Composite material containing self-assembled fullerene, method for producing the same, photothermotherapy drug, photodynamic therapy drug, and photoacoustic contrast agent

Abstract

To provide a composite material containing a self-organized fullerene, a method for producing the same, a photothermal therapy drug, a photodynamic therapy drug, and a photoacoustic contrast medium.SOLUTION: The composite material comprises a solid sphere of fullerene-aliphatic diamine in which fullerene and aliphatic diamine are crosslinked, and gold particles located on the surface of the solid sphere. The photothermotherapeutic agent, the photodynamic therapeutic agent, and the photoacoustic contrast agent include the composite material. The method for producing the composite material includes preparing a solid sphere of fullerene-aliphatic diamine in which fullerene and aliphatic diamine are crosslinked, and mixing the solid sphere with gold particles in a dispersion medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composite material containing self-assembled fullerenes, a method for producing the same, a photothermal therapy agent, a photodynamic therapy agent, and a photoacoustic contrast agent. [Background technology]

[0002] In recent years, self-assembled fullerene-ethylenediamine hollow spheres have been produced by the liquid-liquid interfacial deposition (LLIP) method, and are expected to be applied to supercapacitors (see, for example, Non-Patent Document 1).

[0003] On the other hand, it is known that a composite porous material containing nanoparticles that generate heat upon irradiation with near-infrared rays and a bioabsorbable polymer is used in photothermal therapy (see, for example, Patent Document 1).

[0004] It is expected that self-assembled fullerenes will be used in new applications. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2018-83780 [Non-patent literature]

[0006] [Non-Patent Document 1] Lok Kumar Shrestha et al.,Nanomaterials,2023,13,946 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above, an object of the present invention is to provide a composite material containing self-assembled fullerenes, a method for producing the same, a photothermal therapy agent, a photodynamic therapy agent, and a photoacoustic contrast agent. [Means for solving the problem]

[0008] The composite material of the present invention solves the above-mentioned problems by comprising fullerene-aliphatic diamine solid spheres in which fullerene and aliphatic diamine are crosslinked, and gold particles located on the surfaces of the solid spheres. The gold particles may have a shape selected from the group consisting of spheres, rods, stars, spindles, sea urchins, triangular prisms, rectangular parallelepipeds, and cubes. The fullerene is C 60 Fullerene, C 70 Fullerene, C 76 Fullerene, C 78 Fullerene, C 82 Fullerene, C 84 Fullerene, C 90 Fullerene, C 94 It may be based on a fullerene selected from the group consisting of fullerenes and derivatives thereof. The fullerene is C 60 It may be a fullerene or a derivative thereof. The solid spheres may be amorphous. The aliphatic diamine is -NH-(CH) m It may be represented by -NH2 (m is 1≦m≦10, * is a binding site). The aliphatic diamine may be ethylenediamine where m=2. The mass ratio of the gold particles to the solid spheres may be in the range of 0.5 to 2.0. The composite material may have an average particle size in the range of 320 nm to 380 nm. The gold particles may have an average particle size in the range of 25 nm to 55 nm. In the ultraviolet-visible absorption spectrum, the compound may have peaks in the wavelength ranges of 436 nm to 462 nm, 536 nm to 539 nm, and 675 nm to 705 nm. The absolute value of the zeta potential may be greater than 0 mV and equal to or less than 20 mV. The method for producing a composite material according to the present invention includes preparing solid spheres of fullerene-aliphatic diamine in which a fullerene and an aliphatic diamine are crosslinked, and mixing the solid spheres with gold particles in a dispersion medium, thereby solving the above-mentioned problems. The solid spheres may be prepared by a liquid-liquid interfacial precipitation method using the fullerene, the aliphatic diamine, and an aromatic hydrocarbon. The aromatic hydrocarbon may be selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, and 1,3,5-trimethylbenzene. The aliphatic diamine to fullerene may have a molar ratio of 0.9 to 3.0. It may further comprise post-annealing at a temperature in the range of 500°C to 750°C. The photothermal therapy agent according to the present invention contains the above composite material, thereby solving the above problems. The photodynamic therapy agent according to the present invention comprises the above composite material, thereby solving the above problems. The photoacoustic contrast agent according to the present invention comprises the above composite material, thereby solving the above problems. [Effects of the Invention]

[0009] The composite material of the present invention comprises a fullerene-aliphatic diamine solid sphere in which a fullerene and an aliphatic diamine are crosslinked, and gold particles positioned on the surface of the solid sphere. This composite material exhibits an excellent photothermal effect when irradiated with near-infrared light. This composite material functions as a photothermal therapy agent, a photodynamic therapy agent, and a photoacoustic contrast agent.

[0010] The method for producing a composite material of the present invention is advantageous because it requires only mixing gold particles with solid spheres of fullerene-aliphatic diamine, in which fullerene and aliphatic diamine are crosslinked, and does not require skilled techniques or expensive equipment. [Brief explanation of the drawings]

[0011] [Figure 1]Schematic diagram showing the composite material of the present invention. [Figure 2] Flowchart showing the steps for producing the composite material of the present invention [Figure 3] SEM image of a fullerene-aliphatic diamine solid sphere. [Figure 4] 1 shows SEM images of samples of Examples 1 to 4. [Figure 5] 1 shows STEM images of samples from Examples 1 to 4. [Figure 6] TEM image of the sample in Example 3 [Figure 7] 1 shows the ATR-FTIR spectrum (A), UV-vis spectrum (B), Raman scattering spectrum (C), and XRD pattern (D) of the samples of Examples 1 to 4. [Figure 8] Figure 1 shows the XPS spectra of the sample and solid spheres of Example 3 [Figure 9] Figure 1 shows XPS core level spectra of the sample and solid spheres of Example 3. [Figure 10] Graph showing thermogravimetric changes of the sample in Example 3 [Figure 11] 1 shows an SEM image of the sample of Example 3 after thermogravimetry. [Figure 12] Graph showing the zeta potential of samples in Examples 1 to 4 [Figure 13] Graph showing the photothermal effect of samples from Examples 1 to 4 DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Note that like elements are given like reference numerals and their description will be omitted.

[0013] FIG. 1 is a schematic diagram showing the composite material of the present invention.

[0014] The composite material 100 of the present invention comprises fullerene-aliphatic diamine solid spheres 110 in which fullerene and aliphatic diamine are crosslinked, and gold particles 120 located on the surface of the solid spheres 110. The inventors of the present application have found that such composite material 100 exhibits an excellent photothermal effect when irradiated with near-infrared light. Each component will be described in detail.

[0015] The composite material 100 has a spherical shape, reflecting the shape of the solid spheres 110. A spherical shape means that it can be recognized as a sphere when observed using an electron microscope or the like, and more specifically, in the present specification, a composite material 100 is considered to be spherical if the ratio of the longest diameter to the shortest diameter (aspect ratio), calculated using a scanning electron microscope (SEM), is in the range of 1.0 to 1.5.

[0016] The aspect ratio is calculated as follows: 100 particles are selected from an electron microscope image (for example, 1000x magnification), and their shortest and longest diameters are measured. The aspect ratio of each of the 100 particles is calculated, and the average value is taken as the aspect ratio of the composite material 100.

[0017] The average particle size of the composite material 100 is preferably in the range of 300 nm or more and 600 nm or less. The average particle size of the composite material 100 is more preferably in the range of 320 nm or more and 380 nm or less. If the average particle size is in this range, the composite material 100 can be easily introduced into the body as a photothermal therapy drug. The average particle size is the average of the longest diameters of 100 particles observed in an electron microscope image (e.g., 5000x magnification).

[0018] The solid spheres 110 have an aliphatic diamine on their surfaces. This makes the surfaces of the solid spheres 110 hydrophilic, making them suitable for use in the biomedical field. The aliphatic diamine is introduced during the self-assembly of the solid spheres 110, and is preferably -NH-(CH2) m It is represented by -NH2 (m is 1≦m≦10, * is a bonding site). Among them, ethylenediamine where m=2 is preferred from the viewpoint of yield and safety.

[0019] The fullerene constituting the solid sphere 110 is not particularly limited as long as it is a spherical compound based on fullerene and mainly composed of carbon atoms. 60 Fullerene, C 70 Fullerene, C 76 Fullerene, C 78 Fullerene, C 82 Fullerene, C 84 Fullerene, C 90 Fullerene, C 94 Based on a fullerene selected from the group consisting of fullerenes and derivatives thereof. The fullerene is preferably C 60 The solid spheres 110 are formed efficiently from fullerene or its derivatives by the method described below. The fullerene derivative refers to a compound in which at least a portion of fullerene is modified with a substituent other than ethylenediamine.

[0020] When the fullerene and the aliphatic diamine are crosslinked and self-assembly progresses, solid spheres 110 are formed, and at this time, depending on the degree of crosslinking between the fullerene and the aliphatic diamine, the solid spheres 110 may be amorphous. Naturally, the solid spheres 110 may be a mixture of amorphous and crystalline fullerenes.

[0021] Any particles can be used as the gold particles 120 as long as they contain gold as the main component. The gold particles 120 may have a shape selected from the group consisting of spheres, rods, stars, spindles, sea urchins, triangular prisms, rectangular parallelepipeds, and cubes. The amount of gold as the main component means that the composite material of the present invention contains 50% by mass or more of gold. This allows the composite material of the present invention to exhibit a photothermal effect.

[0022] The average particle size of the gold particles 120 is preferably in the range of 15 nm to 80 nm. The average particle size of the gold particles 120 is more preferably in the range of 25 nm to 55 nm. If the size is in this range, the gold particles 120 are combined with the solid spheres 110 and exhibit excellent photothermal effects. Here, too, the average particle size is the average of the longest diameters of 100 particles observed in an electron microscope image (for example, 20,000 to 30,000 times magnification).

[0023] The mass ratio of gold particles 120 to solid spheres 110 preferably falls within the range of 0.5 or more and 2.0 or less. Within this range, an excellent photothermal effect is exhibited. The mass ratio more preferably falls within the range of 1.0 or more and 2.0 or less. Within this range, an even more excellent photothermal effect is exhibited. The mass ratio even more preferably falls within the range of 1.5 or more and 2.0 or less. Within this range, an even more excellent photothermal effect is exhibited.

[0024] The solid spheres 110 and the gold particles 120 may be composited by conjugation. Conjugation here can mean either a physical bond such as contact or connection, or a chemical bond. However, if an N-C=O bond (approximately 401.7 eV) and an O=C bond (approximately 531.6 eV) are confirmed in the X-ray photoelectron spectroscopy (XPS) spectrum of the composite material in question, it can be said that the material is conjugated.

[0025] The composite material 100 of the present invention preferably has peaks in the wavelength ranges of 436 nm to 462 nm, 536 nm to 539 nm, and 675 nm to 705 nm in the ultraviolet-visible absorption spectrum, which allows the solid spheres 110 and gold particles 120 to be conjugated and exhibits an excellent photothermal effect.

[0026] The composite material 100 of the present invention preferably has a zeta potential in the absolute value range of greater than 0 mV to 20 mV or less. The gold particles 120 suppress the surface charge, which is advantageous for attaching / depositing oppositely charged particles or molecules. The composite material 100 of the present invention more preferably has a zeta potential in the absolute value range of greater than 0 mV to 15 mV or less. This is advantageous for attaching oppositely charged particles or molecules.

[0027] Next, a method for producing the composite material 100 of the present invention will be described. FIG. 2 is a flow chart showing the steps for producing the composite material of the present invention.

[0028] The composite material 100 of the present invention is manufactured by the following process. Step S210: A fullerene-aliphatic diamine solid sphere is prepared in which the fullerene and the aliphatic diamine are crosslinked. Step S220: The solid spheres and gold particles are mixed in a dispersion medium.

[0029] Each step will be described in detail. In step S210, the preparation method is not particularly limited as long as the above-mentioned solid spheres 110 can be obtained, but preferably, a liquid-liquid interfacial precipitation method (LLIP) using fullerene, aliphatic diamine, and aromatic hydrocarbon can be used.

[0030] Here, fullerene is the C 60 Fullerene, C 70 Fullerene, C 76 Fullerene, C 78 Fullerene, C 82 Fullerene, C 84 Fullerene, C 90 Fullerene, C 94 Selected from the group consisting of fullerenes and derivatives thereof. 60 Fullerene or a derivative thereof is preferred.

[0031] The aliphatic diamine is preferably NH2-(CH2) m It is represented by —NH2 (m is 1≦m≦10). Among them, ethylenediamine, where m=2, is preferable as the aliphatic diamine. This promotes self-organization of fullerenes, making it easier to obtain solid spheres having aliphatic diamines on their surfaces. The aliphatic diamine is mixed with fullerenes at a molar ratio of 0.9 to 3. Within this range, fullerenes and aliphatic diamines crosslink, promoting self-organization and producing solid spheres. The aliphatic diamine is preferably mixed with fullerenes at a molar ratio of 0.5 to 1.5. This allows for efficient production of solid spheres. The aliphatic diamine is more preferably mixed with fullerenes at a molar ratio of 0.8 to 1.2. This allows for efficient production of solid spheres.

[0032] The aromatic hydrocarbon is preferably selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, and 1,3,5-trimethylbenzene (mesitylene). These solvents are good solvents for fullerenes. In particular, combining 1,3,5-trimethylbenzene with ethylenediamine promotes self-organization of fullerenes, making it easier to obtain solid spheres.

[0033] The specific preparation can be carried out as follows. First, the selected fullerene (including a fullerene derivative) is dissolved in the aromatic hydrocarbon described above to obtain a saturated fullerene solution. Ultrasonic treatment may be performed during this process. The saturated fullerene solution is then added to a mixed solvent of an aliphatic diamine and an aromatic hydrocarbon, mixed, and incubated. The incubation may be performed at room temperature (15°C to 30°C) for 30 minutes to 10 hours. This results in solid spheres of fullerene-aliphatic diamine, in which the fullerene and the aliphatic diamine are crosslinked. The solid spheres may be centrifuged, washed, and freeze-dried to obtain a powder.

[0034] In step S220, the gold particles can be the gold particles described above, but commercially available gold particles can also be used, or they can be synthesized according to, for example, J. Li et al., Nanoscale, 8, 7992-8007 (2016). By synthesizing them using the above method, gold particles with different sizes and shapes (e.g., rods, stars, etc.) can be obtained.

[0035] In step S220, the dispersion medium may be water or a solvent containing water. Water may be tap water, distilled water, purified water, ultrapure water, deionized water, etc. The water-containing solvent may contain water, and may also contain other solvents. The other solvents are not particularly limited, but are preferably solvents that are miscible with water, such as alcohol (e.g., ethanol).

[0036] In step S220, the solid spheres and gold particles are mixed so that the mass ratio of the gold particles to the solid spheres is preferably in the range of 0.5 to 2.0. This range results in a composite material 100 with excellent photothermal effects. More preferably, the solid spheres and gold particles are mixed so that the mass ratio is in the range of 1.0 to 2.0. This results in a composite material 100 with even better photothermal effects.

[0037] In step S220, the mixture may be ultrasonicated at room temperature (15°C to 30°C) for 10 minutes to 5 hours. This allows the solid spheres and gold particles to undergo a conjugation reaction, resulting in a composite material. The composite material may be centrifuged, washed, and freeze-dried to obtain a powder.

[0038] Following step S220, the product obtained in step S220 may be post-annealed at a temperature ranging from 500°C to 750°C. This allows the gold particles on the surface to aggregate and grow. The heating time is not particularly limited, but may be, for example, in the range of 0.5 hours to 12 hours.

[0039] Next, applications of the composite material 100 of the present invention will be described. The composite material 100 of the present invention can function as a photothermal therapy drug. Photothermal therapy (PTT) is a treatment method that utilizes the fact that cancer cells are more susceptible to heat than normal cells, and causes a photothermal effect of gold particles near the cancer cells to kill them.

[0040] A method (method of use) for treating cancer using a photothermal therapy agent containing the composite material 100 of the present invention administered in vivo includes the following steps. Step S310: A photothermal therapeutic agent is administered into a living body. Step S320: Light is irradiated onto the living body, and cancer cells are destroyed by the heat generated within the living body.

[0041] In step S310, the living body may be a human or a non-human animal. The method of administration may be oral administration, intravenous injection, or direct application to the affected area after surgery. By providing a substance that promotes cellular absorption (e.g., surfactant, cyclodextrin, bioadhesive polymer, etc.) on the surface of the composite material 100 of the present invention, the composite material 100 functions as a photothermal therapy drug that is efficiently absorbed in the small intestine when administered orally. By providing a substance that binds to cancer cells (e.g., antibody, ligand, peptide, etc.) on the surface of the composite material 100 of the present invention, the photothermal therapy drug of the present invention will efficiently accumulate in cancer cells when administered orally or intravenously, allowing the cancer cells to be effectively destroyed by step S320.

[0042] In step S320, the light is preferably near-infrared light, specifically having a wavelength in the range of 700 nm to 1500 nm, more preferably 750 nm to 1300 nm, so that the photothermal therapeutic agent of the present invention can effectively generate heat and destroy cancer cells when irradiated with light.

[0043] The composite material 100 of the present invention can function as a photodynamic therapy drug. Photodynamic therapy (PDT) is a treatment method that utilizes the property of reactive oxygen species to damage the DNA of cancer cells, and generates reactive oxygen species from gold particles near the cancer cells, thereby killing the cancer cells.

[0044] A method for treating cancer using a photodynamic therapy agent containing the composite material 100 of the present invention administered in vivo (method of use) includes the following steps. Step S410: A photodynamic therapy agent is administered into a living body. Step S420: The living body is irradiated with light, and the cancer cells are destroyed by the active oxygen (singlet oxygen) generated in the living body.

[0045] Step S410 is the same as step S310 described above, and therefore a description thereof will be omitted.

[0046] In step S420, the light is preferably near-infrared light, specifically having a wavelength in the range of 700 nm to 1500 nm, more preferably 750 nm to 1300 nm, so that the photodynamic therapy agent of the present invention can effectively generate active oxygen and destroy cancer cells by irradiation with light.

[0047] The composite material 100 of the present invention can function as a photoacoustic imaging agent. Photoacoustic imaging (PAI) is the imaging of cancer, tumors, and other sites using the photoacoustic effect of gold particles.

[0048] A method (method of use) for imaging a site such as a cancer using a photoacoustic contrast agent containing the composite material 100 of the present invention administered into a living body includes the following steps. Step S510: A photoacoustic contrast agent is administered into a living body. Step S520: Light is irradiated onto the living body, and a photoacoustic signal generated within the living body is detected.

[0049] Step S510 is the same as step S310 described above, and therefore a description thereof will be omitted.

[0050] In step S520, the light is preferably near-infrared light, specifically having a wavelength in the range of 700 nm to 1500 nm, more preferably 750 nm to 1300 nm, thereby enabling the photoacoustic imaging agent of the present invention to generate a photoacoustic effect and image sites such as cancers and tumors.

[0051] The photothermal therapy agent, photodynamic therapy agent, and photoacoustic contrast agent of the present invention may contain a dispersion medium together with the composite material 100 of the present invention. The dispersion medium may be, for example, physiological saline, distilled water for injection, phosphate buffer solution, or the like. The photothermal therapy agent, photodynamic therapy agent, and photoacoustic contrast agent may further contain a pharmacologically acceptable additive as necessary. Naturally, the photothermal therapy agent, photodynamic therapy agent, and photoacoustic contrast agent of the present invention may be combined. For example, after imaging the affected area with the photoacoustic contrast agent, cancer cells may be destroyed with a phototherapeutic agent made of the same material as the photoacoustic contrast agent.

[0052] Next, the present invention will be described in detail using specific examples, but it should be noted that the present invention is not limited to these examples. [Example]

[0053] [Preparation of fullerene-aliphatic diamine solid spheres (FE-SS)] Fullerene-aliphatic diamine solid spheres, in which fullerene and aliphatic diamine were crosslinked, were prepared using the dynamic liquid-liquid interfacial precipitation method (DLLIP) (step S210 in Figure 2).

[0054] Specifically, fullerene C is used as the raw material fullerene. 60 140 mg of powder (purity 99.5%, manufactured by MTR Ltd.) was dissolved in 140 mL of 1,3,5-trimethylbenzene (mesitylene) (purity 98.0%, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and ultrasonicated for 60 minutes to prepare a fullerene saturated solution (hereinafter referred to as unit fullerene solution). The fullerene concentration in the fullerene solution was 1 mg / mL.

[0055] 0.55 mL of ethylenediamine (EDA, purity 99.0%, Fujifilm Wako Pure Chemical Industries, Ltd.) as an aromatic diamine and 5 mL of mesitylene as an aromatic hydrocarbon were placed in a glass bottle and ultrasonicated for 30 minutes to prepare 5.55 mL of EDA / mesitylene solution. Ethylenediamine is NH2-(CH2) m In -NH2, m=2.

[0056] 5 mL of the fullerene solution was added to 5.55 mL of the EDA / mesitylene solution at room temperature (25°C). 60 The molar ratio of EDA to fullerene was 1. The mixture was then stirred for 10 seconds and incubated at 25°C for 1 hour, yielding a brown precipitate of fullerene-aliphatic diamine solid spheres (referred to as FE-SS) in which fullerene and aliphatic diamine were crosslinked. The mixture was centrifuged (3000 rpm) for 3 minutes, washed three times with isopropanol (IPA), and freeze-dried for 48 hours to obtain FE-SS powder. The FE-SS powder was observed using a field-emission scanning electron microscope (SEM, Hitachi High-Tech Corporation, S-4800). The observation results are shown in Figure 3.

[0057] FIG. 3 shows an SEM image of a fullerene-aliphatic diamine solid sphere.

[0058] According to Figure 3, fullerene C was synthesized by the liquid-liquid interface deposition method. 60 It was found that the ethylenediamine crosslinked with EDA and self-assembled into a spherical shape, resulting in solid spheres. The aspect ratio of the solid spheres was calculated to be in the range of 1.0 to 1.5, and the particle sizes were all 300 to 600 nm, with an average particle size of 310 nm. X-ray photoelectron spectroscopy (XPS) of the solid spheres confirmed that the solid spheres had ethylenediamine on their surfaces.

[0059] [Preparation of gold nanoparticles (AuNS)] 3 mL of distilled water and 2 mL of HEPES buffer (Dojindo Laboratories, 100 mM, pH 7.4) were mixed for 10 seconds in a glass bottle. Next, this mixture was mixed with 50 μL of chloroauric acid trihydrate solution (Sigma-Aldrich, 20 mM) and incubated at 25°C for 90 minutes to obtain a blue-black dispersion containing gold nanostars (AuNS). The AuNS dispersion was stored at 4°C.

[0060] [Example 1 to Example 4] In Examples 1 to 4, the prepared solid spheres (FE-SS) and gold particles (AuNS) were mixed in a dispersion medium (step S220 in FIG. 2 ), and composite materials with mass ratios of AuNS to FE-SS of 0.5, 1, 1.5, and 2, respectively, were obtained.

[0061] Specifically, as shown in Table 1, solid sphere (FE-SS) powder was added to distilled water and sonicated for 15 minutes to obtain a uniform solid sphere dispersion (1 mg / mL). This solid sphere dispersion was mixed with gold particle (AuNS) dispersions of adjusted concentrations (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL) and sonicated for 30 minutes. The black product formed in the mixture was centrifuged (2000 rpm) for 3 minutes, washed twice with distilled water, and freeze-dried for 48 hours. The products thus obtained are referred to as Sample 1 (FE-SS / AuNS0.5), Sample 2 (FE-SS / AuNS1), Sample 3 (FE-SS / AuNS1.5), and Sample 4 (FE-SS / AuNS2), respectively.

[0062] [Table 1]

[0063] The samples of Examples 1 to 4 were observed using a field emission scanning electron microscope (SEM, manufactured by Hitachi High-Technologies Corporation, S-4800) and a field emission transmission electron microscope (TEM, manufactured by JEOL Ltd., JEM-2100F).

[0064] The surface functional groups of the samples of Examples 1 to 4 were identified using an attenuated total reflection Fourier transform infrared spectrophotometer (ATR-FTIR, Thermo Fisher Scientific, Nexus 670). The ultraviolet-visible (UV-vis) spectra of the samples of Examples 1 to 4 were measured using a spectrophotometer (JASCO Corporation, V-670).

[0065] The Raman spectra of the samples of Examples 1 to 4 were measured using a triple laser Raman spectrometer (Jobin Yvon, T64000). A laser with a wavelength of 514.5 nm and an output of 0.01 mW was used for the measurements. The samples of Examples 1 to 4 were identified using an X-ray diffractometer (XRD, Rigaku Corporation, RINT-Ultima III).

[0066] X-ray photoelectron spectra of the samples in Examples 1 to 4 were measured using a Theta Probe Spectrometer (manufactured by Thermo Electron). Al-Kα monochromatic radiation (energy 15 keV) was used for the measurements. Core-level XPS C1s (energy peak position of the C 1s orbital), O1s (energy peak position of the O 1s orbital), N1s (energy peak position of the N 1s orbital), and Au4f (energy peak position of the Au 4f orbital) were recorded in 0.05 eV steps. A built-in electroflood gun was used for the measurements to prevent charge accumulation on the measurement samples.

[0067] Thermogravimetric analysis of the samples of Examples 1 to 4 was measured using a thermogravimetric analyzer (NETZSCH-Geratebau GmbH, STA2500 Regulus). In addition, SEM images of the samples of Examples 1 to 4 after thermogravimetric analysis were observed.

[0068] The zeta potentials of the samples of Examples 1 to 4 were measured using a zeta potential measuring device (Zetasizer Nano-ZS ZEN 3600, manufactured by Malvern Instruments). The photothermal effects of the samples of Examples 1 to 4 were measured. Specifically, a near-infrared laser (wavelength 808 nm, output 0.8 mW) was irradiated onto the samples (1.0 mg) of Examples 1 to 4 for 4 minutes, and the temperatures were measured before and after irradiation.

[0069] The above results are shown in FIGS. 4 to 13 and will be explained together.

[0070] FIG. 4 shows SEM images of the samples of Examples 1 to 4. As shown in FIG. FIG. 5 shows STEM images of the samples of Examples 1 to 4. As shown in FIG. FIG. 6 shows a TEM image of the sample of Example 3.

[0071] Figures 4(A) to (D) are SEM images of the sample of Example 1 (FE-SS / AuNS0.5), the sample of Example 2 (FE-SS / AuNS1), the sample of Example 3 (FE-SS / AuNS1.5), and the sample of Example 4 (FE-SS / AuNS2), respectively.

[0072] Figures 5(A) to (D) are STEM images of the sample of Example 1 (FE-SS / AuNS0.5), the sample of Example 2 (FE-SS / AuNS1), the sample of Example 3 (FE-SS / AuNS1.5), and the sample of Example 4 (FE-SS / AuNS2), respectively.

[0073] 6(A) and (C), and (B) and (D) are TEM images of the solid sphere (FE-SS) and the sample of Example 3 (FE-SS / AuNS1.5), respectively.

[0074] Figures 4, 5, and 6(B) show that the samples of Examples 1 to 4 were all spheres with particles on their surfaces. Comparing Figures 3 and 6(A) with Figures 4, 5, and 6(B) suggests that mixing solid spheres (FE-SS) and gold particles (AuNS) results in a composite material with gold particles attached to the surface of the solid spheres. Figure 4 shows that the amount of gold particles attached increases as the mass ratio of gold particles to solid spheres increases. However, as shown by the arrows in Figure 4(D) of the sample of Example 4 (FE-SS / AuNS2), aggregation of gold particles was observed, suggesting that a mass ratio of gold particles to solid spheres of 2.0 or less is preferable.

[0075] The particle size of the sample of Example 3 shown in Figure 6(B) was 350 nm, and its average particle size was 340 nm. The average particle sizes of the samples of Examples 1, 2, and 4 also fell within the range of 320 nm to 380 nm. Figure 6(D) shows that the gold particles (AuNS) on the surface of the sample of Example 3 had a star-like shape with multiple protrusions, and their average particle size was 31.2 nm.

[0076] FIG. 7 shows the ATR-FTIR spectrum (A), UV-vis spectrum (B), Raman scattering spectrum (C), and XRD pattern (D) of the samples of Examples 1 to 4.

[0077] Figure 7(A) shows the fullerene C used as the raw material. 60 The ATR-FTIR spectra of the solid spheres (FE-SS) and gold particles (AuNS) are also shown. The ATR-FTIR spectrum of the solid spheres (FE-SS) is consistent with the fullerene C 60 Unlike that of 3100cm -1 ~3500cm -1 A broad NH stretching vibration band at 2867 cm -1 ~2922cm -1 CH stretching vibration band, 1635 cm -1 and 1450 cm -1 NH bending vibration and 1103cm -1 The C—N stretching vibration band was observed.

[0078] Looking at the spectra of samples from Examples 1 to 4 in Figure 7(A), the intensity of the C-N stretching vibration band increased as the mass ratio of gold particles to solid spheres increased, and the intensity was greatest at a mass ratio of 1.5.

[0079] In addition, in the spectra of the samples of Examples 1 to 4, 3299 cm -1 NH stretching vibration, 1658cm -1 The characteristic peaks of gold particles (AuNS), such as the NH bending vibration, overlapped with the characteristic peaks of solid spheres (FE-SS).

[0080] Figure 7(B) also shows the UV-vis spectra of the solid spheres (FE-SS) and gold particles (AuNS). The UV-vis spectrum of the solid spheres (FE-SS) showed a significant absorption peak at 407 nm. This peak was due to fullerene C. 60This is due to the electronic vibrational structure of the nanoparticles and the influence of the surrounding environment. On the other hand, the UV-vis spectrum of gold nanoparticles (AuNSs) showed a high-frequency plasmon mode associated with the core at 533 nm and a low-frequency plasmon mode associated with the star-shaped tip at 638 nm.

[0081] However, the UV-vis spectra of the samples in Examples 1 to 4 showed three peaks (407 nm, 533 nm, and 638 nm), which differed from those of the solid spheres (FE-SS) and gold particles (AuNS). This suggests that the solid spheres and gold particles are not simply composites but are conjugated. Furthermore, when we looked at the intensities of the three new peaks, the peak intensity of the sample in Example 3 (FE-SS / AuNS1.5) was the highest.

[0082] 7C also shows the Raman scattering spectra of the solid spheres (FE-SS) and gold particles (AuNS). The Raman scattering spectra of the samples of Examples 1 to 4 show the peak at 1350 cm based on the solid spheres (FE-SS). -1 The D band and the 1588cm -1 It has a G band near 1465 cm and is based on gold nanoparticles (AuNS). -1 The peak around 1465cm -1 The intensity of the peak near σ increased with increasing mass ratio of gold particles to solid spheres. Specifically, the surface plasmon effect began to increase at mass ratios above 1 and stabilized at a mass ratio of 1.5.

[0083] These findings also suggest that by mixing the solid spheres (FE-SS) with gold particles (AuNS), a composite material in which gold particles are conjugated to the surface of the solid spheres was obtained.

[0084] Figure 7(D) shows the XRD patterns of the sample (FE-SS / AuNS1.5) and the solid sphere (FE-SS) of Example 3. The XRD pattern of the solid sphere (FE-SS) had peaks at 8.32° and 18.58°, which are due to fullerene C with a face-centered cubic structure. 60This indicated that the solid spheres (FE-SS) were amorphous.

[0085] On the other hand, the XRD pattern of the sample (FE-SS / AuNS1.5) in Example 3 showed peaks at 38.2°, 44.4°, and 77.7° corresponding to the (111), (200), and (311) lattice planes of the gold crystal structure, and the other peaks were (8.32, 18.58), similar to the XRD pattern of the solid sphere (FE-SS). This also indicates that the sample (FE-SS / AuNS1.5) in Example 3 is a fullerene C 60 It was found that the gold particles were bonded to the amorphous solid spheres of EDA, and that the composite materials were made of the amorphous solid spheres. Although not shown, the XRD patterns of the samples of Examples 1, 2, and 4 all showed gold peaks at 38.2°, 44.4°, 64.7°, and 77.7°, and the gold peaks were at 38.2°, 44.4°, 64.7°, and 77.7°, respectively, and the fullerene C 60 Some showed peaks of

[0086] FIG. 8 shows the XPS spectra of the sample and solid spheres of Example 3. FIG. 9 shows the XPS core level spectra of the sample and solid spheres of Example 3.

[0087] Figures 9(A) to (D) show the C1s spectrum, N1s spectrum, O1s spectrum, and Au4f spectrum of the sample and solid spheres of Example 3, respectively. The XPS spectrum of the solid spheres (FE-SS) in Figure 8 shows major peaks at 284 eV, 400 eV, and 532 eV. These peaks correspond to carbon, nitrogen, and oxygen on the surface of the solid spheres, respectively. On the other hand, the XPS spectrum of the sample (FE-SS / AuNS1.5) of Example 3 shows, in addition to the three major peaks mentioned above, Au4f at 84 eV, Au4d at 335 eV and 353 eV, and Au4f at 86 eV. p3 / 2 The peaks were 546 eV for Au4p1 / 2, 642 eV for Au4p1 / 2, and 764 eV for Au4s.

[0088] The XPS core spectrum of the solid sphere (FE-SS) in Figure 9(A) shows C=C(sp 2 ) bond state 284.5 eV, CC(sp 3) bonding state and a peak at 284.8 eV for the C=O bonding state, and a peak at 288.3 eV for the C=O bonding state. On the other hand, the XPS core spectrum of the sample (FE-SS / AuNS1.5) of Example 3 in Figure 9(A) showed a peak at 284.5 eV for the C=C bonding state in addition to the above three carbon bonding states.

[0089] The XPS core spectrum of the solid sphere (FE-SS) in Figure 9(B) shows a peak at 399.6 eV for the NH2 bond state and a peak at 400.3 eV for the positively charged nitrogen state. On the other hand, the XPS core spectrum of the sample (FE-SS / AuNS1.5) in Example 3 in Figure 9(B) shows a peak at 401.7 eV for the N-C=O bond state in addition to the two nitrogen bond states mentioned above.

[0090] The XPS core spectrum of the solid sphere (FE-SS) in Figure 9(C) shows peaks at 531.9 eV for the C-OH bond state and 532.2 eV for the C-O-C bond state. On the other hand, the XPS core spectrum of the sample (FE-SS / AuNS1.5) in Example 3 in Figure 9(C) shows a peak at 531.6 eV for the O=C bond state in addition to the two oxygen bond states mentioned above.

[0091] The XPS core spectrum of the sample (FE-SS / AuNS1.5) in Example 3 in Figure 9(D) shows the Au4f 7 / 2 83.7 eV, and Au4f 5 / 2 The peak at 87.7 eV was observed.

[0092] 9(A) to (D), the sample of Example 3 contains fullerene C 60 It was found that the gold particles were conjugated to the solid spheres with EDA on the surface, forming a stabilized composite material.

[0093] FIG. 10 is a diagram showing the thermogravimetric changes of the sample of Example 3. FIG. 11 shows an SEM image of the sample of Example 3 after thermogravimetry.

[0094] The thermogravimetric changes of the solid spheres (FE-SS) and gold particles (AuNS) are also shown in Figure 10. Figures 11(A) and 11(B) are SEM images of the solid spheres (FE-SS) after thermogravimetric measurement, and Figures 11(C) and 11(D) are SEM images of the sample of Example 3 (FE-SS / AuNS1.5) after thermogravimetric measurement.

[0095] In the thermogravimetric analysis of the solid spheres (FE-SS), the mass loss observed below 100 °C was attributed to the evaporation of the dispersion medium (here, water) incorporated into the fullerene structure during self-assembly. The mass loss observed near 200 °C was attributed to the evaporation of mesitylene and unreacted EDA. The significant mass loss observed above 750 °C was attributed to the breaking of C-C bonds and the formation of amorphous carbon. In the thermogravimetric analysis of gold nanoparticles (AuNS), the mass loss observed above 250 °C and below 500 °C was attributed to the burnout of the organic layer (based on HEPES buffer) covering the gold nanoparticles.

[0096] On the other hand, the thermogravimetric change tendency of the sample of Example 3 (FE-SS / AuNS1.5) was similar to that of the solid spheres (FE-SS) and gold particles (AuNS), but the overall mass loss was small. Surprisingly, even when the sample of Example 3 was heated to over 750°C, the mass loss was suppressed to 20%, indicating that the sample of Example 3 has excellent thermal stability. This is because fullerene C 60 C crosslinked with EDA 60 We believe that by conjugating gold particles (AuNS) to the surface of the solid spheres of -EDA, the gold particles also functioned as a barrier layer, effectively suppressing the thermal decomposition of the solid spheres.

[0097] Comparing Figures 11(A)-(B) after thermogravimetry with Figure 3 before thermogravimetry, we see that the size of the solid spheres (FE-SS) did not change with heating. On the other hand, comparing Figures 11(C)-(D) after thermogravimetry with Figure 4 before thermogravimetry, we see that the size of the solid spheres (FE-SS) did not change with heating, but the size of the gold particles (AuNS) did increase. Specifically, the average diameter of the gold particles increased from 31.2 nm to 49.1 nm with heating. This is because the gold particles bonded together to form larger gold particles. Larger gold particles improve the photothermal effect due to increased surface area and improved light absorption, which may lead to more efficient heat generation and cancer cell destruction.

[0098] FIG. 12 is a diagram showing the zeta potentials of the samples of Examples 1 to 4.

[0099] 12, as the mass ratio of gold particles (AuNS) to solid spheres (FE-SS) increases, the absolute value of the zeta potential decreases, falls within the range of greater than 0 mV and less than 20 mV, and stabilizes at -11.0 mV. Having the absolute value of the zeta potential in this range is advantageous for attaching oppositely charged particles or molecules.

[0100] FIG. 13 is a diagram showing the photothermal effect of the samples of Examples 1 to 4.

[0101] Figure 13 also shows the photothermal effects of a glass substrate (manufactured by AS ONE Corporation, 25 mm x 75 mm, 1.0-1.2 mm thick), solid spheres (FE-SS), and gold particles (AuNS). Figure 13 shows that none of the samples showed any temperature change when not irradiated with near-infrared light. Furthermore, the glass substrate did not show any temperature change when irradiated with near-infrared light.

[0102] On the other hand, the solid spheres (FE-SS) and gold particles (AuNS) showed a temperature increase and photothermal effect when irradiated with near-infrared light. The samples in Examples 1 to 4, in which gold particles (AuNS) were conjugated to solid spheres (FE-SS), showed a temperature increase and photothermal effect when irradiated with near-infrared light, but all of them showed a photothermal effect greater than that of the solid spheres (FE-SS) and gold particles (AuNS). In particular, a significant photothermal effect was observed when the mass ratio of gold particles (AuNS) to solid spheres (FE-SS) was in the range of 1.5 to 2.0. Therefore, the composite material of the present invention generates a photothermal effect when irradiated with near-infrared light, and can therefore kill heat-sensitive cancer cells, potentially functioning as a photothermal therapy drug. [Industrial Applicability]

[0103] The composite material of the present invention generates photothermal effect, active oxygen, and photoacoustic effect when irradiated with near-infrared light, and is therefore applicable to photothermal therapy agents, photodynamic therapy agents, and photoacoustic contrast agents. [Explanation of symbols]

[0104] 100 Composite materials 110 solid sphere 120 gold particles

Claims

1. a fullerene-aliphatic diamine solid sphere in which a fullerene and an aliphatic diamine are crosslinked; gold particles located on the surface of the solid sphere; A composite material comprising:

2. 2. The composite material of claim 1, wherein the gold particles have a shape selected from the group consisting of spheres, rods, stars, spindles, sea urchins, triangular prisms, rectangular parallelepipeds, and cubes.

3. The fullerene is C ６０ Fullerene, C ７０ Fullerene, C ７６ Fullerene, C ７８ Fullerene, C ８２ Fullerene, C ８４ Fullerene, C ９０ Fullerene, C ９４ 3. The composite material according to claim 1 or 2, based on a fullerene selected from the group consisting of fullerenes and their derivatives.

4. The fullerene is C ６０ The composite material according to claim 3 , which is a fullerene or a derivative thereof.

5. 5. The composite material according to claim 1, wherein the solid spheres are amorphous.

6. The aliphatic diamine is *-NH-(CH ２ ) ｍ -NH ２ 6. The composite material according to claim 1, wherein m is 1≦m≦10, and * is a bonding site.

7. 7. The composite material of claim 6, wherein the aliphatic diamine is ethylenediamine where m=2.

8. 8. The composite material according to claim 1, wherein the mass ratio of the gold particles to the solid spheres is in the range of 0.5 to 2.

0.

9. The composite material according to any one of claims 1 to 8, wherein the composite material has an average particle size in the range of 320 nm or more and 380 nm or less.

10. The composite material according to any one of claims 1 to 9, wherein the average particle size of the gold particles is in the range of 25 nm to 55 nm.

11. The composite material according to any one of claims 1 to 10, which has peaks in a wavelength range of 436 nm to 462 nm, a wavelength range of 536 nm to 539 nm, and a wavelength range of 675 nm to 705 nm in an ultraviolet-visible absorption spectrum.

12. The composite material according to any one of claims 1 to 11, wherein the absolute value of the zeta potential is greater than 0 mV and not more than 20 mV.

13. preparing a fullerene-aliphatic diamine solid sphere in which the fullerene and the aliphatic diamine are crosslinked; mixing the solid spheres and gold particles in a dispersion medium; A method for producing the composite material according to any one of claims 1 to 12, comprising:

14. The method of claim 13 , wherein the solid spheres are prepared by a liquid-liquid interfacial precipitation method using the fullerene, the aliphatic diamine, and an aromatic hydrocarbon.

15. 15. The method of claim 14, wherein the aromatic hydrocarbon is selected from the group consisting of toluene, o-xylene, m-xylene, p-xylene, and 1,3,5-trimethylbenzene.

16. The method according to claim 14 or 15, wherein the aliphatic diamine has a molar ratio relative to the fullerene of 0.9 to 3.

17. The method according to any one of claims 14 to 16, further comprising post-annealing at a temperature in the range of 500°C to 750°C.

18. A photothermotherapy agent comprising the composite material according to any one of claims 1 to 12.

19. A photodynamic therapy agent comprising the composite material according to any one of claims 1 to 12.

20. A photoacoustic contrast agent comprising the composite material according to any one of claims 1 to 12.

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