Modified carbon nanomaterials, nanoclusters, substance delivery carriers and pharmaceutical compositions

Abstract

To provide a material that can effectively carry a physiologically active substance such as a pharmaceutical product or perfume, and also exhibits excellent solubility in a physiological environment.SOLUTION: The present invention provides a nanocluster in which a carbon nanomaterial modified with a C8-C14 alkyl group or a C8-C18 alkenyl group is self-assembled. The carbon nanomaterial is carbon nanodiamonds or carbon nanodots, with an average particle size of 1-100 nm.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to modified carbon nanomaterials, nanoclusters, substance delivery carriers and pharmaceutical compositions.

[0002] The following abbreviations are used herein: NDc: carboxylated nano diamond ND: Nano diamond NDc-ori: Unmodified carboxylated nanodiamond raw material SP: super particle NDc-SP: Nanoclusters of carboxylated nanodiamonds modified with alkyl groups PEG: polyethylene glycol PEGMEM: polyethylene glycol methyl ether methacrylate DSPE: Distearoylphosphatidylethanolamine F127: Pluronic® F127 CPT: Camptothecin PTX: Paclitaxel [Background technology]

[0003] Carbon nanodiamonds (hereinafter referred to as "CNDs") have the inherent properties of diamond, as well as the characteristics of a small average particle size and a large specific surface area. Furthermore, they have the advantages of being relatively inexpensive and easily available. CNDs can be produced by methods such as explosion and high-temperature and high-pressure methods (Patent Document 1). Because CNDs have low toxicity, excellent biocompatibility, and stable fluorescence properties, their application in the biomedical field has been widely studied. Furthermore, Patent Documents 2 and 3 disclose modified CNDs and methods for producing them.

[0004] As an example of the application of CND in the biomedical field, Non-Patent Document 1 reports that a cisplatin-CND complex was prepared by loading the anticancer drug cisplatin onto CND, that cisplatin can be released from the complex when the pH is in the acidic range, and that the drug released from the complex retains the same level of cytotoxicity as free cisplatin. Non-Patent Document 2 also reports that the anticancer drug epirubicin was adsorbed onto the surface of CND through hydrophobic interaction, and that the epirubicin-CND complex was further encapsulated in a lipid vesicle bound to an antibody (anti-EGFR-PEG) specific to the epidermal growth factor receptor (EGFR) of colon cancer, thereby increasing solubility, and reporting the drug accumulation action and efficacy against cancer cells.

[0005] Conventional complexes in which drugs are loaded onto CNDs have problems such as a low drug loading capacity and a significant decrease in dispersibility in water after complex formation.

[0006] It has been proposed to modify the surface of CND with a polymer in order to improve the solubility, dispersibility, and dispersion stability of CND in water and polar organic solvents. For example, Patent Document 4 reports that modifying the surface of nanodiamonds with specific groups containing polyglycerin chains significantly improves the solubility, dispersibility, and dispersion stability in water and polar organic solvents. However, conventional technology has basically focused on improving the dispersibility of CNDs, and there has been no research on evaluating the self-organizing ability of CNDs after surface chemical modification or on controlling nanostructures for the purpose of drug loading. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-202458 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-186234 [Patent Document 3] Japanese Patent Application Publication No. 2017-186235 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-248023 [Non-patent literature]

[0008] [Non-Patent Document 1] Bo Guan et al., Small 2010, 6, 1514-1519 [Non-patent document 2] Laura Moore et al., Adv. Mater. 2013, 25, 3532-3541 Summary of the Invention [Problem to be solved by the invention]

[0009] A main object of the present invention is to provide a material that can effectively carry physiologically active substances such as medicines and fragrances and that exhibits excellent solubility under physiological conditions. [Means for solving the problem]

[0010] The present invention provides the following modified carbon nanomaterials, nanoclusters, substance delivery carriers and pharmaceutical compositions. Item 1. Nanoclusters formed by self-assembly of carbon nanomaterials modified with higher alkyl or alkenyl groups. Item 2. The nanocluster according to Item 1, wherein the carbon nanomaterial is further modified with polyalkylene glycol. Item 3. The nanocluster according to Item 2, which is modified with polyethylene glycol. Item 4. A nanocluster according to any one of Items 1 to 3, wherein the higher alkyl group, higher alkenyl group, or polyalkylene glycol is linked to the carbon nanomaterial via a linking group selected from the group consisting of -NH-, -O-, -CO-O-, -O-CO-, -CO-NH-, -NH-CO-, -NH-CO-O-, -O-CO-NH-, -O-CO-O-, and -NH-CO-NH-. Item 5. The nanocluster according to any one of Items 1 to 4, wherein the carbon nanomaterial has at least one surface group selected from the group consisting of OH, COOH, and NH2, and a higher alkyl group or a higher alkenyl group is bonded to the carbon nanomaterial via the surface group. Item 6. The nanocluster according to Item 2, wherein the carbon nanomaterial has at least one surface group selected from the group consisting of OH, COOH, and NH2, and the polyalkylene glycol is bonded to the carbon nanomaterial via the surface group. Item 7. The nanocluster according to any one of Items 1 to 5, which is complexed with an active ingredient. Item 8. The nanocluster according to Item 7, wherein the active ingredient is a physiologically active substance, a labeling substance, a fragrance, an essential oil, or an organic dye. Item 9. The nanocluster according to any one of Items 1 to 8, wherein the carbon nanomaterial is a carbon nanodiamond or a carbon nanodot. Item 10. A delivery carrier for an active ingredient, comprising the nanocluster according to any one of items 1 to 9. Item 11. A carbon nanomaterial modified with a higher alkyl group and / or a higher alkenyl group and a polyalkylene glycol. Item 12. The carbon nanomaterial according to Item 11, wherein the higher alkyl group or higher alkenyl group and the polyalkylene glycol are linked by a linking group selected from the group consisting of -NH-, -O-, -CO-O-, -O-CO-, -CO-NH-, -NH-CO-, -NH-CO-O-, -O-CO-NH-, -O-CO-O-, and -NH-CO-NH-. Item 13. The carbon nanomaterial according to Item 11 or 12, wherein the carbon nanomaterial is carbon nanodiamond. Item 14. A pharmaceutical composition comprising the self-assembled nanocluster according to Item 1 complexed with a drug. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide surface-chemically modified carbon nanomaterial clusters that can effectively carry active ingredients such as physiologically active substances, fragrances, and organic dyes and that exhibit excellent solubility under physiological conditions, as well as composite materials such as pharmaceutical compositions containing the nanoclusters. [Brief explanation of the drawings]

[0012] [Figure 1] A method for synthesizing superparticles (SP) (NDc-SP) using nanodiamonds (NDc) with carboxyl groups. The carboxyl group of NDc-ori (a raw material consisting of NDc) and the amino group of an amino-terminated alkyl molecule are covalently bonded by a condensation reaction, and then ultrasonic irradiation is performed to prepare self-assembled NDc-SP nanoclusters. [Figure 2] a) Photographs of various NDc-SP aqueous solutions into which amino-terminated alkyl molecules with different alkyl chain lengths (C8-NH2, C12-NH2, C18-NH2) have been introduced. All ND concentrations are 5.6 mg ml-1. b) Particle size results from DLS measurements of various NDc-SP. It was found that the particle size increases depending on the alkyl chain length. This is thought to be because as the chain length increases, the hydrophobicity of the NDc surface increases, resulting in stronger interactions between NDc particles and resulting in larger particle sizes. c) TEM images of various NDc-SP. High-magnification images are shown in the upper left of each image. As with the results of DLS (dynamic light scattering), it was revealed that the actual particle size also increases as the alkyl chain length increases. Oct(C8) and Oct(C8)-NDc-SP represent nanoclusters self-assembled from NDc modified with an octyl group, Dod(C12) and Dod(C12)-NDc-SP represent nanoclusters self-assembled from NDc modified with a dodecyl group, and Ole(C18) and Ole(C18)-NDc-SP represent nanoclusters self-assembled from NDc modified with an oleyl group. [Figure 3]a) UV-Vis-NIR absorption spectra of various NDc-SPs and the raw material NDc-ori. The ND concentration was adjusted to 56 μg ml-1. b) Thermogravimetric (TGA) measurement results for NDc-ori and various NDc-SPs. These measurement results showed that the proportions of Oct (C8), Dod (C12), and Ole (C18) in the clusters of NDc-SP were approximately 13%, w / w, approximately 17%, w / w, and approximately 35%, w / w, respectively. [Figure 4] a) Cytotoxicity evaluation of various NDc-SPs. The viability of human osteosarcoma cells (U2OS) was measured after 24 hours of exposure to various NDc-SPs. b) UV-Vis-NIR absorption spectra before and after encapsulation of camptothecin (CPT) into Dod(C12)-NDc-SP. After CPT encapsulation, a clear peak derived from CPT was observed, suggesting that CPT was successfully incorporated into NDc-SP. c) Anticancer activity of CPT@NDc-ori, CPT@Oct(C8)-NDc-SP, and CPT@Dod(C12)-NDc-SP. Human U2OS cells were used, and the cell viability after 24 hours of exposure to various nanocomposites was shown. CPT@Dod(C12)-NDc-SP was found to have 10% higher anticancer activity than other nanocomposites. The long alkyl chain derived from Dod (C12) is thought to enhance affinity with cancer cells, resulting in its high anticancer activity. d) Comparative study of the anticancer activity of CPT-incorporated conventional nanomedicines (PEGMEM, F127, DSPE-PEG) and CPT@Dod(C12)-NDc-SP against U2OS after 24 hours of exposure. When CPT@Dod(C12)-NDc-SP was used, approximately 90% of cancer cells were killed. This result showed a maximum 57% improvement in pharmacological activity compared to PEGMEM (approximately 34% death), F127 (approximately 33% death), and DSPE-PEG (approximately 46% death). These results strongly suggest that NDc-SP is useful as a nanocarrier for anticancer drugs. [Figure 5]A method for synthesizing nanoclusters (NDa-SP) using nanodiamonds (NDa) with amino functional groups. The amino group of NDa-ori and the carboxyl group of a compound with a carboxyl-terminated alkyl chain (NaOc (C8), NaLA (C12), NaOle (C18)) are covalently bonded to the amino functional group through a condensation reaction, and self-assembled NDa-SP nanoclusters can be prepared by irradiating with ultrasound. [Figure 6] a) Photographs of aqueous solutions of the raw material NDa-ori and various NDa-SPs (NaOc (C8), NaLA (C12), NaOle (C18)) with different alkyl chains. The ND concentration in each aqueous solution is 3 mg ml-1. b) Particle size measured by DLS for Nda-ori and various NDa-SPs. It was found that particle size can be controlled depending on the alkyl chain length. c) TEM images of various NDa-SPs. It can be seen that particle size increases depending on the alkyl chain length, which complements the DLS results. d) High-magnification TEM images of Nda-ori and various NDa-SPs. NaOc(C8)-NDa-SP represents a nanocluster formed by self-assembly of NDa modified with octyl groups, NaLA(C12)-NDa-SP represents a nanocluster formed by self-assembly of NDa modified with dodecyl groups, and NaOle(C18)-NDa-SP represents a nanocluster formed by self-assembly of NDa modified with oleyl groups. [Figure 7]a) UV-Vis-NIR absorption spectra of NDa-ori and various NDa-SPs (NaOc(C8)-NDa-SP, NaLA(C12)-NDa-SP, NaOle(C18)-NDa-SP). The ND concentration in each aqueous solution was 30 μg ml-1. b) TGA measurements of NDa-ori and various NDa-SPs (NaOc(C8)-NDa-SP, NaLA(C12)-NDa-SP, NaOle(C18)-NDa-SP). The amounts of NaOc (C8), NaLA (C12), and NaOle (C18) in the NDa-SPs were found to be approximately 8%, w / w, approximately 17%, w / w, and approximately 24%, w / w, respectively. c) The amount of amino groups (NH2 loading) and interfacial tension in NDa-ori and various NDa-SPs. The Kaiser test was used to quantify the amino groups, and the amount of amino groups in each nanoparticle was calculated, with NDa-ori being set at 100%. Surface tension was measured for samples at a water temperature of 25°C and an ND concentration of 3 mg ml-1. It can be seen that the introduction of alkyl chains reduces the number of amino groups in the NDa-SPs. Furthermore, a decrease in surface tension was observed for all NDa-SPs compared to NDa-ori. This result suggests that the introduction of alkyl chains gives NDa-SP surfactant-like properties. d) Cytotoxicity evaluation of various NDa-SPs. In this study, U2OS cells were used, and cell viability was measured using WST-8 after 24 hours of exposure to various nanoparticles. Results indicated that all NDa-SPs (NaOc(C8)-NDa-SP, NaLA(C12)-NDa-SP, and NaOle(C18)-NDa-SP) exhibited low cytotoxicity. [Figure 8] Synthesis of polyethylene glycol (PEG)-coated NDa-SP (PEG-coated-NDa-SP or PEG-NDa-SP) and encapsulation of drugs within PEG-NDa-SP using non-covalent bonding. In this study, NaOle (C18)-NDa-SP (average particle size 167 nm), which has a large particle size, was not used for PEG modification. This is because it is said that nanoparticles with a size of 100 nm or less are optimal for the EPR effect, and NaOle (C18)-NDa-SP with a size of 100 nm or more was considered unsuitable for the experiment. [Figure 9]a) Photographs of 050GS-NaLA(C12)-NDa-SP loaded with paclitaxel (PTX) immediately after synthesis and 24 hours later. The ND and PTX concentrations in the nanocomposite were 0.3 and 0.1 mg ml-1, respectively. After 24 hours, a precipitate formed, indicating poor dispersion stability. Therefore, 050GS-NaLA(C12)-NDa-SP was not used in the following experiments. Note that 050GS indicates that PEG was loaded using the commercially available SUNBRIGHT® ME-050GS. b) Photographs of 8Arm-NaOc(C8)-NDa-SP and 8Arm-NaLA(C12)-NDa-SP immediately after synthesis and 24 hours later. Both nanocomposites used NDa-SP with an ND concentration of 3 mg ml-1. The use of 8Arm can improve the dispersibility of NDa-SP. However, the final product, 8Arm-NaOc(C8)-NDa-SP, was too dispersible, resulting in a low concentration of nanoparticles that could be recovered by centrifugation. Based on these results, 8Arm-NaLA (C12)-NDa-SP was used in subsequent experiments. 8-Arm indicates that a PEG group was introduced into NDa using the commercially available product 8-ArmPEG-SCM. [Figure 10]a) Water dispersibility of PTX-loaded PEG-modified NDa-SPs (050GS-NaOc(C8)-NDa-SP and 8Arm-NaLA(C12)-NDa-SP) and PEG-unmodified NDa-SPs (NaOc(C8)-NDa-SP and NaLA(C12)-NDa-SP) after 24 hours. The NDa and PTX concentrations in the nanocomposites were 3 mg ml-1 and 1 mg ml-1, respectively. NDa-SP coated with 050GS and 8Arm maintained high water dispersibility even after PTX loading. Based on these water dispersibility results after drug loading, we decided to use 050GS-NaOc(C8)-NDa-SP and 8Arm-NaLa(C12)-NDa-SP as drug carriers in subsequent experiments. b) Changes in the average particle size of PEG-NDa-SP before and after PTX loading. c) Evaluation of the aqueous dispersion stability of PTX-introduced PEG-NDa-SPs (050GS-NaOc(C8)-NDa-SP and 8Arm-NaLA(C12)-NDa-SP) over a 5-day period. The data show the DLS measurements (particle size) over time for PTX@050GS-NaOc(C8)-NDa-SP (left) and PTX@8Arm-NaLA(C12)-NDa-SP aqueous solutions (right). The internal photographs show the state immediately after synthesis and after 5 days. It can be seen that no aggregates are observed. [Figure 11] a) TEM images of 050GS-NaOc(C8)-NDa-SP and 8Arm-NaLA(C12)-NDa-SP. b) UV-Vis-NIR absorption spectra of PTX@050GS-NaOc(C8)-NDa-SP, PTX@8Arm-NaLA(C12)-NDa-SP, and PTX. Peaks derived from PTX were observed in each nanocomposite, confirming that PTX was successfully encapsulated, as shown in the conceptual diagram. [Figure 12]Anticancer activity evaluation of PTX-loaded PEG-NDa-SP. a) Cytotoxicity evaluation of 050GS-NaOc(C8)-NDa-SP (left) and 8Arm-NaLA(C12)-NDa-SP (right) against SKOV3, U2OS, and TIG3 after 24 hours of exposure. b) Comparative anticancer activity test of PTX@050GS-NaOc(C8)-NDa-SP and PTX@8Arm-NaLA(C12)-NDa-SP with the FDA-approved PTX formulation Abraxane. SKOV3 cells were exposed to various nanocomplexes 24 hours (left) and 48 hours (right). c) Phase contrast and crystal violet staining images of SKOV3 cells exposed to various nanocomplexes for 24 hours. The ND concentration in 050GS-NaOc(C8)-NDa-SP and 8Arm-NaLA(C12)-NDa-SP was 30 μg ml-1. The PTX and ND concentrations in PTX@050GS-NaOc(C8)-NDa-SP and PTX@8Arm-NaLA(C12)-NDa-SP were 10 ng ml-1 and 30 ng ml-1, respectively. The images clearly show that the anticancer activity of PTX@050GS-NaOc(C8)-NDa-SP and PTX@8Arm-NaLA(C12)-NDa-SP is significantly higher. DETAILED DESCRIPTION OF THE INVENTION

[0013] In this specification, carbon nanomaterials include carbon nanodiamonds and carbon nanodots.

[0014] The carbon nanomaterials used to produce nanoclusters are modified with higher alkyl or alkenyl groups. Carbon nanomaterials modified with these functional groups are sometimes referred to as "modified carbon nanomaterials."

[0015] The modified carbon nanomaterial of the present invention contains about 0.0001 to 30 mass %, preferably about 0.001 to 20 mass %, preferably about 0.01 to 15 mass %, preferably about 0.05 to 10 mass % of higher alkyl or higher alkenyl groups.

[0016] The higher alkyl or alkenyl group is linked to the carbon nanomaterial by a divalent linking group, such as -NH-, -O-, -CO-O-, -O-CO-, -CO-NH-, -NH-CO-, -NH-CO-O-, -O-CO-NH-, -O-CO-O-, or -NH-CO-NH-.

[0017] Examples of higher alkyl groups include straight-chain or branched C6-C6 alkyl groups such as hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, isohexadecyl, heptadecyl, octadecyl, isooctadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, and tetracosyl. 24 Alkyl groups, preferably C8-C 18 Alkyl groups, more preferably C8-C 14 alkyl group, more preferably C 10 -C 14 Examples of suitable alkyl groups include:

[0018] Examples of higher alkenyl groups include straight-chain or branched C6-C6 alkenyl groups such as hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl (palmitoleyl), isohexadecenyl, heptadecenyl, octadecenyl (oleyl, Ole), isooctadecenyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl, tricosenyl, and tetracosenyl. 24 Alkenyl groups, preferably C8-C 18 Alkenyl groups, more preferably C 12 -C 18 Alkenyl groups, more preferably C 14 -C 18 An example is an alkenyl group.

[0019] The carbon nanomaterials used in the production of nanoclusters of the present invention may be further modified with polyalkylene glycols, such as polyethylene glycol, polypropylene glycol, polybutylene glycol, and polyethylene glycol-polypropylene glycol block copolymers.

[0020] The average particle size of the primary particles of the carbon nanomaterial before modification with higher alkyl groups and / or higher alkenyl groups and polyalkylene glycol is preferably 10 nm or less, more preferably 1 to 10 nm.

[0021] The average particle size of the primary particles of the carbon nanomaterial modified with a higher alkyl group and / or a higher alkenyl group and a polyalkylene glycol is preferably 12 nm or less, more preferably 1 to 12 nm, and even more preferably 3 to 12 nm.

[0022] The average particle size of the primary particles of the carbon nanomaterial before or after modification may be measured by dynamic light scattering, or may be determined by small-angle X-ray scattering measurement (SAXS method) using an X-ray diffractometer (trade name "Smart Lab", manufactured by Rigaku Corporation).

[0023] The carbon nanomaterial used in producing the nanoclusters of the present invention is preferably a carbon nanomaterial (hereinafter sometimes referred to as "CNM") having many functional groups such as OH, COOH, and NH2 on its surface. Carbon nanomaterials having many groups such as OH, COOH, and NH2 on their surface are known, and such materials can be used to obtain carbon nanomaterials modified with higher alkyl or higher alkenyl groups according to the following schemes (1) to (10). Scheme

[0024] [ka]

[0025] [ka]

[0026] (In the formula, X represents Cl, Br, or I. CNM represents a carbon nanomaterial. n1 represents an integer of 1 or more. n2 represents an integer of 0 or more. R represents a higher alkyl group or a higher alkenyl group.) The reactions of the above schemes (1) to (10) can be carried out according to standard methods, and the target product can be obtained by using 1 mg or more of an excess of any of the compounds R-COX, R-NH2, R-OH, or RX per 1 g of carbon nanomaterial having OH, NH2, or COOH surface groups and reacting for 1 to 24 hours at temperatures ranging from 0°C to the boiling temperature of the solvent. Examples of solvents include halogenated hydrocarbons such as chloroform, methylene chloride, and 1,2-dichloroethane, aromatic hydrocarbons such as toluene, tetrahydrofuran, diethyl ether, and diisopropyl ether.

[0027] The compounds (Ia) to (Ij) obtained in the above schemes (1) to (10) can be further reacted with each unreacted functional group (OH, COOH, NH) to form a polyalkylene glycolation reagent (R 2 -Y 2 -X 2 ;where R 2 is a group containing a polyalkylene glycol moiety, Y 2 represents a single bond or a divalent spacer group, X 2represents NH2, OH, COOH, N-hydroxysuccinimide.) By reacting with , it can be modified with polyalkylene glycol. Divalent spacer groups include methylene, ethylene, propylene, butylene, phenylene (1,2-, 1,3-, 1,4-), -CH2CHO-, -CH2CH2CHO-, -CH2CH2CH2CH2O-, -CH2CH2CH2CH2O-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2CONH-, -CH2CH2CH2CONH-, -CH2CH2CH2CH2CONH-, -CH2CH2NHCO-, -C Examples include H2CH2CH2NHCO-, -CH2CH2CH2CH2NHCO-, -CH2CH2CO-, -CH2CH2CH2CO-, -CH2CH2CH2CO-, -CH2CH2CH2CH2CO-, -CH2CH2CH2CO-, -CH2CH2COO-, -CH2CH2CH2COO-, -CH2CH2CH2CH2COO-, -CH2CH2OCO-, -CH2CH2CH2C0CO-, and the like, and these may be used alone or in combination of two or more. The reaction to introduce polyalkylene glycol uses 100 mg or more in excess of a polyalkylene glycol reagent per 1 g of compounds (Ia) to (Ij), and the reaction is carried out at a temperature from 0°C to the boiling temperature of the solvent for 1 to 24 hours, thereby obtaining a carbon nanomaterial modified with the desired higher alkyl group and / or higher alkenyl group and polyalkylene glycol. Examples of the solvent include halogenated hydrocarbons such as chloroform, methylene chloride, and 1,2-dichloroethane, aromatic hydrocarbons such as toluene, tetrahydrofuran, diethyl ether, and diisopropyl ether.

[0028] The nanoclusters of the present invention can be produced by suspending carbon nanomaterials modified with higher alkyl groups and / or higher alkenyl groups, and optionally with polyalkylene glycol groups, in an appropriate aqueous medium such as water or a buffer solution, and then irradiating the suspension with ultrasound to cause self-assembly. After ultrasound irradiation, unreacted functional group-modified carbon nanomaterials that have not formed clusters can be removed by a purification procedure such as centrifugation, and the self-assembled nanoclusters can be isolated.

[0029] The nanoclusters of the present invention can be complexed with active ingredients by suspending them in a suitable solvent containing the active ingredients. The active ingredients are present on the surface or inside of the nanoclusters. Examples of active ingredients include physiologically active substances, labeling substances, essential oils, fragrances, and organic dyes.

[0030] Examples of labeling substances include fluoresceins such as fluorescein, Oregon Green, eosin, and erythrosine; rhodamines such as tetramethylrhodamine derivatives, Texas Red derivatives, rhodamine B base, Lissamine rhodamine B, and rhodamine 6G; coumarins; dansyl-type (dimethylaminonaphthalenesulfonic acid-type) fluorescent dyes; NBD-type dyes; pyrene; phycobiliproteins such as R-phycoerythrin, phlophycocyanin, and allophycocyanin; BODIPY derivatives; Cy (registered trademark) dyes such as Cy3, Cy3.5, Cy5, and Cy5.5; and Alexa (registered trademark) Flora dyes such as Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 633, 647, 680, 700, and 750. These may be used alone or in combination.

[0031] Examples of essential oils include sweet orange, bitter orange, petitgrain, lemon, grapefruit, lime, bergamot, mandarin, neroli, peppermint, spearmint, lavender, chamomile, rosemary, eucalyptus, sage, basil, rose, geranium, jasmine, ylang-ylang, anise, fennel, star anise, clove, cinnamon, ginger, nutmeg, cardamom, hops, cedar, cypress, vetiver, patchouli, and labdanum, and one or more of these may be used.

[0032] Fragrance ingredients include monoterpenes such as l-menthol, α-pinene, β-pinene, myrcene, camphene, and limonene, sesquiterpenes such as valencene, cedrene, caryophyllene, and longifolene, 1,3,5-undecatriene, butanol, pentanol, isoamyl alcohol, hexanol, prenol, (Z)-3-hexen-1-ol, 2,6-nonadienol, linalool, geraniol, citronellol, tetrahydromyrcenol, farnesol, nerolidol, cedrol, and benzyl alcohol. Alcohol, phenylethyl alcohol, furfuryl alcohol, acetaldehyde, isovaleraldehyde, hexanal, octanal, nonanal, decanal, (E)-2-hexenal, 2,4-octadienal, citronellal, citral, benzaldehyde, cinnamyl aldehyde, vanillin, ethyl vanillin, furfural, heliotropin, 2-heptanone, 2-undecanone, 1-octen-3-one, acetoin, diacetyl, 2,3-pentanedione, maltol, ethyl maltol 2,5-dimethyl-4-hydroxy-3(2H)-furanone, hydroxyketones, terpene ketones such as carvone, menthone, and nootkatone, α-ionone, β-ionone, β-damascenone, raspberry ketone, rose oxide, linalool oxide, menthofuran, theaspirane, methyl chavicol, anethole, ethyl acetate, isoamyl acetate, linalyl acetate, geranyl acetate, lavandulyl acetate, ethyl butyrate, ethyl caproate, benzyl acetate, methyl salicylate, γ-decalactone, and γ-dodecalactone. Examples of suitable alkyl esters include methyl acrylate, δ-decalactone, δ-dodecalactone, 7-decen-4-olide, 2-decen-5-olide, butyric acid, 4-methyl-3-pentenoic acid, octanoic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, indole, skatole, pyridine, alkyl-substituted pyrazine, methyl anthranilate, methanethiol, furfuryl mercaptan, dimethyl sulfide, dimethyl disulfide, difurfuryl disulfide, and allyl isothiocyanate, and two or more of these may be used in combination.

[0033] Organic pigments include annatto pigment, cochineal pigment, red koji pigment, beta-carotene, hibiscus pigment, surf yellow, cocoa pigment, riboflavin, chlorophyll, caramel, annatto, carmine, laccaic acid, brazilin, crocin, shikonin, shisonin, and rutin.

[0034] The nanoclusters of the present invention complexed with an active ingredient gradually release the active ingredient when administered or applied to the body or skin of mammals (e.g., humans, mice, rats, hamsters, horses, cows, pigs, goats, sheep, rabbits, dogs, cats, etc.). Therefore, the nanoclusters of the present invention complexed with an active ingredient are useful as pharmaceuticals or pharmaceutical compositions, cosmetics, and oral compositions. Examples of cosmetics include foundations, face powders, lotions, emulsions, lipsticks, skin toners, serums, massage creams, moisturizing creams, masks, facial cleansers, shampoos, conditioners, hair growth agents, and body powders. Examples of oral compositions include mouthwashes, mouth rinses, toothpastes, chewing gums, candies, gummies, and soda candies. Pharmaceutical dosage forms include tablets, capsules, lozenges, pills, chewable tablets, injections, suppositories, syrups, ointments, and plasters. Furthermore, because the nanoclusters of the present invention gradually release the active ingredient, they are useful as delivery vehicles for active ingredients.

[0035] The physiologically active substance includes a medicine, a nucleic acid, and a protein. Examples of the medicine include an antitumor agent, an antihypertensive agent, an antihypertensive agent, an antipsychotic agent, an analgesic, an antidepressant, an antimanic agent, an anxiolytic agent, a sedative, a hypnotic agent, an antiepileptic agent, an opioid agonist, an antiasthmatic agent, an anesthetic, an antiarrhythmic agent, an arthritis agent, an antispasmodic agent, an ACE inhibitor, a decongestant, an antibiotic, an antianginal agent, a diuretic, an antiparkinsonian agent, a bronchodilator, an antidiuretic, an antihyperlipidemic agent, an immunosuppressant, an immunomodulator, an antiemetic, an antiinfective agent, an antineoplastic agent, an anti Examples of such pharmaceuticals include antifungal agents, antiviral agents, antidiabetic agents, antiallergic agents, antipyretics, antigout agents, antihistamines, antipruritics, bone regulating agents, cardiovascular agents, cholesterol-lowering agents, antimalarials, antitussives, expectorants, mucolytics, nasal decongestant agents, dopaminergic agents, gastrointestinal agents, muscle relaxants, neuromuscular blocking agents, parasympathomimetics, prostaglandins, stimulants, appetite suppressants, thyroid or antithyroid agents, hormones, antimigraine agents, antiobesity agents, anti-inflammatory agents, etc. Preferred pharmaceuticals are antitumor agents. Examples of antitumor agents include hormone therapy agents (e.g., fosfestrol, diethylstilbestrol, chlorotrianicerin, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone, mepartricin, raloxifene, ormeloxifene, levormeloxifene, antiestrogens (e.g., tamoxifen citrate, toremifene citrate, etc.), birth control pills, mepitiostane, testololactone, aminoglutethimide, LH-RH agonists (e.g., goserelin acetate, buserelin, leuprorelin, etc.), droloxifene, epithiostanol, These include ethinyl estradiol sulfonate, aromatase inhibitors (e.g., fadrozole hydrochloride, anastrozole, letrozole, exemestane, vorozole, formestane, etc.), antiandrogens (e.g., flutamide, bicalutamide, nilutamide, etc.), 5α-reductase inhibitors (e.g., finasteride, epristeride, etc.), corticosteroids (e.g., dexamethasone, prednisolone, betamethasone, triamcinolone, etc.), androgen synthesis inhibitors (e.g., abiraterone, etc.), retinoids and drugs that slow the metabolism of retinoids (e.g., liarozole, etc.), and among these, LH-RH agonists (e.g., goserelin acetate,buserelin, leuprorelin, etc.), alkylating agents (e.g., nitrogen mustard, nitrogen mustard-N-oxide hydrochloride, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, carboquone, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine, estramustine sodium phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, etoglucide, carboplatin, cisplatin, miboplatin, Nedaplatin, oxaliplatin, altretamine, ambamustine, dibrospidium hydrochloride, fotemustine, prednimustine, pumitepa, ribomustine, temozolomide, treosulfan, trofosfamide, zinostatin stimalamer, carboquone, adzelesin, systostin, bizelesin), antimetabolites (e.g., mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, enocitabine, cytarabine, cytarabine ocfosfate, ancitabine hydrochloride), 5-FU drugs (e.g., fluorouracil, tegafur), anti-cancer antibiotics (e.g., actinomycin D, actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, etc.), aminopterin, leucovorin calcium, tabloid, butosin, calcium folinate, calcium levofolinate, cladribine, emitefur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim, idoxuridine, mitoguazone, tiazofurin, ambamustine), anti-cancer antibiotics (e.g., actinomycin D, actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, etc.), mycin, peplomycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, neocarzinostatin, mithramycin, sarkomycin, carzinophilin, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride), plant-derived anticancer agents (e.g., etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, vinorelbine, camptothecin, irinotecan hydrochloride), immunotherapeutic agents (BRM) (e.g.,Examples of such drugs include picibanil, krestin, sizofiran, lentinan, ubenimex, interferon, interleukin, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum, levamisole, polysaccharide K, and procodazole, as well as drugs that inhibit the action of cell growth factors and their receptors (for example, antibody drugs such as trastuzumab (Herceptin (trademark); anti-HER2 antibody), ZD1839 (Iressa), and Gleevec). Examples of cancers that can be treated with antitumor agents include colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, gastric cancer, biliary tract cancer, gallbladder and bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, prostate cancer, testicular tumors, bone and soft tissue sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, and brain tumors, with colorectal cancer, gastric cancer, head and neck cancer, lung cancer, breast cancer, pancreatic cancer, biliary tract cancer, and liver cancer being preferred.

[0036] The nucleic acid is not particularly limited and may be any of DNA, RNA, chimeric nucleic acid of DNA and RNA, DNA / RNA hybrid, etc. Furthermore, any one of mono-, tri-, or tri-stranded nucleic acids may be used, but is preferably single- or double-stranded. The nucleic acid may also be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, other oligomers with non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs)), or other oligomers containing special linkages (provided that the oligomer contains nucleotides with configurations that allow base pairing or base attachment as found in DNA or RNA). Furthermore, the nucleic acids may contain known modifications, such as those labeled as known in the art, capped, methylated, those in which one or more natural nucleotides are replaced with analogs, those with intramolecular nucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those with side chains such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) or sugars (e.g., monosaccharides, etc.), those with intercalating compounds (e.g., acridine, psoralen, etc.), those containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, and those with modified linkages (e.g., α-anomeric nucleic acids, etc.). Preferred nucleic acids include RNAs, such as siRNAs.

[0037] siRNA is a double-stranded oligo-RNA consisting of a nucleotide sequence homologous to the nucleotide sequence of the mRNA or initial transcription product of a target gene, or a partial sequence thereof (preferably within the coding region) (including intron portions in the case of initial transcription products), and its complementary strand. The length of the portion homologous to the target nucleotide sequence contained in the siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases), but is not particularly limited as long as it can induce RNA interference. The total length of the siRNA is also usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases), but is not particularly limited as long as it can induce RNA interference.

[0038] The relationship between the target nucleotide sequence and the sequence homologous to it contained in the siRNA may be 100% identical or may include base variations (the sequence may be within the range of at least 70%, preferably 80%, more preferably 90%, and most preferably 95% or more identity).

[0039] siRNA may have an additional base at the 5' or 3' end, consisting of 5 bases or less, preferably 2 bases, that does not form a base pair.The additional base may be DNA or RNA, but using DNA can improve the stability of siRNA.Such additional base sequences include, but are not limited to, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', uuuuu-3', etc.

[0040] The siRNA may be directed against any target gene. Preferably, the siRNA targets a gene whose enhanced expression is involved in the onset and / or exacerbation of the target disease. More specifically, the antisense nucleic acid for the gene may target a gene that is currently in clinical or preclinical development or a newly discovered gene.

[0041] The siRNA may be used alone or in combination of two or more types.

[0042] Examples of proteins include enzymes, receptors, antibodies, antigens, and cytokines such as interferons and interleukins.

[0043] The average particle size of the nanoclusters of the present invention is about 1 to 1000 nm, preferably about 3 to 800 nm, more preferably about 5 to 500 nm, even more preferably about 10 to 300 nm, and particularly preferably about 30 to 250 nm.

[0044] The zeta potential of the nanocluster is preferably about 5 to 30 mV, and more preferably about 10 to 25 mV.

[0045] When the nanocluster of the present invention is complexed with an active ingredient, the active ingredient is contained in an amount of about 5 to 50 parts by mass, preferably about 10 to 20 parts by mass, per 100 parts by mass of the nanocluster. [Example]

[0046] The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples. Example 1 <Experimental Method> 1. Synthesis of supraparticle composites using carboxylated nanodiamonds Carboxylated nanodiamonds (NDc) (particle diameter: 4-5 nm) were prepared by the detonation method according to the description in JP 2017-202940 or JP 2016-113333. The synthesized NDc was purified with nitric acid and calcined under a hydrogen gas atmosphere. Elemental analysis of NDc using an organic elemental analyzer (Micro Corder JM10; J-Science Lab Co., Ltd., Kyoto, Japan) revealed the following: C (86.92%), H (0.44%), and N (2.29%). The purified NDc was dispersed in distilled water using a bead mill (Sand Grinder LSG-4U; Aimex Co., Ltd., Tokyo, Japan). The NDc aqueous dispersion was then centrifuged to remove water-insoluble ND. The resulting supernatant solution (NDc-ori) was used for subsequent experiments. 1 ml of the NDc-ori aqueous dispersion (ND concentration = 56 mg ml) was added. -1), terminally aminated alkyl molecules (50 μl of n-octylamine (Oct (C8)), 50 μl of oleylamine (Ole (C18)), or 10 mg of dodecylamine (Dod (C12)) (all purchased from FUJIFILM Wako Pure Chemical, Osaka, Japan), and 10 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (WSC) (FUJIFILM Wako Pure Chemical) were added to 9 ml of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0, 100 mM) and irradiated for 5 min using a bath-type ultrasonicator (output = 80 W, frequency = 40 kHz) (USD-2R; AS ONE, Osaka, Japan). After 1.5 h of vigorously stirring at room temperature, the mixture was centrifuged and washed three times with Milli-Q water. 10 ml of Milli-Q water was added to the precipitate obtained by centrifugation, and the precipitate was redispersed by 10 minutes of ultrasonic irradiation using a pulsed ultrasonicator (VCX-600; Sonics, Danbury, CT, USA). The resulting carboxylated nanodiamond superparticle (NDc-SP) dispersion was used in subsequent experiments. The resulting carboxylated nanodiamond superparticle (NDc-SP) dispersions were of three types: Oct(C8)-NDc-SP, Dod(C12)-NDc-SP, and Ole(C18)-NDc-SP, all of which constituted nanoclusters (Figure 2(b) and Figure 2(c)). The ND concentration in the final product was approximately 5.6 mg ml -1 The contents of Oct (C8), Dod (C12), and Ole (C18) in the NDc-SP cluster were determined to be approximately 13%, 17%, and 35% w / w, respectively, by thermogravimetric analysis (Q 500; TA Instruments, New Castle, DE, USA).

[0047] CPT@Oct(C8)-NDc-SP and CPT@Dod(C12)-NDc-SP complexes were prepared as follows. The precipitate consisting of Oct(C8)-NDc-SP or Dod(C12)-NDc-SP washed by centrifugation was mixed with 10 mg of CPT (FUJIFILM Wako Pure Chemical) in 10 ml of Milli-Q water and subjected to pulsed ultrasonic irradiation for 10 minutes. CPT@NDc-ori was prepared by mixing 10 mg of CPT, 1 ml of NDc-ori aqueous solution, and 9 ml of Milli-Q water and subjecting the mixture to pulsed ultrasonic irradiation for 10 minutes. Both the CPT@Oct(C8)-NDc-SP and CPT@Dod(C12)-NDc-SP complexes formed nanoclusters (Figure 4(b), Figure 4(c), Figure 4(d)). CPT@PEGMEM, CPT@F127, and CPT@DSPE-PEG were prepared in a similar manner using 56 mg of poly(ethylene glycol) methyl ether methacrylate (PEGMEM) (Sigma-Aldrich, St. Louis, MO, USA), 56 mg of pluronic F127 (F127) (FUJIFILM Wako Pure Chemical), or 56 mg of N-(aminopropyl polyethyleneglycol)carbamyl-distearoylphosphatidyl-ethanolamine (DSPE-PEG) (Sunbright DSPE-020PA; Yuka Sangyo, Tokyo, Japan) instead of the ND precipitate.

[0048] 2. Synthesis of supraparticle composites using aminated nanodiamonds Aminated nanodiamonds (NDa) (particle size: 4-5 nm) were synthesized according to previously reported methods (VV Danilenko, Combust., Explos. Shock Waves 2005, 41, 577; VY Dolmatov, J. Superhard Mater. 2008, 30, 233; VY Dolmatov, V. Myllymaki and A. Vehanen, J. Superhard Mater. 2013, 35, 143). The synthesized NDa was purified with nitric acid and calcined under a hydrogen gas atmosphere. Elemental analysis of NDa [C (92.20%), H (0.74%), and N (2.30%) contents] was performed using an organic elemental analyzer (Micro Corder JM10; J-Science Lab Co., Ltd., Kyoto, Japan). The purified NDa was dispersed in distilled water using a bead mill (Sand Grinder LSG-4U; Aimex Co., Ltd., Tokyo, Japan). The NDa dispersion was then centrifuged to remove water-insoluble ND. The resulting supernatant dispersion (NDa-ori) was used for further experiments. 1 ml of the NDa-ori dispersion (ND concentration = 30 mg ml) was used. -1), 10 mg of carboxyl-terminated alkyl chains (sodium octanoate (NaOc (C8)), sodium laurate (NaLA (C12)), or sodium oleate (NaOle (C18)) (all purchased from FUJIFILM Wako Pure Chemical), and 10 mg of WSC were dissolved in 9 ml of MES buffer (pH 6.0, 100 mM) by sonication for 5 min using a bath-type ultrasonicator (output: 80 W, oscillation frequency: 40 kHz) (USD-2R; AS ONE, Osaka, Japan). The mixture was further stirred vigorously for 1.5 h at room temperature and then centrifuged to wash three times with Mill-Q water to remove unreacted materials. The pellet obtained by centrifugation was sonicated for 10 min using a pulsed ultrasonicator to obtain 10 mg of WSC. The resulting aminated nanodiamond superparticle (NDa-SP) dispersions were NaOc(C8)-NDa-SP, NaLA(C12)-NDa-SP, and NaOle(C18)-NDa-SP, all of which constituted nanoclusters (Figure 6(c) and Figure 6(d)).

[0049] The NDa-SP dispersion was subsequently used in experiments. The ND concentration in the NDa-SP dispersion (~3 mg ml -1 ) was measured using a UV-Vis-NIR spectrophotometer (Figure 7(a)). The concentrations of NaOc (C8) (~8%, w / w), NaLA (C12) (~17%, w / w), and NaOle (C18) (~24%, w / w) in NDa-SP were estimated using thermogravimetry (TGA) (Q 500; TA Instruments, New Castle, DE, USA).

[0050] The amino groups on the ND surface were quantified using a Kaiser test kit (60017-1EA; Sigma-Aldrich) according to a previously published method (Yue Yu et al. Nanoscale 10, 8969-8978 (2018).) (Figure 7(c)).

[0051] The surface tension of various NDa-SPs was measured by Mitsui Chemical Analysis & Consulting Service (Tokyo, Japan) using a surface tensiometer (CBVP-Z; Kyowa Interface Science Co.) ( Figure 7(c) ).

[0052] PEG-modified NDa-SP (PEG-NDa-SP) was synthesized as follows (Figure 8). 1 ml of MES buffer (pH 6.0, 500 mM) was added to 10 ml of NaOc(C8)-NDa-SP aqueous solution or 10 ml of NaLA(C12)-NDa-SP. The mixture was centrifuged (15,000 rpm, 10 min), and the clear supernatant was carefully removed. The pellet was then added to 5 ml of DMSO (FUJIFILM Wako Pure Chemical) containing 20 mg of α-succinimidyloxyglutaryl-ω-methoxy, polyoxyethylene (SUNBRIGHT ME-050GS; Yuka Sangyo, Tokyo, Japan) (050GS) or 20 mg of hexaglycerol octa(succinimidyloxyglutaryl) polyoxyethylene (8-ArmPEG-SCM; Funakoshi, Tokyo, Japan) (8Arm). The mixture was sonicated for 30 minutes and then vigorously stirred overnight at room temperature. Finally, 1 ml of MES buffer (pH 6.0, 100 mM) was added to the reaction mixture, which was then washed three times with Milli-Q water and centrifuged. The resulting pellet was redispersed in 10 ml of Milli-Q water by pulsed sonication for 10 minutes. The synthesized 050GS-coated NaOc(C8)-NDa-SP (050GS-NaOc(C8)-NDa-SP) aqueous solution (ND concentration: ~3 mg ml -1 ) and 8Arm-coated NaLA(C12)-NDa-SP (8Arm-NaLA (C12)-NDa-SP) (ND concentration: ~3 mg ml -1) were used in the subsequent experiments. These constituted nanoclusters (Fig. 10(b), Fig. 11(a), Fig. 11(b)). PTX@050GS-NaOc(C8)-NDa-SP and PTX@8Arm-NaLA(C12)-NDa-SP complexes were prepared as follows. The pellets of 050GS-NaOc(C8)-NDa-SP or 8Arm-NaLA(C12)-NDa-SP were washed by centrifugation, and 10 ml of Milli-Q water containing 10 mg of paclitaxel (PTX) (FUJIFILM Wako Pure Chemical) was added and pulsed sonication was performed for 10 min. The resulting mixture was stored at 4 °C until use. Abraxane was purchased from Taiho Pharmaceutical Co., Ltd. and used as is without chemical modification. These complexes formed nanoclusters (Figures 10(b), 11(a), and 11(b)).

[0053] 3. Physical property analysis of ND-SP The structure and morphology of the synthesized ND-SP were analyzed using a high-resolution transmission electron microscope (TEM) (accelerating voltage: 120 kV) (EM-002B; Topcon, Tokyo, Japan) (Figure 2(c), Figure 6(c), Figure 6(d), Figure 11(a)).

[0054] The particle size (hydrodynamic diameter) of ND-SP was determined by dynamic light scattering (DLS) (Photal FPAR-1000; Otsuka Electronics, Osaka, Japan) (Figures 2(b), 6(b), and 10(b)).

[0055] Spectroscopic analysis of ND-SP and the concentrations of ND, CPT, and PTX in the ND-SP complex were estimated using an ultraviolet-visible-near-infrared spectrometer (V-730 BIO; Jasco, Tokyo, Japan) (Figure 2(a), Figure 3(a), Figure 4(b), Figure 6(a), Figure 7(a), Figure 7(b), Figure 9(a), Figure 9(b), Figure 10(a), Figure 10(c), Figure 11(b)).

[0056] 4. Cell Culture and Cytotoxicity Assessment Human osteosarcoma cells (U2OS), human ovarian adenoma cells (SKOV3), and human normal diploid fibroblasts (TIG3) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan). They were cultured in a nutrient-rich medium containing 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, gentamycin, and penicillin-streptomycin (100 IU ml -1 The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY, USA) containing ATP and Hank's balanced salt solution (Life Technologies, Carlsbad, CA, USA). The cells were cultured at 37°C in a humidified chamber under a 5% CO2 atmosphere.

[0057] Cell viability was assessed using crystal violet staining (FUJIFILM Wako Pure Chemical) and the Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan) according to their respective manuals. Cells were plated in 96-well plates (5 × 10 3 cells well -1 ) and incubated overnight. The cells were then exposed to the drug or nanocomplex dispersion solution, washed with fresh culture medium, and incubated in CCK-8 solution. Finally, the absorbance at 450 nm was measured using a microplate reader (Infinite M200 PRO; Tecan, Mannedorf, Switzerland) to calculate the cell viability (Figures 4(a), 4(c), 4(d), 7(d), 12(a), 12(b), and 12(c)).

[0058] 5. Blood tests Complete blood counts (CBC) and biochemical parameters were measured by Japan SLC and Oriental Yeast Co. (Tokyo, Japan). Specifically, 10-week-old female BALB / cSlc mice (n = 5; average body weight = 21 g; Japan SLC) were injected with 200 μl of each sample [sterilized water containing ND-SP (NDc-SP: 1.12 mg kg )]. -1 ; NDa-SP: 30 mg kg -1 ), PBS buffer, or sterile water was administered via the tail vein. Blood samples were collected 4 weeks after administration of the nanocomplex (Tables 1 to 4).

[0059] 6. Statistical Analysis of Data In the data, ± indicates standard deviation, and n indicates the number of samples used. Statistical analysis of the data was performed using Student's t-test. *, **, and *** indicate p values ​​of < 0.05, < 0.005, and < 0.001, respectively.

[0060] [Table 1]

[0061] [Table 2]

[0062] After administering Dod(C12)-NDc-SP dispersion or PBS to the tail vein of mice, the complete blood count (CBC) and biochemical parameters were investigated 4 weeks later, and no significant difference was observed between the samples. This result indicates that Dod(C12)-NDc-SP has high biocompatibility.

[0063] [Table 3]

[0064] [Table 4]

[0065] Sterile water containing dispersed PEG-NDc-SP or sterile water alone was administered to the tail vein of mice, and the complete blood count (CBC) and biochemical parameters were investigated after 4 weeks. No significant difference was observed between the samples. This result indicates that PEG-NDc-SP has high biocompatibility.

Claims

1. C 8 -C 14 Alkyl group or C 8 -C 18 A nanocluster in which a carbon nanomaterial modified with an alkenyl group and a polyalkylene glycol is self-assembled, the carbon nanomaterial being a carbon nanodiamond or a carbon nanodot, the C8-C14 alkyl group or the C8-C18 alkenyl group being present on the surface and inside of the nanocluster, the polyalkylene glycol being present on the surface of the nanocluster, and the average particle size being 1 to 100 nm.

2. 2. The nanocluster of claim 1, which is modified with polyethylene glycol.

3. C 8 -C 14 Alkyl group or C 8 -C 18 3. The nanocluster according to claim 1 or 2, wherein the alkenyl group or polyalkylene glycol is linked to the carbon nanomaterial via any linking group selected from the group consisting of -NH-, -O-, -CO-O-, -O-CO-, -CO-NH-, -NH-CO-, -NH-CO-O-, -O-CO-NH-, -O-CO-O-, and -NH-CO-NH-.

4. The carbon nanomaterial is OH, COOH, and NH 2 The nanocluster according to any one of claims 1 to 3, having at least one surface group selected from the group consisting of: wherein a C8-C14 alkyl group or a C8-C18 alkenyl group and a polyalkylene glycol are bonded to the carbon nanomaterial via the surface group.

5. 10. A pharmaceutical composition comprising the self-assembled nanocluster of claim 1 complexed with a pharmaceutical.

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