JP2012184199A - Water-soluble photosensitizing material using fullerene derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a water-soluble photosensitizing material containing a fullerene derivative and having high photosensitizing properties.SOLUTION: The water-soluble photosensitizing material having high photosensitizing properties includes: (1) a fullerene-containing liposome wherein a predetermined Cfullerene derivative exists inside the liposome consisting of a lipid molecule including a hydrophilic group and a hydrophobic group; or (2) a fullerene-cyclodextrin complex wherein a predetermined Cfullerene derivative is clathrated in cyclodextrin.

Description

The present invention relates to a photosensitizing material capable of being water-solubilized using a predetermined fullerene derivative, cyclodextrin, and liposome.

Fullerene is attracting attention as a new material in the field of nanotechnology in various fields such as electronics, pharmaceuticals, and cosmetics. For example, there are applications to heat-resistant materials that use the high heat resistance of fullerenes, and solid electrolyte materials for fuel cells that use high charge transport properties. In addition, application to organic solar cells, optical sensors, and the like has been developed as organic photoelectric conversion elements. In addition, research on the construction of artificial photosynthetic systems using the full electron acceptability of fullerene has been reported. In the pharmaceutical field, various applications such as drugs for photodynamic therapy, anti-HIV drugs, and contrast agents for MRI have been developed.

Fullerene is a compound having a spherical structure composed of carbon atoms. A typical example is C ₆₀ fullerene composed of ₆₀ carbon atoms. Refers to one more than the number 60 carbon and higher fullerenes, 70 pieces of C ₇₀ fullerene carbon atoms, 74 carbon carbon atoms, there are various kinds of such 76 amino things. Further, there is a metal-encapsulated fullerene in which a metal is encapsulated in a spherical structure. In particular, inexpensive to manufacture, C ₆₀ fullerene is used in various applications with excellent chemical stability.

Photosensitization refers to the action of inducing energy transfer by light irradiation, such as the ability to generate reactive oxygen species by coexistence with oxygen. Fullerene is known to be a photosensitizer that easily undergoes energy transfer due to light energy when irradiated with light, and produces various actions depending on light energy. For example, active oxygen species is generated by energy transfer to oxygen molecules by light irradiation in some higher fullerenes such as C ₆₀ fullerene and C ₇₀ fullerenes, reactive oxygen species in the higher fullerenes metal endohedral and some It has been reported that oxygen molecules are likely to be generated by energy transfer with singlet oxygen, which is a kind of oxygen (Patent Document 1). The reactive oxygen species refers to singlet oxygen, free radicals, etc., and is chemically unstable and has a high reaction activity. By utilizing such a property and having reaction activity with respect to a predetermined target, it is possible to realize selective exclusion of substances, transmission of information, and the like.

Substances having photosensitivity, particularly the ability to generate reactive oxygen species by light irradiation, can be used as PDT drugs in photodynamic therapy (hereinafter also referred to as “PDT”). PDT introduces PDT drugs directly into cancer tumors, or introduces them into the body by intravenous injection, etc., and then accumulates them in cancer cells. A treatment that kills and treats cancer. Such compatibility with PDT drugs is called photodynamic activity. Fullerene has a high ability to generate active oxygen species such as singlet oxygen and free radicals, and exhibits high photodynamic activity in PDT. As applicable fullerene PDT, and the like C ₆₀ fullerene and C ₇₀ fullerene capable of generating active oxygen species by light irradiation.

Although fullerene exhibits excellent properties such as photosensitization and the like, there is a problem that it is difficult to improve industrial applicability because of its low solubility in water and highly polar organic solvents. In order to solve this problem, various studies have been made, such as derivatization of fullerenes and addition of solubilizers. For example, there is a hydroxylated fullerene derivative in which a hydroxyl group (OH group) that is a polar functional group is introduced into a fullerene skeleton, and a hydroxylated fullerene derivative exhibiting high solubility in an ester solvent has been synthesized (Patent Document 2). . In addition, fullerene derivatives having high solubility in various organic solvents such as ether solvents have been synthesized (Patent Document 3). Furthermore, various fullerene derivatives have been reported. For example, evaluation of electron acceptability of fullerene derivatives in water, or fullerene derivative-DNA hybrid complexes have been reported (Non-Patent Documents 1 to 5).

The present inventors have proposed a method for solubilizing fullerene in water. For example, there is a method of solubilizing fullerene by adding polysaccharides such as cyclodextrin, liposomes and polymers as a solubilizer to fullerene and stirring and mixing them (Patent Documents 4, 5, 6 and 1). ). In Non-Patent Document 6, a complex and a manufacturing method thereof and C ₆₀ fullerene derivative and a cyclodextrin are disclosed. However, none of the prior literatures mentions the photosensitization property of the fullerene derivative-cyclodextrin complex under visible light.

Prior art documents are shown below.

JP2008-74766 JP2010-116380 JP2009-135237 JP 2006-069812 A WO2007 / 132923 WO2008 / 096779

D. M. Guldi, H. Hungerbuhler, K.-D. Asmus, J. Phys. Chem. A, 1997, 101, 1783. A. M. Cassell, W. A. Scrivens, J. M. Tour, Angew. Chem., Int. Ed. 1998, 37, 1528. M. Maggini, A. Karlsson, L. Pasimeni, G. Scorrano, M. Prato, L. Valli, Tetrahedron Lett., 1994, 35, 2985. I. Lamparth, A. Hirsch, J. Chem. Soc., Chem. Commun. 1994, 1727. C. Bingel, Chem. Ber. 1993, 126, 1957. A. Ikeda, T. Genmoto, N. Maekubo, J. Kikuchi, M. Akiyama, T. Mochizuki, S. Kotani, T. Konishi, Chem. Lett. 2010, 39, 12, 1256.

C ₆₀ fullerene and C ₇₀ fullerene unmodified known not completely soluble in water. Most fullerene derivatives having a hydrophilic substituent introduced self-associate to become micelles and become water-soluble. Such micelle formation causes a problem that, when a fullerene derivative is used as a photosensitizer, self-deactivation is caused by the photoexcited fullerene derivative. That is, since micelles are formed in an aqueous solution, the light energy received by the fullerene derivative is lost as thermal energy before being transmitted to others, leading to a decrease in photosensitization and a decrease in photoactivity.

In order to solve this problem, a method for solubilizing unmodified fullerene using a solubilizer has been proposed. However, there is a problem that the solubilizing agent sterically hinders the energy transfer between the substrate including unmodified fullerene photoexcited and dissolved oxygen. Therefore, unmodified fullerene solubilized with a solubilizing agent also has a problem of low photosensitization.

The inventors have solved the above-mentioned problems by using a C ₆₀ fullerene derivative having a predetermined substituent, and have developed a photosensitizing material containing a C ₆₀ fullerene derivative having high photoactivity and capable of forming an aqueous solution. . The present invention can be water-soluble by using the combined liposome or cyclodextrin, by developing a predetermined C ₆₀ fullerene derivatives showing high photoactivity was achieved wide photosensitizing material application range.

Photosensitizing material of the present invention is an inclusion material include C ₆₀ fullerene derivative represented by the following formula (1) or (2), composed of lipid molecules containing a hydrophilic group and a hydrophobic group A fullerene-containing liposome in which the C ₆₀ fullerene derivative is present inside the liposome, or a fullerene-cyclodextrin complex in which the C ₆₀ fullerene derivative is included in a cyclodextrin.

(R1 is hydrogen or, .R2 and R3 represents a substituent group or atom group having a hydrophilic functional group identical or may be different, .X representing hydrogen, a substituent or group ^- represents a counter anion .)

Photosensitizing material of the present invention, the predetermined C ₆₀ fullerene derivatives, inclusion fullerene derivative cyclodextrin - photosensitizing material cyclodextrin complex or a predetermined C ₆₀ fullerene derivative, and a hydrophilic group It is a photosensitizing material including a fullerene derivative-containing liposome present inside a liposome composed of a lipid molecule containing a hydrophobic group. C ₆₀ fullerene derivative of the predetermined present invention is the use of a cyclodextrin or liposomes is encompassed material can be readily water-solubilizing. Furthermore, according to the present invention a predetermined C ₆₀ fullerene derivatives include materials which are water-soluble, it is possible to obtain a photosensitizing of C ₆₀ fullerene derivative at a high efficiency, high performance can be obtained of photosensitizing material.

Photosensitizing material of the present invention, R1, R2 and R3 of the C ₆₀ fullerene derivative is contained in inclusion material contained in the photosensitizing material is hydrogen or a substituted having 10 or less carbon atoms including at least one carbon It is preferably a group or an atomic group. The inclusion in the inclusion material means inclusion in the cyclodextrin when the inclusion material is cyclodextrin, and means that the inclusion material exists inside the liposome when the inclusion material is liposome.

Photosensitizing material of the present invention, R1 of C ₆₀ fullerene derivatives encompassed by the inclusion material contained in the photosensitizing material is an alkyl group having a hydrophilic functional group, an ester group, an ether group, an amide group or an acyl Preferably any of the groups. By having a hydrophilic functional group, water-solubility can be more easily achieved by combination with inclusion materials.

Photosensitizing material of the present invention, R2 and R3 of the C ₆₀ fullerene derivative is contained in inclusion material contained in the photosensitizing material, or an alkyl group having 6 or less carbon atoms, ethylene glycol chain having 10 or less carbon atoms It is preferable that The nitrogen atom to which R2 and R3 are bonded has a positive charge, and the performance of water solubilization becomes higher by combination with the inclusion material. As the counter anion (X ⁻ ), a negatively charged ion can be used as appropriate.

In the photosensitizing material of the present invention, it is preferable that the C ₆₀ fullerene derivative present in the fullerene derivative-containing liposome contained in the photosensitizing material is present in the vicinity of the hydrophilic group of the lipid molecule constituting the liposome. . By being present in the vicinity, the contact between the dissolved oxygen present in the solution and the C ₆₀ fullerene derivative present in the liposome is facilitated, and the active oxygen species generation efficiency of the C ₆₀ fullerene derivative can be increased.

In the photosensitizing material of the present invention, it is preferable that a fluorescent substance having an absorption wavelength in the visible light region is present inside the fullerene derivative-containing liposome contained in the photosensitizing material. The presence of the fluorescent substance in combination with the C ₆₀ fullerene derivative can increase the energy transfer efficiency of the C ₆₀ fullerene derivative and increase the generation efficiency of active oxygen species.

In the photosensitizing material of the present invention, the fluorescent substance contained in the photosensitizing material is preferably present in the vicinity of the hydrophilic group of the lipid molecule constituting the liposome. In the liposome, the C ₆₀ fullerene derivative or the fluorescent substance is present in the vicinity of the hydrophilic group of the lipid molecule constituting the liposome, whereby the reactive oxygen species generation efficiency can be increased.

According to the photosensitizing material of the present invention, a material having high water solubility and high photosensitization can be obtained. In particular, an improvement in cell lethality due to light irradiation was confirmed as compared with a case where a containing material containing unmodified fullerene was introduced into cells. (1) In the system of the cyclodextrin complex was achieved improved light cell mortality compared to the unmodified C ₆₀ fullerene, C ₇₀ fullerene. Further, (2) in the system of the liposomes increased the amount of generation of singlet oxygen as compared to the unmodified C ₆₀ fullerenes, and based thereon, to achieve improved light cell lethality.

FIG. 1 is a diagram showing C ₆₀ fullerene derivatives used in Examples and Comparative Examples of the present invention. FIG. 2 is a diagram schematically showing the mechanism of singlet oxygen generation in fullerene derivative-containing liposomes. FIG. 3 is a schematic diagram of a method for producing the fullerene derivative-containing liposome of the present invention. FIG. 4 is a view showing an absorption spectrum in the visible to ultraviolet region of the fullerene derivative used in the photosensitizing material of the present invention. FIG. 5 is a diagram showing a ¹ H NMR spectrum of a fullerene derivative used in the photosensitizing material of the present invention. Figure 6 is C ₆₀ fullerene derivative of the present invention - is a diagram showing the photodynamic activity of cyclodextrin complexes. FIG. 7 is a graph showing the photodynamic activity of the C ₆₀ fullerene derivative-containing liposome of the present invention. FIG. 8 is a graph showing the ability to generate singlet oxygen of the C ₆₀ fullerene derivative-cyclodextrin complex of the present invention. FIG. 9 is a diagram showing the singlet oxygen generation ability of the C ₆₀ fullerene derivative-containing liposome of the present invention.

As the fullerene of the present invention, a fullerene having 60 carbon atoms is used. As such fullerene, a commercially available product can be used as appropriate.

An example of the C ₆₀ fullerene derivative used in the present invention is shown below.
(R1 is hydrogen or, .R2 and R3 represents a substituent group or atom group having a hydrophilic functional group identical or may be different, .X representing hydrogen, a substituent or group ^- represents a counter anion .)

R1, R2 and R3 represent a substituent or an atomic group having a hydrophilic functional group. This substituent or atomic group is preferably a substituent or atomic group containing at least one carbon and having 10 or less carbon atoms. The substituent of the C ₆₀ fullerene derivative needs to be hydrophilic. In the substituent of the C ₆₀ fullerene derivative of the formula (1) include hydrophilic functional groups. In the substituent of the C ₆₀ fullerene derivative of the formula (2), the nitrogen atom in the substituent (N) has a positive charge, by the effect of the presence of this positive charge, hydrophilicity is ensured . The counter cation can be appropriately selected.

In particular, R1 is preferably an alkyl group, ester group, ether group, amide group or acyl group having a hydrophilic functional group. A part of the substituent or atomic group may be substituted with a hydrophilic functional group. The hydrophilic functional group may be cationic, anionic, or neutral. Examples of the cationic hydrophilic functional group include an ammonium group and a pyridinium group. Examples of the anionic hydrophilic functional group include a carbonate group (carboxylate ion), a sulfonate group (sulfonate ion), and a phosphonate group (phosphate acid). Neutral hydrophilic functional groups such as ions) are preferably oligoethylene glycol chains and polyethylene glycol chains. R2 and R3 may be the same or different and are preferably a lower alkyl group having 6 or less carbon atoms or an ethylene glycol chain having 10 or less carbon atoms. In addition, the substituent or atomic group of the C ₆₀ fullerene derivative may be linear or branched.

The substituent introduced into C ₆₀ fullerene is preferably a methyl group in the compound of the above formula (2), for example. It can manufacture more easily by making a substituent into a methyl group. The position at which the substituent is introduced into C ₆₀ fullerene is not particularly limited. Although it is possible to introduce a plurality of substituents, the number of substituents introduced into C ₆₀ fullerene is preferably 1. When a plurality of substituents are introduced, it becomes difficult to control the number of substituents and the introduction positions for realizing high water solubility and high photosensitization.

The cyclodextrin used in the present invention is not limited as long as 5 or more glucoses are bonded, and α-cyclodextrin (cyclohexaamylose) having 6 glucoses bonded, β-cyclodextrin (cycloheptaamyloses) having 7 glucoses bonded thereto. ), 8 linked γ-cyclodextrins (cyclooctaamylose), 9 linked δ-cyclodextrins, and the like. Particularly preferred is γ-cyclodextrin. By using γ-cyclodextrin, the C ₆₀ fullerene derivative of the present invention can be more reliably included.

The liposome used in the present invention is a complex composed of a lipid bilayer, and the lipid bilayer is composed of lipid molecules containing a hydrophilic group and a hydrophobic group. The specific composition of the liposome is not particularly limited, and the lipid molecule can be used alone or in combination of a cationic lipid, an anionic lipid and a neutral lipid depending on the application and purpose. As the photosensitizing material of the present invention, the liposome has a diameter of 40 to 500 nm, preferably 50 to 200 nm. The film thickness of the liposome varies depending on the lipid molecule to be selected, but is 3 to 10 nm, preferably 4 to 7 nm.

In the fullerene derivative containing liposomes of the present invention, the C ₆₀ fullerene derivative or a fluorescent substance is present inside the liposome, it means that present in the membrane interior of the lipid bilayer constituting the liposome. Incidentally, C ₆₀ position of the membrane inside the fullerene derivative or a fluorescent substance is present lipid bilayer may be any position from the membrane center facing to each other hydrophobic group to the vicinity of the surface of the liposome close to the hydrophilic group . In particular, it is preferable that C ₆₀ fullerene derivative or a fluorescent substance is present in proximity to the hydrophilic groups at the internal membrane. By being present at such a position, it becomes easy to transfer energy from the photoexcited C ₆₀ fullerene derivative to dissolved oxygen, so that active oxygen species can be generated more efficiently.

FIG. 2 is a schematic diagram of singlet oxygen generation when a C ₆₀ derivative is in contact with the hydrophilic group inside the liposome. In FIG. 2 (A), unmodified C ₆₀ fullerene exists between the hydrophobic groups of the lipid molecules constituting the liposome within the liposome membrane, and in FIG. 2 (B), the C ₆₀ fullerene derivative of the present invention. Is present in the vicinity of the hydrophilic group of the lipid molecule constituting the liposome within the liposome membrane. That C ₆₀ fullerene derivative in the vicinity of the liposome surface are present, it can be said that contribute to efficient generation.

The fluorescent substance having an absorption wavelength in the visible light region used in the present invention is a substance that can absorb light having a wavelength in the range of 500 nm to 800 nm, and the substance absorbs light and generates light energy. Features. The light energy generated by the substance is fluorescence. Examples of such substances include carbocyanine dyes and rhodamine dyes. Light energy fluorescent material occurs, it is possible to exert a higher photosensitizing and C ₆₀ fullerene derivative absorption of the present invention. Fluorescent substances are present inside the liposomes as above C ₆₀ fullerene derivative is preferably present in proximity to the hydrophilic groups of the lipid molecules making up the liposome.

C ₆₀ fullerene derivative of the present invention - a method for manufacturing a cyclodextrin complex is not particularly limited, it can be produced, for example, by high vibration grinding method. C ₆₀ fullerene derivative using fast vibration pulverization method - an example of a manufacturing method of a cyclodextrin complex below. C ₆₀ fullerene derivative and cyclodextrin are placed in a high-speed vibration pulverization vessel and subjected to high-speed vibration pulverization. For example, the frequency can be 10 to 60 Hz, preferably 20 to 40 Hz, and the vibration time can be 10 to 60 minutes, preferably 15 to 45 minutes. The frequency and vibration time can be appropriately selected depending on the performance of the high-speed vibration device, the vibration method, and the like. Then, water is added and stirred, insoluble components are removed by centrifugation, and a brown supernatant is collected. The supernatant, C ₆₀ fullerene derivative - cyclodextrin complex solution. Regarding the high-speed vibration pulverization method, the method can be used with reference to JP-A-2009-23886.

The method for manufacturing a C ₆₀ fullerene derivative-containing liposomes of the present invention is not particularly limited, C ₆₀ fullerene derivative of the present invention - can be prepared by an exchange reaction of a cyclodextrin complex and liposomes. The exchange reaction is achieved by mixing the C ₆₀ fullerene derivative-cyclodextrin complex and liposome in water and stirring. The water temperature can be 50 to 100 degrees, preferably 70 to 90 degrees, stirring time 10 minutes to 3 hours, preferably 30 minutes to 2 hours. The water temperature and the stirring time can be appropriately selected depending on the stirring method and the like. The C ₆₀ fullerene derivative-containing liposome aqueous solution thus prepared can be used as the photosensitizing material of the present invention.

Furthermore, for the above C ₆₀ fullerene derivative containing liposomes solution by centrifugation using a membrane filter with centrifugation tube, it is possible to remove the cyclodextrin. Includes a cyclodextrin filtrate after centrifugation, which was collected by filtration is C ₆₀ fullerene derivative-containing liposomes. By dispersing the filtered C ₆₀ fullerene derivative-containing liposomes in water, a C ₆₀ fullerene derivative-containing liposome aqueous solution can be obtained and used as a photosensitizing material.

Photosensitizing material of the present invention can include materials other than cyclodextrins containing liposomes or C ₆₀ fullerene derivative containing C ₆₀ fullerene derivatives. For example, as a substance for introducing a photosensitizing material to a target that should exhibit photosensitization, a low molecule such as sugar, folic acid or 5-aminolevulinic acid, an antibody, a peptide, a protein, or the like can be introduced. Moreover, cyclodextrin may be contained as a mixture. The photosensitizing material of the present invention can be contained in various substances. Further, the photosensitizing material of the present invention can be used in combination with other drugs. Further, the photosensitizing material of the present invention can be used by dehydrating and drying to solidify or gelling with a gelling agent.

By <1> C ₆₀ synthesis following method of the fullerene derivative, to give a C ₆₀ fullerene derivative used in the embodiment of the present invention. The C ₆₀ fullerene derivative and C ₆₀ fullerene synthesized in Example 1 and Comparative Example 1 are shown in FIG.

In FIG. 1, the C ₆₀ fullerene derivatives of Compound 11 to Compound 13 were synthesized with reference to Non-Patent Documents 1 to 3, and the C ₆₀ fullerene derivative of Compound 15 was synthesized with reference to Non-Patent Document 3. Compound 14 was synthesized as follows. 30.0 mg (0.037 mmol) of fulleropyrrolidine (Compound 15) was dissolved in 50 ml of toluene, and 37.0 mg (0.37 mmol) of succinic anhydride was added thereto and refluxed. After 6 hours, the mixture was allowed to cool to room temperature, and the precipitate was collected by filtration. Washing with toluene gave a brown solid. Compounds 16 and 17 were synthesized as follows. 1M hydrochloric acid was added to the cyclodextrin complex of compound 15 or 11 or an aqueous solution contained in liposomes. The synthesis route is shown below.

As a result, fullerene derivatives represented by the formulas (11) to (17) were obtained as shown below. The counter anion of formula (12) is I ⁻ (iodine ion). As the counter anion, in addition to I (iodine), Cl, Br, NO ₃ , BF ₄ , PF ₆ , ClO ₄ , CH ₃ OSO _{3 and the} like can be used, but the present invention is not limited to these, and various types are used. be able to.

(Me represents a methyl group.)

<2> C ₆₀ fullerene derivatives - prepared C ₆₀ fullerene derivative of cyclodextrin complexes - preparing cyclodextrin complexes by high vibration grinding method. For each _{C 60} fullerene derivative of the formula (14) from equation (11) obtained by the above method, the 6.94 mmol, .gamma.-cyclodextrin 27.8 mmol (Wako Pure Chemical), placed in a high speed vibration grinding container High-speed vibration pulverization was performed at 30 Hz for 20 minutes. Thereafter, 1.5 ml of water was added to the obtained mixture and stirred, insoluble components were removed by centrifugation, and a brown supernatant was collected. The supernatant, C ₆₀ fullerene derivative - cyclodextrin complex solution. This aqueous solution can be used as the photosensitizing material of the present invention.

_{<3> C 60 C 60} fullerene derivative produced _{C 60} fullerene-containing liposomes of the fullerene-containing liposome - made by exchange reaction of cyclodextrin complexes with liposomes. The liposome used here is 9: 1 (molar ratio) of a cationic lipid synthesized with Dipalmitoylphosphatidylcholine (DPPC) manufactured by NOF Corporation. From obtained by the aforementioned method (of 4) (11) _{C 60} fullerene derivative of the formula (14) - for each of the cyclodextrin complexes, 0.2 mM, and 0.5 mL, liposomes 2 mM (lipid concentration), 0. Mix 5 mL. The mixture was stirred at 80 degrees for 1 hour. Thus the C ₆₀ fullerene derivative containing liposomes solution was obtained. This aqueous solution can be used as the photosensitizing material of the present invention. FIG. 3 shows a schematic diagram of the manufacturing method. In FIG. 3, “12-cyclodextrin complex” refers to a complex of (12) fullerene derivative of formula (12) and γ-cyclodextrin, and “12-containing liposome” refers to (formula 4) of formula (12). 2 shows liposomes containing fullerene derivatives.

When the temperature condition was 30 ° C., the exchange reaction of the C ₆₀ fullerene derivative to the liposome did not occur. Similarly, when the temperature condition is 30 ° C., the unmodified C ₆₀ fullerene does not undergo an exchange reaction, and the unmodified C ₇₀ fullerene undergoes an exchange reaction, and is a liposome that is a pseudo cell membrane that is detached from the cyclodextrin. Moved to. Incidentally, the unmodified C ₆₀ cyclodextrin complex and the C ₇₀ cyclodextrin direct cell introduction previously reported are (A. Ikeda complex, M. Matsumoto, M. Akiyama, J. Kikuchi,
T. Ogawa, T. Takeya, Chem. Commun., 2009, 1547.).
<Comparative Example 1>
_<1> Synthesis of _{C 60} fullerene derivatives

For C ₆₀ fullerene derivative of the compound 21 and compound 22 in Figure 1, with reference to Non-patent Documents 4 and 5, were synthesized. As a result, fullerene derivatives represented by the following (Chemical Formula 5) Formula (21) and Formula (22) were obtained.

(Et represents an ethyl group.)

<2> C ₆₀ fullerene derivatives - prepared C ₆₀ fullerene derivative of cyclodextrin complexes - preparing cyclodextrin complexes by high vibration grinding method. For each _{C 60} fullerene derivative obtained by the method (of 5) (21) and (22), like the _{C 60} fullerene derivative of the formula (14) from the (Formula 4) (11) A C ₆₀ fullerene derivative-cyclodextrin mixed aqueous solution was obtained by a high-speed vibration pulverization method. As for the fullerene derivative (21), it can be seen from FIG. 5F that a peak based on the fullerene derivative-cyclodextrin complex was not observed in the ¹ H NMR spectrum, so that no complex was formed. On the other hand, it can be seen that the fullerene derivative (22) is not dissolved since absorption based on fullerene is hardly seen in FIG. Accordingly, _{C 60} fullerene derivative from _{C 60} fullerene derivative (Formula 5) (21) and (22) - it was not possible to produce a cyclodextrin complex.

From the above Examples and Comparative Examples by fullerene derivative 1 was obtained (11) (14), (21), fullerene for (22) and _{C 60} fullerene - Figure 4 the ultraviolet absorption spectrum - visible cyclodextrin complex Show. From this figure, because it can ensure a characteristic absorption clearly fullerene derivative in the vicinity of 430 nm, the fullerene derivative (11) from (14) and C ₆₀ fullerene it can be seen that the solubilized in the dispersed state without meeting. On the other hand, it can be seen that the fullerene derivative (21) is associated because the absorption is broad. Furthermore, it can be seen that the fullerene derivative (22) is not dissolved because absorption based on fullerene is hardly observed.

FIG. 5 shows the ¹ H NMR spectrum of the fullerene-clodextrin complex for C ₆₀ fullerene and fullerene derivatives (11) to (14) and (21) obtained in the above Examples and Comparative Examples. In the figure, white circles indicate peaks due to γ-cyclodextrin not forming a complex, and black circles indicate peaks due to γ-cyclodextrin forming a complex with fullerene. In the figure, (A) is a C ₆₀ -cyclodextrin complex, (B) is a fullerene derivative (11) -cyclodextrin complex, (C) is a fullerene derivative (12) -cyclodextrin complex, and (D) is a fullerene derivative (13). ) -Cyclodextrin complex, (E) shows the spectrum of the fullerene derivative (14) -cyclodextrin complex, and (F) shows the spectrum of the fullerene derivative (21) -cyclodextrin mixture. From this figure, it can be seen that the fullerene derivative of Comparative Example (21) does not form a complex with γ-cyclodextrin. _Thus, by using a _{C 60} fullerene derivative of the _{C 60} fullerene derivative (11) to (14), it can be seen that complexes with cyclodextrin are formed.

For the photosensitized material prepared in Example 1 <2> and <3> and a material containing unmodified fullerene, an evaluation experiment of photodynamic activity using HeLa cells was performed. The results are shown in FIGS. The photosensitizing material was introduced into HeLa cells as follows. A photosensitizing material was added to the culture solution of HeLa cells cultured in a petri dish and treated for 24 hours. The photosensitized material that was not taken up by the cells was removed by washing with PBS (phosphate buffered saline) at pH 7.4. C ₆₀ fullerene derivatives to be introduced into HeLa cells have been subjected to experiments at various concentrations, and the concentrations on the horizontal axis in FIGS. 6 and 7 indicate the concentrations of fullerene derivatives. The HeLa cells into which the photosensitizing material was introduced were irradiated with light having a wavelength of 400 to 740 nm and an irradiation light amount of 37 mWcm ⁻² for 30 minutes under a temperature condition of 25 ° C., and the cell viability was measured after 24 hours. The photodynamic activity of the sensitive material was evaluated.

FIG. 6 and Table 1 show the evaluation results for the C ₆₀ fullerene derivative-cyclodextrin complex and the C ₆₀ fullerene-cyclodextrin complex, which are photosensitizing materials prepared in Example 1 <2>. According to FIG. 6, after introducing cyclodextrin complexes of C ₆₀ derivatives (12), (13) and (14) into HeLa cells, cell viability was dependent on concentration when irradiated with light having a wavelength of 400 to 740 nm. It is understood that a decrease in cell viability (or “cell death”) is confirmed, and in the dark, there is no change in cell viability. In contrast, the cyclodextrin complex of C ₆₀ fullerene, C ₇₀ fullerene and C ₆₀ fullerene derivative (11) showed a decrease in cell viability even when irradiated with light under the same conditions. C ₆₀ derivative fullerene (12), (13) It was found that this occurs less than the cyclodextrin complex of (14) and (14). Accordingly, C ₆₀ derivative (12) can be confirmed to have (13) and (14) that cyclodextrin complex was introduced into the cells, and these photosensitizing material photodynamic activity.

The above evaluation results are shown in FIG. 7 and Table for the C ₆₀ fullerene derivative-containing (11) (12) liposome, the C ₆₀ fullerene-containing liposome, and the C ₇₀ fullerene-containing liposome, which are photosensitizing materials prepared in Example 1 <3>. It is shown in 2. In FIG. 7, (A) and (B) differ in the value of the density on the x-axis.

IC ₅₀ is the concentration at which cell viability is inhibited by 50%, that is, the concentration of fullerene or fullerene derivative added at 50% mortality. The photosensitizing material of the present invention has an IC ₅₀ value in photokill experiments using HeLa cells of (i) cyclodextrin / C ₆₀ fullerene derivative (12) and C ₆₀ fullerene derivative (14) -cyclodextrin complex. Compared with C ₆₀ fullerene-cyclodextrin complex by about 35 times and C ₇₀ fullerene-cyclodextrin complex by about 16 times, (ii) C ₆₀ fullerene derivative (12) -containing liposome is about 66 compared with C ₆₀ fullerene-containing liposome. fold, it was improved by about 9 times compared to the C ₇₀ fullerene-containing liposomes.

An experiment was conducted to confirm the generation of reactive oxygen species in the C ₆₀ fullerene derivative-cyclodextrin complex and the C ₆₀ fullerene derivative-containing liposome, which are the photosensitizing materials of the present invention. The experimental method is as follows. The photosensitizing material of the present invention and 9,10-anthracenedipropionic acid disodium salt (ADPA) are added to heavy water (D ₂ O). Concentrations are 15 μM for unmodified fullerene photosensitizing material and fullerene derivative photosensitizing material, and 25 μM for ADPA. The solution is irradiated with light having a wavelength of 400 nm or more at an irradiation light amount of 15 mWcm ⁻² , and the generation amount of active oxygen species from the photosensitizing material for each irradiation time is measured. ADPA has a property of absorbing light having a wavelength of 400 nm in a normal state. Due to the presence of singlet oxygen, the structure of ADPA changes, and the structure does not absorb the light. By utilizing this, the amount of generated reactive oxygen species in the system can be measured by measuring the amount of light absorbed in the solution for each irradiation time.

The results of the experiment are shown in FIGS. 8 and 9, the graphs are normalized with the amount of absorption before irradiating light as 1. FIG. 8 shows the results for the C ₆₀ fullerene derivative-cyclodextrin complex and the C ₆₀ fullerene-cyclodextrin complex, which are photosensitizing materials prepared in Example 1 <2>, and FIG. 1 <3> _{C 60} fullerene derivative (11) or (12) liposomes and _{C 60} fullerene-containing liposomes containing a photosensitizing material produced _in, shows results for _{C 70} fullerene-containing liposomes. Thereby, it can be seen that the amount of active oxygen species generated in the photosensitizing material of the present invention is remarkably large.

The C ₆₀ fullerene derivative of the present invention can use C ₆₀ fullerene as an active oxygen generating species under visible light in various fields, so that it is possible to widen the choice of applications utilizing the C ₆₀ fullerene, Various devices and systems can be advanced. For example, in PDT, it is possible to realize PDT that effectively destroys target cells in a wavelength region safe for the human body.

Claims (7)

A inclusion materials include C ₆₀ fullerene derivative represented by the following formula (1) or (2), the C ₆₀ fullerene derivative in the interior of liposomes composed of lipid molecules containing a hydrophilic group and a hydrophobic group photosensitizing material comprising cyclodextrin complexes - fullerene-containing liposomes or fullerenes the C ₆₀ fullerene derivative is inclusion in cyclodextrins, but there.
(R1 is hydrogen or, .R2 and R3 represents a substituent group or atom group having a hydrophilic functional group identical or may be different, .X representing hydrogen, a substituent or group ^- represents a counter anion .) 2. The photosensitizing material according to claim 1, wherein R 1, R 2, and R 3 are hydrogen or a substituent or atomic group containing at least one carbon and having 10 or less carbon atoms. 3. The photosensitizing material according to claim 2, wherein R1 is any one of an alkyl group, ester group, ether group, amide group or acyl group having a hydrophilic functional group. The photosensitizing material according to claim 2, wherein R2 and R3 are alkyl groups having 6 or less carbon atoms or ethylene glycol chains having 10 or less carbon atoms. A photosensitizing material 4 according to claims 1 and C ₆₀ fullerene derivative present in the interior of the fullerene-containing liposomes contained in the photosensitizing material is close to the hydrophilic groups of the lipid molecules forming the liposomes Existing photosensitizing material. 6. The photosensitizing material according to claim 1, wherein a fluorescent substance having an absorption wavelength in the visible light region is present inside the fullerene-containing liposome contained in the photosensitizing material. 3. The photosensitizing material according to claim 1, wherein the fluorescent substance present in the fullerene-containing liposome contained in the photosensitizing material is close to the hydrophilic group of the lipid molecule constituting the liposome. Existing photosensitizing material.