[0114] The administration route of the surface-modified ND is preferably intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraspinal administration, or intraperitoneal administration.

[0115] The surface modified ND can be administered to a mammal suffering from a tumor disease within 72 hours (preferably 5 minutes to 48 hours before, more preferably 30 minutes to 30 hours before) before irradiation with a thermal neutron beam or an epithermal neutron beam. In addition, as required, it can also be administered during irradiation with a thermal neutron beam or an epithermal neutron beam. The representative dosage of the surface modified ND is preferably in the range of 0.01 mg to 5000 mg / kg body weight relative to one irradiation with a thermal neutron beam or an epithermal neutron beam. Boron-10 ( 10 B) conversion, preferably within the range of 0.005 mg to 1000 mg / kg body weight relative to the irradiation of a thermal neutron beam or an epithermal neutron beam. The irradiation time of a thermal neutron beam or an epithermal neutron beam is preferably 1 minute to 5 hours, more preferably 10 minutes to 2 hours. The irradiation dose of a thermal neutron beam or an epithermal neutron beam is not particularly limited, and generally, it can be any irradiation dose used in neutron capture therapy.

[0116] Boron neutron capture therapy using the surface-modified NDs can be applied as a single treatment or in combination with conventional surgery or chemotherapy. If desired, the tumor can be removed to the extent that it can be surgically removed, and then the remaining tumor can be destroyed by boron neutron capture therapy using the surface-modified NDs.

[0117] The composition containing the surface-modified ND can be used for boron neutron capture therapy. The composition can use the surface-modified ND as an active ingredient as it is, or can be formulated by adding pharmaceutically acceptable additives. In addition, as described above, other boron compounds such as BPA and BSH can also be included.

[0118] The additive is not particularly limited, and examples thereof include: pharmaceutically acceptable organic solvents such as purified water, saline, phosphate buffer, dextrose, glycerol, and ethanol; animal and vegetable oils, glucose, mannose, fructose, galactose, sorbitol, mannitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, gum tragacanth, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin.

[0119] The surface-modified ND can be formulated into a non-oral dosage form such as an injection solution, suspension, emulsion, paste, ointment, inhalant, suppository, etc. together with a pharmaceutically acceptable carrier such as a pharmaceutically acceptable solvent, excipient, binder, stabilizer, dispersant, etc. The surface-modified ND is preferably formulated into an injection solution.

[0120] The injection solution can be prepared in the form of a solution of the surface-modified ND in a pharmaceutically acceptable solvent. Their solution may also contain a stabilizing component and / or a buffering component. In addition, the injection solution may also be a dry preparation added with an appropriate solvent before use.

[0121] [Method for producing surface-modified nanodiamond]

[0122] The surface modified ND disclosed in the present invention can be manufactured by reacting ND particles, boron clusters, and molecules having the ability to recognize cancer cells. In addition, the surface modified ND disclosed in the present invention is preferably manufactured by reacting ND particles, hydrophilic polymers, boron clusters, and molecules having the ability to recognize cancer cells. The reaction of the hydrophilic polymer, the boron cluster, and the molecules having the ability to recognize cancer cells with the ND particles can be carried out simultaneously or separately. From the perspective of easily obtaining the desired surface modified ND, it is preferred that after reacting the ND particles with the hydrophilic polymer, the resulting reaction product is reacted with the boron cluster and the molecules having the ability to recognize cancer cells. It should be noted that the order in which the boron clusters and the molecules having the ability to recognize cancer cells react relative to the reaction product of the ND particles with the hydrophilic polymer is not particularly limited.

[0123] The hydrophilic polymer is not particularly limited as long as it is a polymer containing a structural unit derived from a monomer having a hydrophilic group, and examples thereof include polyethers (polyoxyethylene, polyoxypropylene, copolymers thereof, etc.), polyglycerol (C 3H 6 O(CH 2 CH(OH)CH 2 It should be noted that the polyglycerol may be a polyglycerol in which a part or all of the hydroxyl groups are substituted with amino groups.

[0124] The reaction between the ND particles and the hydrophilic polymer is not particularly limited and can be carried out by a known and conventional method. The case where polyglycerol is used as the hydrophilic polymer is described in detail below.

[0125] The surface modification method of ND particles using polyglycerol is not particularly limited, and can be obtained, for example, by ring-opening polymerization of glycidol and ND particles. ND particles naturally have carboxyl groups, hydroxyl groups, etc. on the particle surface during their manufacturing process. By reacting these functional groups with glycidol, the surface of ND particles can be modified with polyglycerol chains.

[0126] The amount of glycidol used is, for example, 0.5 to 100 parts by weight, preferably 10 to 50 parts by weight, based on 1 part by weight of the ND particles. By adjusting the amount of glycidol used, the chain length of the polyglyceryl group and the bonding density to the ND particle surface can be controlled.

[0127] The ring-opening polymerization reaction can be carried out, for example, by heating a mixed solution of a solvent and ND particles and sequentially adding glycidol thereto at a heating temperature of, for example, 40 to 180°C, preferably 60 to 150°C, and particularly preferably 80 to 125°C.

[0128] The solvent is not particularly limited, and preferably a solvent with low reactivity with glycidol and high solubility in polyglycerol is used. As the solvent, alcohol is preferred, and aliphatic alcohol is particularly preferred. As aliphatic alcohol, for example, aliphatic monohydric alcohols having 2 to 4 carbon atoms such as ethanol, propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol; aliphatic polyhydric alcohols having 2 to 4 carbon atoms such as ethylene glycol, propylene glycol, 1,3-propylene glycol, 1,3-butanediol, 1,4-butanediol, and glycerol can be listed. They can be used alone or in combination of two or more.

[0129] A catalyst may be used in the ring-opening polymerization reaction. The catalyst includes an acidic catalyst and a basic catalyst. Examples of the acidic catalyst include trifluoroborane, acetic acid, phosphoric acid, etc. Examples of the basic catalyst include triethylamine, pyridine, dimethylaminopyridine, triphenylphosphine, etc.

[0130] The ring-opening polymerization reaction can be stopped by cooling the reaction solution (for example, by cooling by adding water to the reaction system). In addition, the product can be purified by known or conventional methods (for example, washing with water, centrifugal separation, membrane filtration, etc.). Through this purification operation, the solvent, the remaining glycidol, and the by-products (polyglycerol that is not bonded to the ND particles and is in a free state, etc.) can be removed. In this way, ND particles having a group containing a polyglycerol chain are obtained in the form of an aqueous dispersion.

[0131] ND particles surface-modified with polyglycerol chains can be prepared by converting the CH 2 At least one of the OH groups is oxidized to be substituted with a carboxyl group.

[0132] The CH 2 When at least one of the OH groups is replaced by a carboxyl group, the carboxyl group is more 2 The OH group has high reactivity, so it is easy to introduce boron clusters and molecules with cancer cell recognition ability into the polyglycerol chain.

[0133] In this case, it is preferable to include the step of: using N-oxoammonium ions to convert the CH 2 A step of oxidizing at least one of the OH groups to convert it into an aldehyde group or its equivalent (aldehyde group conversion step); and a step of oxidizing the aldehyde group or its equivalent to convert it into a carboxyl group (carboxyl group conversion step).

[0134] In the aldehyde group conversion step, the CH 2 At least one of the OH groups is oxidized and converted to an aldehyde group or its equivalent (e.g. -CH(OH) 2 base).

[0135] N-oxoammonium ion is an active substance that promotes the oxidation reaction and has the function of converting CH 2 The OH group is oxidized and converted into an aldehyde group or its equivalent.

[0136] N-oxoammonium ions are generated, for example, by oxidizing a nitroxyl radical compound with hypochlorite.

[0137] From the viewpoint of increasing the reaction rate, the oxidation reaction of the nitroxyl radical compound with hypochlorite is preferably carried out in the presence of an alkali metal bromide such as sodium bromide.

[0138] The amount of the alkali metal bromide used is, for example, 1 to 40 times the molar amount of the nitroxyl radical compound used, and preferably 10 to 20 times the molar amount.

[0139] Examples of the nitroxyl radical compound include compounds represented by the following formulae (c-1) to (c-3) or precursors of the compounds. These compounds may be used alone or in combination of two or more.

[0140]

[0141] In formula (c-1), R c1 ~R c4 The same or different alkyl groups, R c5 represents a hydroxyl group, a carboxyl group, an acyl group, a substituted or unsubstituted amino group, an amide group, an alkoxy group, an aryloxy group or an aralkyloxy group. s represents an integer of 0-4.

[0142] In formula (c-2), R c6 represents an alkyl group, a substituted or unsubstituted amino group, an amide group, a halogen atom or a hydroxyl group. t represents an integer of 0-4.

[0143] In formula (c-3), R c7 represents an alkyl group, a halogen atom or a hydroxyl group. u represents an integer of 0-4.

[0144] Examples of the alkyl group include alkyl groups having 1 to 4 carbon atoms, such as a methyl group and an ethyl group.

[0145] Examples of the acyl group include C 2-hydroxy-2-nitropropanoyl, ... 1-4 Aliphatic acyl; acetoacetyl; benzoyl, etc. 7-14 Aromatic acyl, etc.

[0146] The substituted amino group includes a monosubstituted amino group and a disubstituted amino group. Examples of the substituents that the substituted amino group may have include C 1-5 Alkyl, C 3-6 Cycloalkyl, C 6-10 Examples of the acyl group include the same examples as those for the acyl group described above. Examples of the substituted amino group include acetylamino and the like.

[0147] The amide group is a group represented by -NHCOR', wherein R' represents a hydrocarbon group. Examples of the hydrocarbon group include an alkyl group having 1 to 4 carbon atoms, a halogenated alkyl group having 1 to 4 carbon atoms, an aryl group having 6 to 14 carbon atoms, and an aralkyl group having 4 to 15 carbon atoms. The amide group includes an acetamide group, a benzamide group, and the like.

[0148] Examples of the halogenated alkyl group include trifluoromethyl and trichloromethyl. 1-4 Halogenated alkyl.

[0149] Examples of the alkoxy group include alkoxy groups having 1 to 4 carbon atoms, such as a methoxy group.

[0150] Examples of the aryloxy group include C 6-10 Aryloxy, etc.

[0151] Examples of the aralkyloxy group include C 7-16 Aralkyloxy.

[0152] s, t, and u represent the number of groups shown in the brackets, and are the same or different and are integers of 0 to 4. When s, t, and u are integers of 2 or more, two or more of the groups may be the same or different.

[0153] Examples of the compound represented by formula (c-1) include 2,2,6,6-tetramethylpiperidine-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-benzoyloxy-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-acetamide-2,2,6,6-tetramethylpiperidine-1-oxyl, and 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxyl represented by the following formula (c-1-1).

[0154]

[0155] Examples of the compound represented by formula (c-2) include 2-azaadamantane-N-oxy represented by the following formula (c-2-1), 1-methyl-2-azaadamantane-N-oxy represented by the following formula (c-2-2), 1,5-dimethyl-2-azaadamantane-N-oxy represented by the following formula (c-2-3), 5-amino-2-azaadamantane-N-oxy, and 5-acetylamino-2-azaadamantane-N-oxy.

[0156]

[0157] Examples of the precursor of the compound represented by the formula (c-2) include 2-hydroxy-2-azaadamantane represented by the following formula (c-2-1′).

[0158]

[0159] Examples of the compound represented by formula (c-3) include 9-azanoradamantane-N-oxyl represented by the following formula (c-3-1) and 1,5-dimethyl-9-azanoradamantane-N-oxyl represented by the following formula (c-3-2).

[0160]

[0161] The amount of hypochlorous acid used is, for example, 5 to 50 mol, preferably 10 to 30 mol, particularly preferably 20 to 30 mol, based on 1 mol of the nitroxyl radical compound.

[0162] The nitroxyl free radical compound can be used directly or after being supported on a carrier.

[0163] The usage-amount of the nitroxyl radical compound is, for example, 0.005 to 1 part by weight, and preferably 0.01 to 0.5 part by weight, based on 1 part by weight of the surface modification group of the ND particle having a polyglycerol group.

[0164] In addition, the N-oxoammonium ion generated by oxidation of the nitroxyl radical compound is converted into a 2 The OH group is oxidized and reduced to N-hydroxylamine, which loses its oxidizing ability. N-hydroxylamine is converted back to N-oxoammonium ion (active form) by oxidation with a co-oxidant and can be reused in the oxidation reaction.

[0165] Examples of the co-oxidant include: hypochlorites (= salts of hypochlorous acid and alkali metals or alkaline earth metals) such as sodium hypochlorite, potassium hypochlorite, and calcium hypochlorite; chlorites (= salts of chlorous acid and alkali metals or alkaline earth metals) such as sodium chlorite, potassium hypochlorite, and calcium hypochlorite; hydrogen peroxide, tert-butyl hydroperoxide, trichloroisocyanuric acid, peracetic acid, performic acid, trichloroperacetic acid, trifluoroperacetic acid, iodophenyl diacetate, and the like.

[0166] As the co-oxidant, from the viewpoint of being able to efficiently reactivate N-hydroxyamine, it is preferable to use hypochlorites such as sodium hypochlorite, potassium hypochlorite, and calcium hypochlorite.

[0167] The carboxyl group conversion step is a step of oxidizing the aldehyde group or its equivalent formed in the aldehyde group conversion step to convert it into a carboxyl group.

[0168] An oxidant may be used in the reaction of the carboxyl conversion step. Examples of the oxidant include hypochlorites (= salts of hypochlorous acid and alkali metals or alkaline earth metals) such as sodium hypochlorite, potassium hypochlorite, and calcium hypochlorite; and chlorites (= salts of chlorous acid and alkali metals or alkaline earth metals) such as sodium chlorite, potassium chlorite, and calcium chlorite. These may be used alone or in combination of two or more.

[0169] When hypochlorite is used as a co-oxidant in the aldehyde group conversion step and hypochlorite is used as the oxidant in the carboxyl group conversion step (Case 1), CH contained in the polyglycerol chain can be efficiently converted by a one-pot method. 2 A series of reactions that convert OH groups to carboxyl groups (CH 2OH group → aldehyde group or its equivalent → carboxyl group).

[0170] In Case 1, the amount of hypochlorite used is, for example, 5 to 50 mol, preferably 10 to 30 mol, particularly preferably 20 to 30 mol, based on 1 mol of N-oxoammonium ion.

[0171] In Case 1, the pH of the reaction system is preferably adjusted to a region where hypochlorite is stably present and can well exert its oxidizing ability, for example, preferably adjusted to a range of pH 8 to 11.

[0172] When hypochlorite is used as a co-oxidant in the aldehyde group conversion step and chlorite is used as the oxidant in the carboxyl group conversion step (Case 2), the amount of carboxyl groups contained in the surface-modified ND particles can be controlled by adjusting the amount of chlorite used and the reaction time. For example, by reducing the amount of chlorite used, surface-modified ND particles with low carboxylation ratios can be selectively produced.

[0173] In Case 2, when chlorite is used in the carboxyl conversion step, if the chlorite oxidizes the aldehyde group or its equivalent and converts it into a carboxyl group, the chlorite itself is reduced to become hypochlorite. The hypochlorite thus generated can be used as a co-oxidant in the conversion step such as the aldehyde group.

[0174] In Case 2, the amount of hypochlorite used is, for example, 0.1 to 10 mol, preferably 0.3 to 3 mol, particularly preferably 0.5 to 1.5 mol, relative to 1 mol of N-oxoammonium ion.

[0175] In Case 2, the amount of chlorite used is, for example, 3 to 100 mol, preferably 4 to 75 mol, particularly preferably 5 to 50 mol, relative to 1 mol of the N-oxoammonium ion.

[0176] In Case 2, the usage ratio of chlorite to hypochlorite (the former / the latter; molar ratio) is, for example, 2 to 100, preferably 3 to 75, and particularly preferably 5 to 50.

[0177] In Case 2, the total amount of hypochlorite and chlorite used is, for example, 5 to 100 mol, preferably 5 to 75 mol, particularly preferably 5 to 50 mol, relative to 1 mol of N-oxoammonium ion.

[0178] In Case 2, the reaction can be carried out under mild conditions. The pH of the reaction system is, for example, 3-11, preferably 4-9.

[0179] An example of the reaction of Case 2 is shown below.

[0180]

[0181]

[0182] The reaction between ND particles and boron clusters is not particularly limited and can be carried out by a known and conventional method. As in the case where polyglycerol as a hydrophilic polymer is reacted with ND particles, a group containing a boron cluster can be introduced by reacting functional groups such as carboxyl groups and hydroxyl groups present on the surface of ND particles with boron clusters.

[0183] In the case where the surface-modified ND particles of the present disclosure are produced by reacting ND particles with a hydrophilic polymer and then reacting the obtained reaction product with a boron cluster, the boron cluster may react with the hydrophilic polymer chain in the reaction product or with the functional group present on the surface of the ND particles. In the case of the former reaction, the surface-modified ND of the present disclosure has a group containing a boron cluster via the hydrophilic polymer chain, and in the case of the latter reaction, the surface-modified ND of the present disclosure has a group containing a boron cluster as a surface modification group on the surface of the ND particles.

[0184] The reaction between ND particles and molecules having cancer cell recognition ability is not particularly limited and can be implemented by known conventional methods. As in the case where polyglycerol as a hydrophilic polymer is reacted with ND particles, groups having cancer cell recognition ability can be introduced by reacting functional groups such as carboxyl groups and hydroxyl groups present on the surface of ND particles with molecules having cancer cell recognition ability.

[0185] In the case where the surface-modified ND particles of the present disclosure are manufactured by reacting the ND particles with a hydrophilic polymer and then reacting the obtained reaction product with a molecule having the ability to recognize cancer cells, the molecule having the ability to recognize cancer cells may react with the hydrophilic polymer chain in the reaction product or with the functional group present on the surface of the ND particles. In the case of the former reaction, the surface-modified ND of the present disclosure has a group having the ability to recognize cancer cells via the hydrophilic polymer chain, and in the case of the latter reaction, the surface-modified ND of the present disclosure has a group having the ability to recognize cancer cells as a surface modification group on the surface of the ND particles.

[0186] It should be noted that, as ND particles before surface modification, there is no particular limitation on their manufacturing method, for example, detonation method ND (i.e., ND generated by detonation method, sometimes referred to as "DND"), high temperature and high pressure method ND (i.e., ND generated by high temperature and high pressure method), chemical vapor deposition method ND (ND formed by crushing diamond film by CVD method), etc. can be used. Among them, detonation method ND is preferably used from the perspective of being more excellent in dispersibility in the dispersion medium and the particle size of the primary particles being a single-digit nanometer scale.

[0187] As the detonation method ND, for example, a detonation method ND produced through the following generation step, acid treatment step, oxidation treatment step, drying step, surface functional group adjustment step, and pulverization / classification treatment step can be used.

[0188] The production process is to put explosives into a pressure-resistant container for detonation, seal the container in a state where the explosives coexist with the gas of the atmosphere, and detonate the explosives in the container. As the explosive, for example, a mixture of TNT and RDX can be used.

[0189] During detonation, explosives are used with partially incompletely burned and freed carbon as raw material, and the pressure and energy of the shock wave generated by the explosion are used to obtain a crude nanodiamond product (including nanodiamond aggregates, coal, and metal oxides such as Fe, Co, Ni derived from containers, etc.).

[0190] The acid treatment step is a step of removing metal oxides by causing a strong acid to act on the nanodiamond crude product in an aqueous solvent. Examples of the strong acid include hydrochloric acid, hydrofluoric acid, sulfuric acid, nitric acid, and mixtures thereof. The acid treatment temperature is, for example, 70 to 150° C. The acid treatment time is, for example, 0.1 to 24 hours. After the acid treatment, it is preferred that the solid component is washed with water, for example, by decantation, until the pH of the precipitate reaches, for example, 2 to 3.

[0191] The oxidation treatment step is a step of removing graphite from the raw nanodiamond product using an oxidant. As the oxidant, for example, a mixed acid (particularly a sulfuric acid / nitric acid weight ratio of, for example, 60 / 40 to 95 / 5) can be preferably used.

[0192] The amount of the oxidizing agent (particularly the mixed acid) used is, for example, 20 to 40 parts by weight relative to 1 part by weight of the crude nanodiamond product.

[0193] In addition, when the mixed acid is used as an oxidant, a catalyst may be used together with the mixed acid. By using a catalyst, the removal efficiency of graphite can be further improved. As the catalyst, for example, copper (II) carbonate and the like can be cited. The amount of the catalyst used is, for example, about 0.01 to 10 parts by weight relative to 100 parts by weight of the crude nanodiamond product.

[0194] The oxidation treatment temperature is, for example, 100 to 200° C. The oxidation treatment time is, for example, 1 to 24 hours.

[0195] The drying step is a step of evaporating the liquid component from the nano-diamond-containing solution obtained in the oxidation treatment step. For example, a spray drying device, an evaporator, a drying oven, etc. can be used to implement the drying step. Nano-diamond powder is obtained through this step.

[0196] The surface functional group adjustment step is a step for adjusting the surface functional groups of the nano-diamond powder obtained through the drying step. This step includes hydrogenation treatment and oxygen oxidation treatment. These treatments can be selected and implemented according to the purpose.

[0197] Hydrogenation treatment is a process in which nanodiamond powder is heated under a hydrogen-containing gas atmosphere. The temperature conditions of the hydrogenation treatment are, for example, 400 to 800° C. In addition, the hydrogen-containing gas may also be a mixed gas containing an inert gas in addition to hydrogen. As the inert gas, for example, nitrogen, argon, carbon dioxide, helium, etc. may be listed. The hydrogen concentration of the hydrogen-containing gas is preferably 0.1 to 99.9% by volume, more preferably 0.5 to 50% by volume, and more preferably 1 to 10% by volume. By implementing the hydrogenation treatment, the carboxyl group present on the nanodiamond surface may be removed.

[0198] Oxygen oxidation treatment is a treatment of heating nanodiamond powder in an oxygen-containing gas atmosphere. The oxygen oxidation treatment temperature is, for example, 250 to 500° C. The oxygen-containing gas is, for example, a mixed gas in which an inert gas is added to oxygen. The oxygen concentration of the mixed gas is, for example, 1 to 35% by volume. By implementing oxygen oxidation treatment, functional groups (such as hydroxyl groups, etc.) reactive with glycidol can be imparted to the surface of the nanodiamond.

[0199] When the nanodiamond obtained through the surface functional group adjustment step is in the form of aggregates (secondary particles), it is preferably subjected to a crushing treatment or a classification treatment. Examples of the crushing treatment include ultrasonic treatment, bead milling (eg, bead milling using zirconia beads), and the like.

[0200] The average particle size of the ND particles before surface modification is not particularly limited, for example, 100 nm or less, preferably 50 nm or less, more preferably 30 nm or less, and particularly preferably 10 nm or less. The lower limit of the average particle size of the ND particles before surface modification is, for example, 0.1 nm. The average particle size of the surface-modified ND can be measured by a dynamic light scattering method.

[0201] Each scheme disclosed in this specification may also be combined with any other feature disclosed in this specification. Each configuration and combination thereof in each embodiment is an example, and the addition, omission, substitution and other changes of the configuration may be appropriately made within the scope of the subject matter of this disclosure. In addition, each invention disclosed in this disclosure is not limited by the embodiment or the following examples, but only by the patent claims.

[0202] Example

[0203] Hereinafter, the present disclosure will be described in more detail based on examples, but the present disclosure is not limited to the examples.

[0204] [Example 1]

[0205] (Detonation ND, the production of "DND")

[0206] The formed explosives and electric detonators are placed inside a pressure-resistant container for detonation, and the container is sealed. The container is made of iron and has a volume of 15m 3 . As an explosive, 0.50 kg of a mixture of TNT and RDX was used. The mass ratio of TNT to RDX in the explosive (TNT / RDX) was 50 / 50. Next, the electric detonator was detonated to cause the explosive to detonate in the container (generation of ND and DND based on the detonation method). Next, the container and its interior were cooled by leaving it at room temperature for 24 hours. After the cooling, the crude DND product (containing aggregates of DND particles and coal) attached to the inner wall of the container was scraped off with a scraper, and the crude DND product was recovered.

[0207] The crude DND product obtained by repeatedly performing the above-mentioned steps was subjected to an acid treatment step. Specifically, 6 L of 10% by mass hydrochloric acid was added to 200 g of the crude DND product to obtain a slurry, and the slurry was heat-treated under reflux at normal pressure for 1 hour. The heating temperature in the acid treatment was 85°C to 100°C. Then, after cooling, the solid components (including DND aggregates and coal) were washed with water by decantation. The washing of the solid components by decantation was repeated until the pH of the precipitate was reduced from the low pH side to 2.

[0208] Next, an oxidation treatment process was performed. Specifically, after adding 6L of 98 mass % sulfuric acid and 1L of 69 mass % nitric acid to the precipitate (containing DND aggregates) obtained by decantation after the acid treatment to prepare a slurry, the slurry was heated under reflux at normal pressure for 48 hours. The heating temperature in the oxidation treatment was 140°C to 160°C. Then, after cooling, the solid component (containing DND aggregates) was washed with water by decantation. The initial supernatant after washing was colored, but the washing of the solid component by decantation was repeated until the supernatant became transparent by visual observation.

[0209] Next, the precipitate liquid (liquid containing DND aggregates) obtained by the water washing process was subjected to a drying process to obtain a dry powder (DND aggregates). As a drying process method in the drying process, evaporation to dryness using an evaporator was used.

[0210] Next, 4.5 g of the dry powder (DND aggregate) obtained through the above-mentioned drying process was placed in the furnace core tube of an atmosphere furnace (trade name "atmosphere tube furnace KTF045N1", manufactured by Koyo Thermo System Co., Ltd.), and after nitrogen was continuously circulated in the furnace core tube at a flow rate of 1 L / min for 30 minutes, the circulating gas was switched from nitrogen to a mixed gas of oxygen and nitrogen, and the mixed gas was continuously circulated in the furnace core tube at a flow rate of 1 L / min. The oxygen concentration in the mixed gas was 4% by volume. After switching to the mixed gas, the temperature in the furnace was raised to the heating setting temperature, i.e., 400°C. In terms of the heating rate, it was set to 10°C / min until 380°C, which was 20°C lower than the heating setting temperature, and then set to 1°C / min from 380°C to 400°C. Then, the temperature condition in the furnace was maintained at 400°C, and the DND powder in the furnace was subjected to oxygen oxidation treatment. The treatment time was set to 3 hours.

[0211] Next, the hydrogenation process was continued using the above-mentioned atmosphere furnace. Specifically, after nitrogen was continuously circulated at a flow rate of 1L / min for 30 minutes in an atmosphere furnace equipped with DND powder that had undergone an oxygen oxidation process, the circulating gas was switched from nitrogen to a mixed gas of hydrogen and nitrogen, and the mixed gas was continuously circulated in the furnace core tube at a flow rate of 1L / min. The hydrogen concentration in the mixed gas was 2% by volume. After switching to the mixed gas, the temperature in the furnace was raised to a heating set temperature of 600°C. The heating rate was set to 10°C / min. Then, the temperature condition in the furnace was maintained at 600°C, and the DND powder in the furnace was hydrogenated. The treatment time was set to 5 hours. As described above, hydrogenated DND powder was obtained.

[0212] Next, a crushing process was performed. Specifically, first, 0.9 g of the DND powder that had undergone the above-mentioned hydrogenation process and 29.1 ml of pure water were added to a 50 ml sample bottle and mixed to obtain about 30 ml of slurry. After adjusting the pH to 4 using 1 equivalent of hydrochloric acid, the slurry was subjected to ultrasonic treatment. During the ultrasonic treatment, an ultrasonic irradiator (trade name "ultrasonic cleaning machine AS-3", manufactured by AS ONE) was used to irradiate the slurry with ultrasonic waves for 2 hours. Then, a bead milling device (trade name "parallel four-cylinder sand mill LSG-4U-2L type", manufactured by IMEX Co., Ltd.) was used to perform bead milling. Specifically, 30 ml of the slurry after ultrasonic irradiation and zirconia beads with a diameter of 30 μm were added to a 100 ml volumetric flask (manufactured by IMEX Co., Ltd.) as a grinding container and sealed, and the device was driven to perform bead milling. In this bead mill, the amount of zirconium oxide beads introduced is, for example, 33% by volume relative to the volume of the grinding container, the rotation speed of the grinding container is 2570 rpm, and the grinding time is 2 hours.

[0213] Next, the slurry that has undergone the crushing process as described above was subjected to centrifugal separation treatment (classification operation) using a centrifugal separation device. The centrifugal force in the centrifugal separation treatment was set to 20,000×g, and the centrifugal time was set to 10 minutes. Next, 10 ml of the supernatant of the DND-containing solution that had undergone the centrifugal separation treatment was recovered. In this way, a DND dispersion in which DND particles were dispersed in pure water was obtained. Regarding the aqueous dispersion, the solid content concentration was 2.1% by mass and the pH was 5.40. The median diameter (particle size D50) of the DND aqueous dispersion obtained as described above was 35.8 nm.

[0214] (DND surface modified with polyglycerol, production of "DND-PG")

[0215] The DND aqueous dispersion (DND D50: 4.5 nm, trade name "Dinnovare ζ+ nano diamond aqueous dispersion", manufactured by DAICEL) was concentrated under reduced pressure using an evaporator and further dried at 105°C for 2 hours. The obtained DND powder (1.0 g) was suspended in ethylene glycol (15.0 g), and glycidol (45.1 g, 0.61 mol) was added dropwise over 160 minutes, maintaining the temperature in the range of 95-100°C. The obtained black dispersion was stirred at the same temperature for 4 hours. Water (40 mL) was slowly added to decompose the unreacted glycidol, and then the dispersion was diluted with water to 400 mL. Afterwards, an ultrafiltration membrane (Ultracel Membrane, 30kDa) was concentrated to less than 20mL. The concentrate was diluted and concentrated again, and this operation was repeated 5 times to wash, and purified DND-PG was obtained in the form of a black aqueous dispersion. The yield was 100.0g (DND-PG content was 4.10wt%, 4.10g).

[0216] A portion of the dispersion was dried on a heated PTFE sheet to obtain a sample for analysis. The analysis results are shown below.

[0217] FT-IR (diffuse reflectance method, cm -1 ): 3332, 2918, 2875, 1456, 1118, 1078.

[0218] 1 H-NMR (500 MHz, D 2 O): δppm 3.42, 3.50, 3.58, 3.75, 3.88.

[0219] TGA (under air atmosphere, 20°C / min) measurement was performed. The results showed that the weight loss (%) was as follows.

[0220] 374-530℃; 72.3%.

[0221] 530-798°C; 23.6%.

[0222] From this, the weight ratio of PG / DND (including free PG impurities) can be estimated to be 3.1.

[0223] (Removal of free PG impurities by ultracentrifugation)

[0224] The aqueous dispersion of DND-PG (30.0 g, solid content concentration 4.10%, DND-PG content 1.23 g) was subjected to ultracentrifugation (50000 rpm (183400×g)×2 hours) by ultracentrifugation (HIMAC CP 70MX ultracentrifuge). About 20 mL of the supernatant was extracted, and 20 mL of water was added to the remaining lower layer to make a uniform dispersion, and the operation of ultracentrifugation under the same conditions was repeated twice. The supernatant was extracted, and water was added to the remaining lower layer to remove free PG to obtain 30.8 g of a black aqueous dispersion of DND-PG. Among them, after adjusting the pH of 15.8 g of the dispersion to 1.93, it was diluted 5 times and concentrated using an ultrafiltration membrane (400 mL was concentrated to less than 10 mL) to obtain 15.77 g of a black dispersion. The content of DND-PG was 2.87% by weight (0.45 g as a solid content).

[0225] This revealed that the content of DND-PG in 30.8 g of the black aqueous dispersion of DND-PG was 0.88 g, and the recovery rate from the aqueous dispersion of DND-PG before ultracentrifugation was 71.5% by weight.

[0226] The zeta potential of the DND-PG aqueous dispersion at pH 8.1 was +18.0 mV.

[0227] A portion of the obtained black aqueous dispersion of DND-PG was dried on a heated PTFE sheet to obtain a sample. The obtained sample was used to perform DND-PG 13 C-NMR measurement. The results are shown in Figure 1 .

[0228] The TGA measurement of DND-PG (under air atmosphere, 20°C / min) was performed. As a result, the weight loss (%) was as follows. From this, the PG modification rate (PG / DND weight ratio) was estimated to be 2.0.

[0229] 360-528℃: 63.6%.

[0230] 528-613℃: 31.8%.

[0231] The obtained DND-PG was analyzed by FT-IR and the 1Its structure was confirmed by H-NMR. In the FT-IR spectrum, a -1 Absorption based on OH stretching at 2918 cm -1 and 2876cm -1 Asymmetric and symmetric stretching of CH at 1118 cm -1 and 1078cm -1 The COC asymmetry and CO stretching of the PG chain at 1 In H-NMR, the signal at 3 ppm to 4 ppm indicates that all hydrogen atoms in the PG chain are located on carbon atoms adjacent to oxygen functional groups such as hydroxyl groups and ether bonds.

[0232] (Production of DND-PG-COOH)

[0233] To 7.4 g of DND-PG aqueous dispersion (DND-PG content: 0.25 g) was added 12.47 g of water, and further 0.4 M sodium acetate buffer (pH 4.7, 12.51 g) was added. Sodium chlorite (51.2 mg, purity 81%, 0.46 mmol) and 4-acetamide-2,2,6,6-tetramethylpiperidinyl 1-oxyl radical (4-acetamide-TEMPO, 13.5 mg, 0.063 mmol) were added thereto, and further sodium hypochlorite aqueous solution (37.5 uL, concentration 12%, 0.060 mmol) was added, and stirred at 50°C for 24 hours. After adding ethanol to the reaction solution to decompose the remaining oxidant, it was filtered twice using an ultrafiltration membrane (diluted with water to 400 mL and concentrated to less than 10 mL). After adding 6 M hydrochloric acid (2.0 mL) to the concentrate to make it acidic, it was filtered and washed twice using an ultrafiltration membrane. The yield of the obtained DND-PG-COOH was 239 mg. In addition, by acid-base titration (indicator: phenolphthalein, titration using 0.05 M KOH alcohol solution), it was found that the -COOH content was 1.17 mmol / g.

[0234] The results of the various analyses of the obtained DND-PG-COOH are shown below. Figure 2 and Figure 3 .

[0235] FT-IR (KBr, diffuse reflectance, cm -1 ): 3273,2906,2878,1456,1734,1122,1080.

[0236] 1 H-NMR (500MHz, 0.5% NaOD): δppm 4.13, 3.97, 3.90, 3.58.

[0237] 13C-NMR (125MHz, 0.5% NaOD): δppm 179.1, 78.9, 72.8, 63.7, 61.5, 35.1.

[0238] Elemental analysis: C; 59.36%, H; 5.63%, N; 0.73%, O; 34.16%.

[0239] (Preparation of DND-PG(OTs)-COOH)

[0240] 10.6 g of the aqueous dispersion of DND-PG-COOH (DND-PG-COOH content is 0.15 g) was concentrated to dryness under reduced pressure. The residue was subjected to azeotropic dehydration with pyridine (10 mL) twice, and then the residue was dispersed in pyridine (5.0 mL). p-Toluenesulfonyl chloride (TsCl, 28.4 mg, 1.49 mmol) and N,N-dimethylaminopyridine (8.1 mg, 0.066 mmol) were added under ice cooling, and stirred at room temperature for 14 hours. Water was added to the reaction solution and centrifuged (30000 g × 10 min). The precipitate was dispersed in a small amount of tetrahydrofuran (THF), reprecipitated by adding water thereto, centrifuged, and the operation was repeated twice, thereby washing. The precipitate was further dispersed with a small amount of THF, and toluene was added thereto, reprecipitated, and centrifuged, and the operation was performed twice. The precipitate was heated and dried at 40°C under reduced pressure. DND-PG(OTs)-COOH was obtained as 194.3 mg of a black solid substance.

[0241] The analysis results of the obtained DND-PG(OTs)-COOH are shown below and Figure 4 .

[0242] FT-IR (KBr, diffuse reflectance, cm -1 ): 3408,2911,2875,1747,1597,1359,1176,1097,929,815,687,556.

[0243] (Preparation of DND-PG(BSH)-COOH)

[0244] Disodium mercaptoundecahydrododecaborate-10B (BSH, 91.1 mg, 0.43 mmol) was dissolved in a mixed solution of water (2.0 mL) and 10% (w / v) sodium hydroxide aqueous solution (0.18 mL), and acrylonitrile (0.05 mL, 0.76 mmol) was added, and stirred at room temperature for 3 hours. 1M hydrochloric acid (0.44 mL) was added to adjust the pH to 7-8, and then concentrated to dryness under reduced pressure. Acetonitrile (3.0 mL) was added to the residue, and concentrated to dryness. This operation was repeated twice, and the water was removed, and then dissolved in N,N-dimethylformamide (DMF, 2.0 mL) to obtain an acrylonitrile adduct solution of BSH.

[0245] DND-PG(OTs)-COOH (129.5 mg, estimated to contain 0.19 mmol of -OTs group) was dispersed in DMF (4.0 mL), and the total amount of the acrylonitrile adduct solution of BSH obtained by the above operation was added. The reaction solution was heated and stirred at 90°C for 48 hours, and then heated and stirred at 120°C for 2 hours. During the reaction, an acrylonitrile adduct solution of BSH prepared in the same manner as above from BSH (39.5 mg, 0.19 mmol) was added, and tetrabutylammonium iodide (21.4 mg, 0.058 mmol) was also added. After cooling, water (5.0 mL) was added to the reaction solution, and centrifugation was performed (30000 g×10 min). The precipitate was suspended in water and washed by centrifugation. Next, DMF (3.0 mL) and a 2% aqueous sodium hydroxide solution (1.25 mL) were added to the precipitate, and the mixture was stirred at 60°C for 4.5 hours. Water (15 mL) and 6 M hydrochloric acid (0.20 mL) were added to the reaction solution, and ultracentrifugation was performed (183400 g×10 minutes). Water (15 mL) was added to the precipitate to disperse it, and ultracentrifugation was performed to wash it, thereby obtaining DND-PG(BSH)-COOH.

[0246] The obtained DND-PG(BSH)-COOH 1 The results of H-NMR and FT-IR are shown below and Figure 5 and Figure 6 .

[0247] In DND-PG(BSH)-COOH 1 In H-NMR, multiple lines of BH derived from BSH (boron cluster) were confirmed at around 1.0 ppm. In addition, in FT-IR, multiple lines of BH derived from BSH (boron cluster) were confirmed at around 2501 cm -1 A peak derived from BH stretching vibration was confirmed at .

[0248] (Production of DND-PG(BSH)-PBA)

[0249] 64.9 mg of DND-PG(BSH)-COOH derived from DND-PG(OTs)-COOH (net content of about 39 mg) was dispersed in 3.0 mL of water, and 3.0 mL of methanol was added thereto. 3-Aminophenylboronic acid hydrate (7.3 mg, 0.047 mmol) and 4-methylmorpholine (25 uL, 0.23 mmol) were added, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM, 12.8 mg, 0.046 mmol) was further added, and stirred at room temperature for 13 hours. After adding 6M hydrochloric acid to the reaction solution to make it acidic, centrifugal separation was performed (30000g×10 minutes). The precipitate was dispersed with 10 mL of water and 0.20 mL of 1M sodium hydroxide aqueous solution, and concentrated by centrifugal filtration using Amicon Ultra15. The residue on the filter was dispersed in 15 mL of water and centrifuged for cleaning. Next, the mixture was washed once with a mixture of 10 mL of water and 1 mL of dimethyl sulfoxide, and then washed three times with water. The volume of the target product was adjusted to 5.0 mL with water, and the insoluble matter was removed by cotton plug filtration to obtain DND-PG(BSH)-PBA.

[0250] The obtained DND-PG(BSH)-PBA 1 The results of H-NMR are shown below and Figure 7 .

[0251] 1 H-NMR: A signal of BH derived from the boron cluster was observed at around 1.0 ppm, and a signal derived from the phenyl group of phenylboronic acid was observed at around 7.5 ppm.

[0252] It should be noted that the 1 H-NMR analysis was performed using ECX500 NMR spectrometer (JEOL). FT-IR analysis was performed using a Fourier transform infrared spectrophotometer "IRTracer" (manufactured by Shimadzu Corporation) equipped with a heated vacuum stirring reflection "Heat Chamber Type-1000°C" (manufactured by ST Japan Co., Ltd.). The same applies to the following experiments.

[0253] [Example 2]

[0254] DND-PG-COOH was prepared in the same manner as in Example 1.

[0255] (Preparation of DND-PG(OTs)-COOH)

[0256] 13.5 g of a DND-PG-COOH aqueous dispersion (DND-PG-COOH content: 0.40 g, 2.95% (w / w), PG / DND weight ratio = 3.85, -COOH content: 1.01 mmol / g), 2.45 mL of water, and 8 M NaOH (3.0 mL, 24 mmol) were added, and the mixture was cooled in an ice bath. While stirring vigorously, 0.58 g (3.0 mmol) of a solution of p-toluenesulfonyl chloride (TsCl) in 3.6 mL of tetrahydrofuran (THF) was added intermittently for about 1 hour. The reaction mixture was further stirred for 2 hours under ice-cooling conditions, and then 4.0 mL of 6 M hydrochloric acid was added for acidification, and stirred for several hours. The obtained reaction mixture was centrifuged (3000 rpm, 30 minutes), and a small amount of THF (2-3 mL) and water (10-15 mL) were added to the precipitate to wash it three times. Ultracentrifugation (183400 g × 10 minutes) was performed according to the dispersion state of the supernatant to obtain a precipitate. The precipitate was dispersed in 20 mL of N,N-dimethylformamide (DMF) and ultracentrifuged (183400 g × 30 minutes). This operation was repeated twice as a solvent replacement to obtain 16.5 g of DND-PG(OTs)-COOH DMF dispersion. The yield of DND-PG(OTs)-COOH was 0.27 g, and the concentration was 1.4% (w / w).

[0257] The analysis results of the obtained DND-PG(OTs)-COOH are shown below. Figure 8 (a) shows the result of FT-IR spectrum.

[0258] Elemental analysis: C; 56.85%, H: 6.53%, N; 0.51%, O; 29.08%, S; 4.08%. (DND-PG(N 3 )-COOH production

[0259] 196 mg (3.0 mmol) of sodium azide (NaN) was added to 13.8 g of DMF dispersion of DND-PG(OTs)-COOH (DND-PG(OTs)-COOH content was 0.20 g, 1.48% (w / w)). 3 ), 3.0 mL of water and 0.30 mL of 1 M NaOH (0.30 mmol). The resulting reaction mixture was stirred at 60°C for 0.5 hours, then the temperature was raised to 90°C and stirred for 18 hours. The resulting suspension was subjected to ultracentrifugation (183400 g × 2 hours), and the precipitate was washed three times with water by dilution and ultracentrifugation (183400 g × 30 minutes). The precipitate was dispersed in 30.0 g of water. DND-PG (N3 The yield of )-COOH was 0.17 g and the concentration was 0.57% (w / w).

[0260] The obtained DND-PG (N 3 The analysis results of )-COOH are shown below. Figure 8 (b) shows the result of FT-IR spectrum.

[0261] Elemental analysis: C; 55.28%, H: 5.54%, N; 5.46%, O; 30.96%.

[0262] ( 10 Production of BSH(Pgy)

[0263] 192 μL of 10 wt% NaOH aqueous solution (NaOH content: 19.2 mg, 0.48 mmol) was added to 4.0 mL of water, and the mixture was degassed under vacuum while being irradiated with ultrasound. 100.9 mg (0.48 mmol) of sodium undecylhydrazine-closed-dodecaboride ( 10 B)(Sodium mercaptoundecahydro-closo-dodecaborate( 10 B), 10 BSH) was dissolved in the NaOH aqueous solution obtained by the above operation, and then 60 μL of acrylonitrile (0.92 mmol) was added. The mixture was stirred at room temperature for 5 hours under a nitrogen atmosphere. The stirred mixture was washed three times with about 10 mL of ethyl acetate (AcOEt), and the water layer was evaporated to obtain 204 mg of S-(2-cyanoethyl)thioundecane-closed-dodecaborate ( 10 B)(S-(2-cyanoethyl)-thioundecahydro-closo-dodecaborate( 10 B), 10 BSH (AN)) as a colorless oil.

[0264] What you get 10BSH(AN) was dissolved in a mixture of 20.0 mL of acetonitrile (MeCN) and 5.0 mL of water, and 199 μL of a mixed solution of propargyl bromide (314 mg, 2.64 mmol) in MeCN (2.0 mL) / water (0.50 mL) was slowly added, followed by stirring at room temperature overnight. The reaction product was evaporated to dryness, MeCN was added to the residue, and it was evaporated again to remove the remaining water. About 10 mL of MeCN was added to the residue, and insoluble salts were removed by filtration. The filtrate was concentrated to obtain S,S-[(2-cyanoethyl)-(2-propynyl)]sulfonyl undecanoate-closed-dodecaborate ( 10 B)(S,S-[(2-cyanoethyl)-(2-propynyl)]sulfonioundecahydro-closo-dodecaborate( 10 B), 10 The crude product of BSH(AN)(Pgy)).

[0265] The obtained rough 10 BSH(AN)(Pgy) was dissolved in 2.0 mL of acetone, and 1.04 mL (0.96 mmol) of a 10 wt% tetramethylammonium hydroxide methanol solution was added to obtain a white precipitate, which was recovered by filtration. The residue was washed with a small amount of acetone and vacuum dried at room temperature to obtain 151 mg (theoretical yield: 167 mg) of ditetramethylammonium S-(2-propynyl)thioundecane-closed-dodecaborate ( 10 B)(bis-tetramethylammonium S-(2-propynyl)thioundecahydro-closo-dodecaborate( 10 B), 10 BSH(Pgy)·2TMA). The product may contain inorganic salts (NaBr) as impurities, but it was used in the subsequent click reaction without further purification. It should be noted that the above product is referred to as "crude 10 BSH(Pgy)·2TMA”.

[0266] Fig.10 Shown in 10 BSH(Pgy) (a) 1 H-NMR spectrum, (b) 10 B-NMR spectrum. The solvent used for the determination was DMSO-d6. 10 The synthesis scheme of BSH(Pgy) is shown below.

[0267]

[0268]

[0269] (DND-PG-( 10 Preparation of BSH)-COOH

[0270] To DND-PG(N 3 )-COOH aqueous dispersion 8.84 g (0.57% (w / w), DND-PG (N 3 4.0 mL of acetonitrile (MeCN) and 2.5 mL of 0.4 M phosphate buffer (pH 7.4) were added to 42.2 mg (0.21 mmol) of sodium ascorbate and 50.3 mg of crude 10 The mixture of BSH(Pgy)·2TMA was vacuum degassed. CuSO was slowly added to the mixture. 4 Aqueous solution (CuSO 4 The reaction was carried out by FT-IR (at 2102 cm -1 and 2503cm -1 Azide N 3 During the reaction, 24.8 mg (total amount of 3 additions) of crude 10 BSH(Pgy)·2TMA, 10.3 mg sodium ascorbate, 3.4 mg CuSO 4 , and further stirred for 2 days. The obtained dark brown dispersion was subjected to ultracentrifugation separation (183400g×30 minutes), and the precipitate was washed twice with 10mL of water, 10mL of an aqueous solution of ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt (EDTA·2Na) (0.3%), and 10mL of water. The concentrate on the filter was diluted with water to obtain 10.1g of brown dispersion. The concentration was calculated based on the weight after freeze-drying and was 0.50% (w / w), DND-PG ( 10 The yield of BSH)-COOH was 50.5 mg.

[0271] The obtained DND-PG( 10 The analysis results of BSH)-COOH are shown below. Figure 8 (c) shows the result of FT-IR spectrum. Fig. 9 (a) shows 1 H-NMR spectrum, Fig. 9(b) shows 13 C-NMR spectroscopy, Fig. 9 (c) shows 10 B-NMR spectrum. It should be noted that the measurement solvent is D 2 O.

[0272] Elemental analysis: C; 48.22%, H: 5.33%, N; 3.78%.

[0273] The sample was subjected to alkali fusion treatment, and the boron content was measured by ICP atomic emission spectrometry (ICP-AES) using SPS3100 (manufactured by SII Nano Technology Co., Ltd.), and was found to be 7.97%.

[0274] (DND-PG( 10 Production of BSH-PBA

[0275] To DND-PG( 10 BSH)-COOH aqueous dispersion 5.0g (DND-PG ( 10 2 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) aqueous solution (pH 4.5), 0.22 mL of 3-aminophenylboronic acid monohydrate DMF solution (3-aminophenylboronic acid monohydrate content 2.2 mg, 1.0 wt%, 0.014 mmol) and 0.50 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) aqueous solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride content 5.0 mg, 0.026 mmol) were added to a 1 M NaOH solution and stirred at room temperature for 15 hours. The reaction mixture was adjusted to pH 10 with 1 M NaOH and stirred for 45 minutes. The dispersion was filtered through a centrifugal filter (Amicon Ultra 30 kDa) and then washed with water 5 times. The concentrate on the filter was diluted with water to obtain 4.91 g of a brown dispersion. DND-PG( 10 The concentration of BSH)-PBA was calculated based on the weight after lyophilization to be 0.50% (wt / wt), and the yield was 24.6 mg.

[0276] The obtained DND-PG( 10 The analysis results of BSH)-PBA are shown below. Figure 8 (d) shows the result of FT-IR spectrum. Fig.11 (a) shows 1 The results of H-NMR spectrum. The solvent was determined to be D 2 O.

[0277] Elemental analysis: C; 51.35%, H: 5.10%, N; 4.16%.

[0278] From the change in nitrogen content in elemental analysis, the amount of PBA groups introduced was estimated to be 0.40 mmol / g.

[0279] (Production of DND-PG-PBA)

[0280] In addition to using DND-PG-COOH instead of DND-PG ( 10 BSH)-COOH, and the above-mentioned "DND-PG( 10 DND-PG-PBA was prepared in the same manner as described in "Preparation of BSH)-PBA".

[0281] (DND-PG-( 10 Preparation of BSH-c(RGDyK)

[0282] To 3.4g of DND-PG ( 10 BSH)-COOH aqueous dispersion (DND-PG( 10 2 mL of 0.4 M phosphate buffer (pH 7.4), an aqueous solution of N-hydroxysuccinimide (1.0 wt%, 0.47 mL), a content of N-hydroxysuccinimide of 4.7 mg), and an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (1.0%, 0.39 mL, and a content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride of 3.9 mg) were added to the mixture, and the mixture was stirred at room temperature for 30 minutes. Then, 7.4 mg of cyclic pentapeptide cyclo-(L-Arg-Gly-L-Asp-D-Tyr-L-Lys)(c(RGDyK)) was added, and the mixture was stirred at room temperature for 24 hours. The dispersion was filtered through a centrifugal filter (Amicon Ultra 30 kDa) and then washed with water 6 times. The concentrate on the filter was diluted with water to obtain 1.70 g of a dark brown dispersion. DND-PG( 10 The concentration of BSH)-c(RGDyK) was calculated based on the weight after freeze-drying to be 0.85% (wt / wt), and the yield was 14.5 mg. The introduction of c(RGDyK) was carried out by 1 The presence of two doublets (signals of Tyr aromatic rings) detected at 7.48 and 7.78 ppm in H-NMR was confirmed ( Fig.11 (b)). The amount of introduction was estimated to be 0.0053 mmol / g based on the integral value of the Tyr signal.

[0283] The obtained DND-PG(10 The analysis results of BSH)-c(RGDyK) are shown below. Figure 8 (e) shows the result of FT-IR spectrum. Fig.11 (b) shows 1 The results of H-NMR spectrum. The solvent was determined to be D 2 O.

[0284] (Production of DND-PG-c(RGDyK))

[0285] In addition to using DND-PG-COOH instead of DND-PG ( 10 BSH)-COOH, and the above-mentioned "DND-PG( 10 DND-PG-c(RGDyK) was prepared in the same manner as described in "Preparation of BSH)-c(RGDyK)".

[0286] The synthesis scheme of Example 2 is shown below.

[0287]

[0288]

[0289] The DND-PG-COOH obtained in Example 1 and the DND-PG-PBA, DND-PG-c(RGDyK), DND-PG( 10 BSH)-COOH, DND-PG( 10 BSH)-PBA, DND-PG( 10 BSH)-c(RGDyK) (hereinafter sometimes referred to as "nanoparticles") were evaluated as follows.

[0290] [Evaluation 1: Cytotoxicity test]

[0291] B16 mouse melanoma cells were cultured at 3 × 10 3 Cells were seeded in 96-well microplates in 100 μL Dulbecco's modified Eagle's medium (DMEM, glucose 4.5 g / L, 10% fetal bovine serum (FBS) and 1% 100× penicillin-streptomycin-amphotericin B) at 4 °C. 2The cells were cultured in an incubator at 37°C for 3 days. The culture medium (100 μL) was replaced, and phosphate buffered saline (PBS, pH 7.4, control) or PBS dispersion of nanoparticles (25 μL for 2500 μg / mL, 1250 μg / mL, and 625 μg / mL, respectively, and the concentrations in the culture medium were 500 μg / mL, 250 μg / mL, and 125 μg / mL, respectively) was added, and the cells were further cultured for 24 hours. After washing the cells with the culture medium (twice) and PBS, 100 μL of the culture medium was added, and 10 μL of CCK-8 (a mixture of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt (WST-8) and 1-methoxy-5-methylphenazine methylsulfate (1-methoxy PMS), manufactured by Dojindo Chemical Laboratories) was added. After 0.5 hours, the absorbance of each well at 450 nm was measured using a microplate reader. Fig.12 ).

[0292] [Evaluation 2: Confirmation of the BNCT effect of thermal neutron irradiation on cells]

[0293] The BNCT effect of nanoparticles was evaluated by thermal neutron irradiation of B16 mouse melanoma cells. The sample concentration of nanoparticles was determined based on the results of the above cytotoxicity test. The concentration of each nanoparticle was 500 μg / mL ( Fig.13 ). In DND-PG-c (RGDyK), it is 125 μg / mL ( Fig.13 ). In DND-PG( 10 BSH)-PBA: 125 μg / mL, 250 μg / mL ( Fig.13 )、100μg / mL、200μg / mL( Fig.14 ). In DND-PG( 10 BSH)-c(RGDyK) was 125 μg / mL ( Fig.13 )、100μg / mL、200μg / mL( Fig.14 ).

[0294] B16 mouse melanoma cells (8.8×10 5 or 1.0×10 6 ) were inoculated in a 100 nmφ dish containing 10 mL of DMEM medium (supplemented with 4.5 g / L glucose, 10% FBS, and antibiotics / antimycotics) and incubated at 37°C in a CO 2 The cells were cultured in an incubator for 2 days. The culture medium (10 mL, with or without FBS) was replaced once, and PBS or a PBS dispersion of nanoparticles (1.0 mL) was added to set the concentrations as specified above.2 After culturing at 37°C in an incubator for 22 to 24 hours, the cells were washed with PBS, and trypsin (0.5%, 2.0 mL) was added and treated at 37°C for 5 minutes to detach the cells. After stopping the trypsin treatment in the culture medium (with FBS added), the number of cells was counted, and after adjusting the concentration to 100,000 cells / mL, 1 mL of the cell suspension was dispensed into each of the four plastic tubes. The thermal neutron beam was irradiated with an irradiation dose in three stages (e.g., irradiation time of 5 minutes, 10 minutes, 15 minutes, and no irradiation (i.e., control)). It should be noted that the irradiation time of 5 minutes, 10 minutes, and 15 minutes was 4.09×10 11 ,7.48×10 11 and 1.17×10 12 neutrons / cm 2 The thermal neutron dose was determined by averaging the gold foil attached to the tube surface along the injection direction of the thermal neutrons. The cells were seeded in a 60 mmφ dish with 5 mL of culture medium (supplemented with FBS) at a specified number (300 to 10,000 cells / dish). 2 After culturing at 37°C in an incubator for 9 to 11 days, the obtained colonies were fixed with 70% ethanol and stained with 0.1% crystal violet solution. The number of colonies was counted by visual inspection (with the naked eye).

[0295] According to the above results, without 10 The colony growth was observed to be equivalent to that of the control group in the BSH nanoparticles, confirming the effectiveness of BNCT. 10 Only a small amount of colonies were formed in the BSH nanoparticles. Moreover, at high concentrations or high irradiation doses, no colonies were formed or only a small amount of colonies were formed ( Fig.13 ).

[0296] In addition, at low concentrations or low irradiation doses, 10 All samples in the BSH section passed 1.17×10 12 cm -2 The thermal neutrons under the condition of 200 μg / mL showed a good BNCT effect with a survival rate of less than 0.01 at a nanoparticle concentration of 200 μg / mL ( Fig.14 ). In addition, a concentration dependence on the injection volume was observed. DND-PG at 200 μg / mL ( 10 BSH)-PBA and DND-PG( 10 The gradient difference between BSH)-c(RGDyK) was significant at the same concentration.

[0297] [Evaluation 3: Observation of Nanoparticle Introduction into Cells Using Transmission Electron Microscopy (TEM)]

[0298] B16 cells were cultured in 400 μL of DMEM (containing 4.5 g / L glucose and 10% FBS) at a rate of 2.5 × 10 4 pcs / hole(3.0×10 4 cells / cm 2 ) were inoculated on chamber slides (Nagel Nunc177445). 2 After culturing at 37°C in an incubator for 3 days, the culture medium (400 μL) was replaced once, and PBS (control) or a PBS dispersion of nanoparticles at a specified concentration was added, and the cells were further cultured for 24 hours. The cells were washed with culture medium (twice) and PBS, and a mixture of 25% glutaraldehyde and 4% paraformaldehyde in 0.1M phosphate buffer (1 / 10 (v / v)) was added, and fixed in a refrigerator for several days. Next, the fixed cells were kept at 4°C together with 1.5% potassium ferrocyanide and 2% osmium tetroxide in deionized water (DW). After 1 hour, the cells were washed with DW and fixed with 2% osmium tetroxide in DW at room temperature for 1 hour. Next, the samples were stained overnight with a solution of 4% uranyl acetate dissolved in DW, and then washed with DW. The samples were then further stained with Walton's lead aspartate solution for 2 hours, dehydrated with a series of ethanol dilutions (60%, 70%, 80%, 90%, 99%, and 100%), and embedded in Epon 812. Thin sections were prepared using an ultramicrotome (Leica UC7). The sections were stained with uranyl acetate and lead citrate, and the introduction of nanoparticles into cells was confirmed by TEM observation using an H-7650 (transmission electron microscope, manufactured by Hitachi High-Tech Co., Ltd.) ( Fig.15 and Fig.16 ). In the figure, white arrows indicate the presence of vesicles caused by nanoparticles introduced into cells.

[0299] Fig.15 Indicates a) control, b) DND-PG ( 10 BSH)-PBA, c)DND-PG( 10 TEM images of BSH)-c(RGDyK) introduced into B16 cells. Fig.16 TEM images are shown when d) DND-PG-COOH, e) DND-PG-PBA, and f) DND-PG-c(RGDyK) were introduced into B16 cells.

[0300] In the TEM images b) and c) of "nanoparticles having a group containing a boron cluster and a group having the ability to recognize cancer cells", vesicles were significantly observed, which indicates that the corresponding nanoparticles were introduced into the cells. It should be noted that in the TEM images e) and f) of "nanoparticles having a group having the ability to recognize cancer cells", vesicles were significantly observed, which indicates that the corresponding nanoparticles were introduced into the cells. On the other hand, in the TEM image d) of "nanoparticles not having a group having the ability to recognize cancer cells", it was confirmed that the corresponding nanoparticles were not introduced into the cells at all. It should be noted that when b) and c) are compared with e) and f), an increase in the amount of the corresponding nanoparticles introduced into the cells is observed, and it is believed that the reason for this is the presence of boron clusters ( 10 The contribution of BSH) to non-specific introduction into cancer cells.

[0301] According to the above evaluations 1 to 3 (especially evaluations 2 and 3), it was confirmed that: 10 Nanodiamond particles with a group containing BSH) and a group with cancer cell recognition ability (PBA, c(RGDyK)) recognize specific cancer cells and are introduced into the cells, thereby exerting a BNCT effect due to the introduction into the cancer cells ( Fig.13 , Fig.14 , Fig.15 b) and c)). In addition, it was confirmed that there are no boron clusters ( 10 Nanodiamond particles with a group that has cancer cell recognition ability (PBA, c(RGDyK)) but not a group that has ... Fig.13 , Fig.16 e) and f)).

[0302] As a summary of the above, the configuration of the present disclosure and its modifications are described below.

[0303] [1] A surface-modified nanodiamond comprising: a nanodiamond particle; and a group containing a boron cluster and a group having cancer cell recognition ability as a surface modification group of the nanodiamond particle.

[0304] [2] The surface-modified nanodiamond according to [1], wherein the group containing boron clusters and the group having cancer cell recognition ability are present in the same surface modification group or in different surface modification groups.

[0305] [3] The surface-modified nanodiamond according to [1] or [2], wherein the surface-modified nanodiamond further has a group containing a hydrophilic polymer chain as the surface modification group.

[0306] [4] The surface-modified nanodiamond according to [3], wherein the hydrophilic polymer chain in the group containing a hydrophilic polymer chain is a polyglycerol chain.

[0307] [5] The surface-modified nanodiamond according to [3] or [4], wherein the group containing boron clusters, the group having cancer cell recognition ability, and the group containing hydrophilic polymer chains are present in the same surface modification group, or in different surface modification groups.

[0308] [6] The surface-modified nanodiamond according to any one of [1] to [5], wherein the group containing a boron cluster is bonded to the nanodiamond particle via a hydrophilic polymer chain.

[0309] [7] The surface-modified nanodiamond according to any one of [1] to [6], wherein the group containing the boron cluster is bonded to the hydrophilic polymer chain via an alkenyl group, a thioether bond, a sulfonium bond, a sulfoxide group, a secondary amine group, a tertiary amine group, a quaternary amine group (quaternary ammonium cation group), an amide bond, an ether bond, an ester bond, a thioester bond, a heterocycle (e.g., a 1,2,3-triazole ring), a carbonyl group, an acetal bond, or a group formed by bonding two or more thereof, and the hydrophilic polymer chain is bonded to the nanodiamond particle.

[0310] [8] The surface-modified nanodiamond according to any one of [1] to [7], wherein the group having cancer cell recognition ability is bonded to the nanodiamond particle via a hydrophilic polymer chain.

[0311] [9] The surface-modified nanodiamond according to any one of [1] to [8], wherein the group having cancer cell recognition ability is bonded to a hydrophilic polymer chain via an amide bond, an ester bond, or a thioester bond, and the hydrophilic polymer chain is bonded to the nanodiamond particle.

[0312]

[10] The surface-modified nanodiamond according to any one of [1] to [9], wherein the group having cancer cell recognition ability is a group derived from a molecule having cancer cell recognition ability, and the molecule having cancer cell recognition ability is boric acid, amino acid, peptide, protein, folic acid or porphyrin.

[0313]

[11] The surface-modified nanodiamond according to

[10] , wherein the boric acid is a compound represented by the following formula (A).

[0314] R a2 -B(-R a1 ) 2 (A)

[0315] (Where R a1represents a hydroxyl group which is optionally protected by a protecting group. a2 represents a hydrogen atom or a monovalent organic group).

[0316]

[12] The surface-modified nanodiamond according to

[11] , wherein the R a2 The monovalent organic group is a substituted or unsubstituted hydrocarbon group (monovalent hydrocarbon group, especially a monovalent aliphatic or aromatic hydrocarbon group); a substituted or unsubstituted heterocyclic group (monovalent heterocyclic group); a group formed by bonding two or more of the monovalent hydrocarbon group and / or the monovalent heterocyclic group.

[0317]

[13] The surface-modified nanodiamond according to any one of

[10] to

[12] , wherein the peptide is a peptide having an RGD sequence.

[0318]

[14] The surface-modified nanodiamond according to any one of

[10] to

[13] , wherein the peptide is at least one selected from the group consisting of GRGDNP (Gly-Arg-Gly-Asp-Asn-Pro), cyclo(-Arg-Gly-Asp-D-Phe-Cys), cyclo[-Arg-Gly-Asp-D-Phe-Lys(Cys)], cyclo(-Arg-Gly-Asp-D-Tyr-Lys) and cyclo(-Arg-Gly-Asp-D-Phe-Lys).

[0319]

[15] The surface-modified nanodiamond according to any one of [4] to

[14] , wherein the number average degree of polymerization of glycerol in the polyglycerol chain is 3 to 2000, 5 to 500 or 10 to 200.

[0320]

[16] The surface-modified nanodiamond according to any one of [1] to

[15] , wherein the boron cluster is selected from carborane, dodecaborate, 10 H 10 The boron ion cluster shown, and the molecular formula M(C 2 B 9 H 10 ) 2 At least one of the group consisting of the sandwich-type ion clusters shown (M is a transition element).

[0321]

[17] The surface-modified nanodiamond according to any one of [1] to

[16] , wherein the boron atoms in the group containing boron clusters are concentrated to 10 B.

[0322]

[18] The surface-modified nanodiamond according to any one of [1] to

[17] , wherein the nanodiamond particles include primary particles of nanodiamond or secondary particles formed by agglomeration (agglomeration) of a plurality of the primary particles.

[0323]

[19] The surface-modified nanodiamond according to any one of [1] to

[18] , wherein the nanodiamond particles are detonation-processed nanodiamonds.

[0324]

[20] A composition for boron neutron capture therapy comprising the surface-modified nanodiamond according to any one of [1] to

[19] .

[0325] Industrial Applicability

[0326] The surface-modified nanodiamond disclosed in the present invention can selectively and sufficiently accumulate boron atoms in cancer tissues and cancer cells, and has a high degree of lethality to cancer cells.

Claims

1. A surface-modified nanodiamond, the surface-modified nanodiamond having: Nanodiamond particles; and The surface modification groups of the nano-diamond particles include a group containing a boron cluster and a group having cancer cell recognition ability.

2. The surface-modified nanodiamond according to claim 1, wherein The surface-modified nanodiamond further has a group including a hydrophilic polymer chain as the surface-modifying group.

3. The surface-modified nanodiamond according to claim 2, wherein: The hydrophilic polymer chain in the group comprising a hydrophilic polymer chain is a polyglycerol chain.

4. The surface-modified nanodiamond according to claim 1 or 2, wherein: The group comprising boron clusters is bonded to the nanodiamond particles via a hydrophilic polymer chain.

5. The surface-modified nanodiamond according to claim 1 or 2, wherein: The group having cancer cell recognition ability is bonded to the nano-diamond particle via a hydrophilic polymer chain.

6. The surface-modified nanodiamond according to claim 1 or 2, wherein: The group having cancer cell recognition ability is bonded to the hydrophilic polymer chain via an amide bond, an ester bond or a thioester bond, The hydrophilic polymer chains are bonded to the nanodiamond particles.

7. The surface-modified nanodiamond according to claim 1 or 2, wherein: The group having the ability to recognize cancer cells is a group derived from a molecule having the ability to recognize cancer cells, The molecule having cancer cell recognition ability is boric acid, amino acid, peptide, protein, folic acid or porphyrin.

8. The surface-modified nanodiamond according to claim 1 or 2, wherein: The boron atoms in the group containing the boron cluster are condensed to 10 B.

9. A composition for boron neutron capture therapy comprising the surface-modified nanodiamond according to claim 1 or 2.

Images