JP2026013513A - Composite material containing self-assembled fullerene, method for producing the same, photothermal therapy agent, photodynamic therapy agent, and photoacoustic contrast agent - Google Patents

Abstract

The present invention provides 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.
[Solution] The composite material of the present invention comprises fullerene-aliphatic diamine solid spheres in which a fullerene and an aliphatic diamine are crosslinked, and gold particles located on the surfaces of the solid spheres. The photothermal therapy agent, photodynamic therapy agent, and photoacoustic contrast agent of the present invention include the above composite material. The method for producing the composite material of the present invention includes preparing fullerene-aliphatic diamine solid spheres in which a fullerene and an aliphatic diamine are crosslinked, and mixing the solid spheres with gold particles in a dispersion medium.
[Selected Figure] Figure 1

Description

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.

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

On the other hand, it is known that composite porous materials containing nanoparticles that generate heat when irradiated with near-infrared light and bioabsorbable polymers can be used in photothermal therapy (see, for example, Patent Document 1).

Self-assembled fullerenes are expected to be used in new applications.

JP 2018-83780 A

Lok Kumar Shrestha et al., Nanomaterials, 2023, 13, 946

Based on the above, the 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.

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 may be based on a fullerene selected from the group consisting of _C60 fullerene, _C70 fullerene _{, C76} fullerene, _C78 fullerene, _C82 fullerene, _C84 fullerene, C90 fullerene, _C94 fullerene, and derivatives thereof.
The fullerene may be a _C60 fullerene or a derivative thereof.
The solid spheres may be amorphous.
The aliphatic diamine may be represented by *-NH-(CH ₂ ) _m -NH ₂ (m is 1≦m≦10, * is a bonding 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 less than or equal to 20 mV.
The method for producing a composite material according to the present invention comprises preparing fullerene-aliphatic diamine solid spheres 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 may have a molar ratio relative to the fullerene of 0.9 or more and 3 or less.
The method may further include 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.

The composite material of the present invention comprises fullerene-aliphatic diamine solid spheres in which fullerene and aliphatic diamine are crosslinked, and gold particles positioned on the surface of the spheres. 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.

The method for producing the composite material of the present invention is advantageous because it simply requires mixing solid spheres of fullerene-aliphatic diamine, in which fullerene and aliphatic diamine are crosslinked, with gold particles, eliminating the need for skilled techniques or expensive equipment.

Schematic diagram showing the composite material of the present invention. Flowchart showing the steps for producing the composite material of the present invention SEM image of a fullerene-aliphatic diamine solid sphere. 1 shows SEM images of samples of Examples 1 to 4. 1 shows STEM images of samples from Examples 1 to 4. TEM image of the sample of Example 3. 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 1 shows XPS spectra of the sample and solid spheres of Example 3. Figure 1 shows XPS core level spectra of the sample and solid spheres of Example 3. Graph showing thermogravimetric changes of samples of Example 3 FIG. 1 shows an SEM image of the sample of Example 3 after thermogravimetry. Graph showing the zeta potential of samples from Examples 1 to 4 Graph showing the photothermal effect of samples from Examples 1 to 4

Embodiments of the present invention will be described below with reference to the drawings. Note that similar elements will be given similar reference numerals and their description will be omitted.

Figure 1 is a schematic diagram showing the composite material of the present invention.

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 have discovered that such a composite material 100 exhibits an excellent photothermal effect when irradiated with near-infrared light. Each component will be described in detail below.

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. Specifically, in this 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.

The aspect ratio is calculated as follows: 100 particles are selected from an electron microscope image (e.g., 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 used as the aspect ratio of the composite material 100.

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. Within 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).

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 as the solid spheres 110 self-assemble, and is preferably represented by *-NH-(CH ₂ ) _m -NH ₂ (where m is 1≦m≦10, * is a bonding site). Of these, ethylenediamine, where m=2, is preferred from the standpoints of yield and safety.

The fullerenes constituting the solid spheres 110 are not particularly limited as long as they are spherical compounds based on fullerenes and primarily comprise carbon atoms. For example, the fullerenes are based on fullerenes selected from the group consisting of _C60 fullerene, _C70 fullerene, _{C76 fullerene} , _C78 fullerene, _C82 fullerene, C84 fullerene, _C90 fullerene, _C94 fullerene, and derivatives thereof. The fullerene is preferably _C60 fullerene or a derivative thereof. This allows the solid spheres 110 to be efficiently produced by the method described below. The fullerene derivative refers to a compound in which at least a portion of a fullerene is modified with a substituent other than ethylenediamine.

When the fullerene and the aliphatic diamine crosslink and self-organization progresses, solid spheres 110 are formed. 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 also be a mixture of amorphous and crystalline fullerenes.

Any particles containing gold as the main component can be used as the gold particles 120. Illustratively, the gold particles 120 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.

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. Within this range, the particles are combined with the solid spheres 110 and exhibit excellent photothermal effects. Again, the average particle size is the average of the longest diameters of 100 particles observed in an electron microscope image (e.g., 20,000x to 30,000x magnification).

The mass ratio of gold particles 120 to solid spheres 110 preferably falls within the range of 0.5 to 2.0. This range provides an excellent photothermal effect. The mass ratio more preferably falls within the range of 1.0 to 2.0. This range provides an even more excellent photothermal effect. The mass ratio even more preferably falls within the range of 1.5 to 2.0. This range provides an even more excellent photothermal effect.

The solid spheres 110 and 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 to be conjugated.

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

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. Because the gold particles 120 suppress the surface charge, this 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. This is advantageous for attaching oppositely charged particles or molecules.

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.

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: Mix the solid spheres and gold particles in a dispersion medium.

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.

Here, the fullerene is selected from the group consisting of the above-mentioned _C60 fullerene, _C70 fullerene, _C76 fullerene, _C78 fullerene, _C82 fullerene, _C84 fullerene, _C90 fullerene, _C94 fullerene, and derivatives thereof. Among them, _C60 fullerene or its derivatives are preferred.

The aliphatic diamine is preferably represented by NH ₂ —(CH ₂ ) _m —NH ₂ (m is 1≦m≦10). Among these, 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 3.0. Within this range, crosslinking occurs between the fullerenes and the aliphatic diamine, 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.

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.

The specific preparation can be carried out as follows.
First, a 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. Next, the saturated fullerene solution is 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 or higher and 30°C or lower) 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.

In step S220, the gold particles can be those 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). Gold particles synthesized using the above method can be obtained with different sizes and shapes (e.g., rods, stars, etc.).

In step S220, the dispersion medium may be water or a solvent containing water. Examples of water include tap water, distilled water, purified water, ultrapure water, and deionized water. The water-containing solvent may contain water, and may also contain other solvents. While there are no particular limitations on the other solvents, solvents that are miscible with water are preferred, such as alcohol (e.g., ethanol).

In step S220, the solid spheres and gold particles are mixed so that the mass ratio of gold particles to 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.

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 obtained as a powder by centrifugation, washing, and freeze-drying.

Following step S220, the product obtained in step S220 may be post-annealed at a temperature in the range of 500°C to 750°C. This allows the gold particles on the surface to aggregate and grow. There are no particular restrictions on the heating time, but it may be, for example, in the range of 0.5 hours to 12 hours.

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 the cancer cells.

A method for treating cancer using a photothermal therapy drug containing the composite material 100 of the present invention administered in vivo (method of use) 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.

In step S310, the living organism 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. If a substance that promotes cellular absorption (e.g., surfactant, cyclodextrin, bioadhesive polymer, etc.) is applied to the surface of the composite material 100 of the present invention, it will function as a photothermal therapy drug that is efficiently absorbed in the small intestine when administered orally. If a substance that binds to cancer cells (e.g., antibody, ligand, peptide, etc.) is applied to 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 for effective destruction of cancer cells in step S320.

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

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

A method for treating cancer using a photodynamic therapy drug containing the composite material 100 of the present invention administered in vivo (method of use) includes the following steps.
Step S410: A photodynamic therapy drug 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.

Step S410 is similar to step S310 described above, so its explanation will be omitted.

In step S420, the light is preferably near-infrared light, specifically having a wavelength in the range of 700 nm to 1500 nm, and more preferably in the range of 750 nm to 1300 nm. This allows the photodynamic therapy agent of the present invention to effectively generate reactive oxygen species and destroy cancer cells when irradiated with light.

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.

A method for imaging a site such as a cancer (method of use) 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.

Step S510 is similar to step S310 described above, so its explanation will be omitted.

In step S520, the light is preferably near-infrared light, specifically having a wavelength in the range of 700 nm to 1500 nm, and more preferably in the range of 750 nm to 1300 nm. This allows the photoacoustic contrast agent of the present invention to produce a photoacoustic effect and image sites such as cancers and tumors.

The photothermal therapy agent, photodynamic therapy agent, and photoacoustic contrast agent of the present invention may contain a dispersion medium in addition to the composite material 100 of the present invention. The dispersion medium may be, for example, physiological saline, distilled water for injection, phosphate buffer solution, etc. 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 also be combined. For example, after imaging the affected area with a photoacoustic contrast agent, cancer cells may be destroyed with a phototherapy agent made of the same material as the photoacoustic contrast agent.

Next, the present invention will be described in detail using specific examples, but please note that the present invention is not limited to these examples.

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

Specifically, 140 mg of fullerene _C60 powder (purity 99.5%, manufactured by MTR Ltd.) as the raw fullerene 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 a unit fullerene solution). The fullerene concentration in the fullerene solution was 1 mg/mL.

0.55 mL of ethylenediamine (EDA, purity 99.0%, manufactured by 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 an EDA/mesitylene solution. Ethylenediamine is expressed as NH ₂ —(CH ₂ ) _m —NH ₂ , where m = 2.

Five mL of the fullerene solution was added to 5.55 mL of the EDA/mesitylene solution at room temperature (25°C). The molar ratio of EDA to fullerene _C60 was 1. The mixture was then stirred for 10 seconds and incubated at 25°C for 1 hour to obtain a brown precipitate of fullerene-aliphatic diamine solid spheres (referred to as FE-SS) in which the fullerene and the 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.

Figure 3 shows an SEM image of a fullerene-aliphatic diamine solid sphere.

3, it was found that fullerene _C60 was crosslinked with EDA and self-assembled into a spherical shape by the liquid-liquid interface deposition method, resulting in the production of 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. From the spectra of the solid spheres obtained by X-ray photoelectron spectroscopy (XPS), it was confirmed that the solid spheres had ethylenediamine on their surfaces.

[Preparation of gold particles (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 aqueous solution (Sigma-Aldrich, 20 mM) and incubated at 25°C for 90 minutes to obtain a bluish-black dispersion containing gold nanostars (AuNS). The AuNS dispersion was stored at 4°C.

[Examples 1 to 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.

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 concentration-adjusted gold particle (AuNS) dispersions (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.

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

The surface functional groups of the samples in 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 in Examples 1 to 4 were measured using a spectrophotometer (JASCO Corporation, V-670).

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

X-ray photoelectron spectra of the samples in Examples 1 to 4 were measured using a Theta Probe Spectrometer (Thermo Electron). Measurements were performed using Al-Kα monochromatic radiation (energy 15 keV). 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. To avoid charge accumulation on the measurement sample, a built-in electroflood gun was used for the measurements.

Thermogravimetric analyses of the samples of Examples 1 to 4 were performed using a thermogravimetric analyzer (NETZSCH-Geratebau GmbH, STA2500 Regulus). SEM images of the samples of Examples 1 to 4 after the thermogravimetric analyses were also observed.

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

The above results are shown in Figures 4 to 13 and will be explained together.

FIG. 4 shows SEM images of the samples of Examples 1 to 4.
FIG. 5 shows STEM images of the samples of Examples 1 to 4.
FIG. 6 shows a TEM image of the sample of Example 3.

Figures 4(A) to (D) are SEM images of Sample 1 (FE-SS/AuNS0.5), Sample 2 (FE-SS/AuNS1), Sample 3 (FE-SS/AuNS1.5), and Sample 4 (FE-SS/AuNS2), respectively.

Figures 5(A) to 5(D) are STEM images of Sample 1 (FE-SS/AuNS0.5), Sample 2 (FE-SS/AuNS1), Sample 3 (FE-SS/AuNS1.5), and Sample 4 (FE-SS/AuNS2), respectively.

Figures 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.

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 in which gold particles are attached to the surface of the solid spheres. Figure 4 shows that the amount of attached gold particles 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.

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.

Figure 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.

7(A) also shows the ATR-FTIR spectra of the raw materials, fullerene _C60 , solid spheres (FE-SS), and gold particles (AuNS). The ATR-FTIR spectrum of the solid spheres (FE-SS) differs from that of fullerene _C60 , exhibiting a broad N-H stretching vibration band from 3100 cm ^-1 to 3500 cm ^-1 , a C-H stretching vibration band from 2867 cm ^-1 to 2922 cm ^-1 , N-H bending vibrations at 1635 cm ^-1 and 1450 cm ^-1 , and a C-N stretching vibration band at 1103 cm ^-1 .

Looking at the spectra of samples from Examples 1 to 4 in Figure 7(A), the intensity of the C-N stretching vibration band increases as the mass ratio of gold particles to solid spheres increases, with the highest intensity at a mass ratio of 1.5.

In addition, in the spectra of the samples of Examples 1 to 4, characteristic peaks of gold particles (AuNS), such as the N-H stretching vibration at 3299 cm ^-1 and the N-H bending vibration at 1658 cm ^-1 , overlapped with peaks characteristic of solid spheres (FE-SS).

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 prominent absorption peak at 407 nm. This peak is attributed to the electronic vibrational structure of fullerene _C60 and the influence of the surrounding environment. On the other hand, the UV-vis spectrum of the gold particles (AuNS) 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.

However, the UV-vis spectra of the samples in Examples 1 to 4 showed three peaks (407 nm, 533 nm, and 638 nm), unlike 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 focusing on the intensities of the three new peaks, the peak intensity of the sample in Example 3 (FE-SS/AuNS1.5) was the highest.

Figure 7(C) also shows the Raman scattering spectra of the solid spheres (FE-SS) and gold particles (AuNS). The Raman scattering spectra of the samples in Examples 1 to 4 exhibited a D band near 1350 cm ^-1 due to the solid spheres (FE-SS) and a G band near 1588 cm ^-1 due to the gold particles (AuNS), as well as a peak near 1465 cm ^-1 due to the gold particles (AuNS). The intensity of the peak near 1465 cm ^-1 increased as the mass ratio of gold particles to solid spheres increased. Specifically, the surface plasmon effect began to increase at mass ratios of 1 or more and stabilized at a mass ratio of 1.5.

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

7D 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°, but these peaks did not match those of fullerene _C60 , which has a face-centered cubic structure. This indicated that the solid sphere (FE-SS) was amorphous.

On the other hand, the XRD pattern of the sample of Example 3 (FE-SS/AuNS1.5) 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 remaining peaks were similar to the XRD pattern of the solid spheres (FE-SS) (8.32, 18.58). This also indicated that the sample of Example 3 (FE-SS/AuNS1.5) was a composite material in which fullerene _C60 and EDA were crosslinked, and gold particles were bonded to 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 some also showed a peak of fullerene _C60 .

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.

Figures 9(A) to 9(D) show the C1s, N1s, O1s, and Au4f spectra of the sample and solid spheres of Example 3, respectively. The XPS spectrum of the solid spheres (FE-SS) in Figure 8 showed major peaks at 284 eV, 400 eV, and 532 eV. These peaks corresponded to carbon, nitrogen, and oxygen on the surface of the solid spheres, respectively. Meanwhile, the XPS spectrum of the sample (FE-SS/AuNS1.5) in Example 3 showed, in addition to the three major peaks mentioned above, peaks at 84 eV for Au4f, 335 eV and 353 eV for Au4d, 546 eV for _Au4p3/2 , 642 eV for Au4p1/2, and 764 eV for Au4s.

The XPS core spectrum of the solid sphere (FE-SS) in Figure 9(A) showed peaks at 284.5 eV for the C=C (sp ² ) bonding state, 284.8 eV for the C-C (sp ³ ) bonding state, and 288.3 eV for the C=O bonding state. On the other hand, the XPS core spectrum of the sample of Example 3 (FE-SS/AuNS1.5) 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.

The XPS core spectrum of the solid sphere (FE-SS) in Figure 9(B) showed 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 of Example 3 (FE-SS/AuNS1.5) in Figure 9(B) showed a peak at 401.7 eV for the N-C=O bond state in addition to the two nitrogen bond states mentioned above.

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

The XPS core spectrum of the sample (FE-SS/AuNS1.5) of Example 3 in FIG. 9(D) showed peaks at 83.7 eV for Au4f _7/2 and 87.7 eV for Au4f _5/2 .

9(A) to (D), it was found that the sample of Example 3 was a stabilized composite material in which fullerene _C60 and EDA were crosslinked to form solid spheres having EDA on the surface, and gold particles were conjugated to these solid spheres.

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.

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

In the thermogravimetric changes of 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 changes of gold particles (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 particles.

On the other hand, the thermogravimetric change trend 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 750°C or higher, the mass loss was suppressed to 20%, indicating that the sample of Example 3 has excellent thermal stability. This is thought to be because the gold particles (AuNS) are conjugated to the surface of the solid spheres of _{C60 -} EDA, in which fullerene C60 and EDA are crosslinked, and the gold particles also function as a barrier layer, effectively suppressing thermal decomposition of the solid spheres.

Comparing Figures 11(A) and (B) after thermogravimetry with Figure 3 before thermogravimetry, no change in the size of the solid spheres (FE-SS) was observed even with heating. On the other hand, comparing Figures 11(C) and (D) after thermogravimetry with Figure 4 before thermogravimetry, no change in the size of the solid spheres (FE-SS) was observed even with heating, but the size of the gold particles (AuNS) increased. Specifically, the average particle size 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 upon heating. 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 destruction of cancer cells.

Figure 12 shows the zeta potential of the samples in Examples 1 to 4.

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

Figure 13 shows the photothermal effect of samples from Examples 1 to 4.

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 a temperature change when not irradiated with near-infrared light. Furthermore, the glass substrate did not show a temperature change when irradiated with near-infrared light.

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. This suggests that the composite material of the present invention, which produces a photothermal effect when irradiated with near-infrared light, can kill heat-sensitive cancer cells and function as a photothermal therapy drug.

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

100 Composite material 110 Solid sphere 120 Gold particles

Claims (20)

a fullerene-aliphatic diamine solid sphere in which a fullerene and an aliphatic diamine are crosslinked;
and gold particles located on the surface of said solid sphere. 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 composite material according to claim 1 or 2 _, wherein the fullerene is based on a fullerene selected from the group consisting of _C60 fullerene, _C70 fullerene, _C76 fullerene, _C78 fullerene, _C82 fullerene, _C84 fullerene, C90 fullerene, _C94 fullerene, and derivatives thereof. 4. The composite material of claim 3, wherein the fullerene is _C60 fullerene or a derivative thereof. The composite material described in any one of claims 1 to 4, wherein the solid spheres are amorphous. 6. The composite material according to claim 1, wherein the aliphatic diamine is represented by *-NH-(CH ₂ ) _m -NH ₂ (where m is 1≦m≦10, and * is a bonding site). The composite material described in claim 6, wherein the aliphatic diamine is ethylenediamine where m = 2. The composite material described in any one of claims 1 to 7, wherein the mass ratio of the gold particles to the solid spheres is in the range of 0.5 to 2.0. The composite material described in any one of claims 1 to 8, wherein the average particle size of the composite material is in the range of 320 nm or more and 380 nm or less. The composite material described in 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. The composite material described in any one of claims 1 to 10 has peaks in the ultraviolet-visible absorption spectrum in the wavelength ranges of 436 nm to 462 nm, 536 nm to 539 nm, and 675 nm to 705 nm. The composite material described in any one of claims 1 to 11, wherein the absolute value of the zeta potential is greater than 0 mV and less than or equal to 20 mV. preparing a fullerene-aliphatic diamine solid sphere in which the fullerene and the aliphatic diamine are crosslinked;
and mixing the solid spheres and gold particles in a dispersion medium. 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. 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. The method of claim 14 or 15, wherein the aliphatic diamine to fullerene molar ratio is in the range of 0.9 to 3. The method of any one of claims 14 to 16, further comprising post-annealing at a temperature in the range of 500°C or higher and 750°C or lower. A photothermal therapy drug comprising the composite material described in any one of claims 1 to 12. A photodynamic therapy drug comprising the composite material described in any one of claims 1 to 12. A photoacoustic contrast agent comprising the composite material described in any one of claims 1 to 12.