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• W2085703722 abstract "The objective of this study is to evaluate the ability of C60/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) naonparticles to generate reactive oxygen species (ROS) and to induce cell toxicity by the photoirradiation. C60 nanoparticles were prepared by cogrinding with HP-β-CyD for 3 h at 4°C under reduced pressure. The photodynamic activity of C60/HP-β-CyD nanoparticles was evaluated by spectroscopic methods, including the electron spin resonance spin-trapping method, and by the cell viability test using Hela cells. C60/HP-β-CyD nanoparticles efficiently generated not only superoxide anion radical (O2·−) and hydroxyl radical (·OH), but also singlet oxygen (1O2) through photoirradiation. The ROS generation was enhanced by decreasing the mean particle diameter of C60 nanoparticles, and the particle size smaller than 90 nm showed a high generation of ·OH and 1O2. In addition, HP-β-CyD enhanced the generation of 1O2, compared with polyvinylpyrrolidone (an effective solubillizer for C60), due to partial disposition of C60 in the hydrophobic CyD cavity. Furthermore, C60/HP-β-CyD nanoparticles showed cell toxicity after the light irradiation, but no toxicity was observed without the light irradiation. Therefore, HP-β-CyD is useful for the preparation of stable C60 nanoparticles with high ROS generation ability, and C60/HP-β-CyD nanoparticles are a promising photosensitizer for photodynamic therapy. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3390–3397, 2012 The objective of this study is to evaluate the ability of C60/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) naonparticles to generate reactive oxygen species (ROS) and to induce cell toxicity by the photoirradiation. C60 nanoparticles were prepared by cogrinding with HP-β-CyD for 3 h at 4°C under reduced pressure. The photodynamic activity of C60/HP-β-CyD nanoparticles was evaluated by spectroscopic methods, including the electron spin resonance spin-trapping method, and by the cell viability test using Hela cells. C60/HP-β-CyD nanoparticles efficiently generated not only superoxide anion radical (O2·−) and hydroxyl radical (·OH), but also singlet oxygen (1O2) through photoirradiation. The ROS generation was enhanced by decreasing the mean particle diameter of C60 nanoparticles, and the particle size smaller than 90 nm showed a high generation of ·OH and 1O2. In addition, HP-β-CyD enhanced the generation of 1O2, compared with polyvinylpyrrolidone (an effective solubillizer for C60), due to partial disposition of C60 in the hydrophobic CyD cavity. Furthermore, C60/HP-β-CyD nanoparticles showed cell toxicity after the light irradiation, but no toxicity was observed without the light irradiation. Therefore, HP-β-CyD is useful for the preparation of stable C60 nanoparticles with high ROS generation ability, and C60/HP-β-CyD nanoparticles are a promising photosensitizer for photodynamic therapy. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3390–3397, 2012 INTRODUCTIONPhotodynamic therapy (PDT) is a next-generation cancer treatment based on reactive oxygen species (ROS). In PDT, a photosensitizer is systemically, locally, or topically administered, and tumor sites are irradiated by visible lights to generate ROS site-selectively, leading to cell deaths and tissue destructions.1.Castano A.P. Demidova T.N. Hamblin M.R. Mechanisms in photodynamic therapy: Part 1—Photosensitizers, photochemistry and cellular localization.Photodiagn Photodyn Ther. 2005; 1: 279-293Abstract Full Text Full Text PDF Scopus (1470) Google Scholar, 2.Castano A.P. Demidova T.N. Hamblin M.R. Mechanisms in photodynamic therapy: Part 2—Cellular signaling, cell metabolism and modes of cell death.Photodiagn Photodyn Ther. 2005; 2: 1-23Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 3.Castano A.P. Demidova T.N. Hamblin M.R. Mechanisms in photodynamic therapy: Part 3—Photosensitizer pharmacokinetics, biodistribution, tumor localization and modes of tumor destruction.Photodiagn Photodyn Ther. 2005; 2: 91-106Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar Therefore, PDT is an effective method for destroying diseased tissues without damaging the surrounding healthy tissues. The ideal photosensitizer for PDT is required to have high quantum yields in the generation of ROS, effective absorption of long-wavelength lights, and low toxicity under nonphotoirradiation. Many clinically employed photosensitizers are based on porphyrin molecules such as a hematoporphyrin derivative, Photofrin®. However, these compounds have several disadvantages such as prolonged skin sensitivity necessitating avoidance of sunlight for few weeks,4.Baas P. van Mansom I. van Tinteren H. Stewart F.A. van Zandwijk N. Effect of N-acetylcysteine on Photofrin-induced skin photosensitivity in patients.Lasers Surg Med. 1995; 16: 359-367Crossref PubMed Scopus (38) Google Scholar suboptimal tumor selectivity,5.Orenstein A. Kostenich G. Roitman L. Shechtman Y. Kopolovic Y. Ehrenberg B. Malik Z. A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model.Br J Cancer. 1996; 73: 937-944Crossref PubMed Scopus (110) Google Scholar and poor light penetration into tumors6.Spikes JD. Chlorins as photosensitizers in biology and medicine.J Photochem Photobiol B. 1990; 6: 259-274Crossref PubMed Scopus (199) Google Scholar due to the absorption of the relatively short wavelengths (630 or 664 nm). Therefore, more efficient photosensitizers are in various stages of development to improve their usefulness.7.Detty M.R. Gibson S.L. Wagner S.J. Current clinical and preclinical photosensitizers for use in photodynamic therapy.J Med Chem. 2004; 47: 3897-3915Crossref PubMed Scopus (929) Google ScholarFullerenes are currently of great interest for practical applications that take advantage of their unique electronic properties and biological activities.8.Jensen A.W. Wilson S.R. Schuster D.I. Biological applications of fullerenes.Bioorg Med Chem. 1996; 4: 767-779Crossref PubMed Scopus (613) Google Scholar The fullerene family, especially C60, has appealing photochemical, electrochemical, and physical properties, which can be exploited in various medical fields.9.Bakry R. Vallant R.M. Najam-ul-Haq M. Rainer M. Szabo Z. Huck C.W. Bonn G.K. Medicinal applications of fullerenes.Int J Nanomed. 2007; 2: 639-649PubMed Google Scholar Specifically, C60 has been regarded as an efficient photosensitizer for PDT10.Liu J. Ohta S.-.I. Sonoda A. Yamada M. Yamamoto M. Nitta N. Murata K. Tabata Y. Preparation of PEG-conjugated fullerene containing Gd3+ions for photodynamic therapy.J Controlled Release. 2007; 117: 104-110Crossref PubMed Scopus (116) Google Scholar11.Hamblin M.R. Mroz P. Tegos G.P. Gali H. Wharton T. Sarna T. Pawlak A. Photodynamic therapy with fullerenes.Fullerene Res Adv. 2007; : 1-31Google Scholar due to the light absorption of relatively long wavelengths (S–S absorption: 530 and 620 nm; T–T absorption: 400 and 740 nm) and the high quantum yield of photoexcitation reactions.12.Arbogast J.W. Darmanyan A.P. Foote C.S. Diederich F.N. Whetten R.L. Rubin Y. Alvarez M.M. Anz S.J. Photophysical properties of sixty atom carbon molecule (C60).J Phys Chem. 1991; 95: 11-12Crossref Scopus (1152) Google Scholar13.Nagano T. Arakane K. Ryu A. Masunaga T. Shinmoto K. Mashiko S. Hirobe M. Comparison of singlet oxygen production efficiency of C60with other photosensitizers, based on 1268 nm emission.Chem Pharm Bull. 1994; 42: 2291-2294Crossref Scopus (71) Google Scholar Photoirradiation of C60 results in the formation of the singlet excited state, 1C60*, followed by conversion to a triplet state, 3C60*, through the intersystem crossing with the high quantum yield (Fig. 1). Because the reduction potential of 3C60* is significantly high,14.Hwang K.C. Mauzerall D. Vectorial electron transfer from an interfacial photoexcited porphyrin to ground-state fullerene C60and C70and from ascorbate to triplet C60and C70in a lipid bilayer.J Am Chem Soc. 1992; 114: 9705-9706Crossref Scopus (86) Google Scholar an electron transfer from reductants such as amines and NADH to 3C60* occurs to give the C60·− radical anion, subsequently reducing oxygen to superoxide anion radial (O2·−) (type I pathway).15.Arbogast J.W. Foote C.S. Kao M. Electron transfer to triplet fullerene C60.J Am Chem Soc. 1992; 114: 2277-2279Crossref Scopus (429) Google Scholar16.Yamakoshi Y. Sueyoshi S. Fukuhara K. Miyata N. Masumizu T. Kohno M. ·OH and O2⋅− generation in aqueous C60and C70solutions by photoirradiation: An EPR study.J Am Chem Soc. 1998; 120: 12363-12364Crossref Scopus (136) Google Scholar In addition, the energy transfer reactions (type II pathway) of photoexcited C60 have been reported,12.Arbogast J.W. Darmanyan A.P. Foote C.S. Diederich F.N. Whetten R.L. Rubin Y. Alvarez M.M. Anz S.J. Photophysical properties of sixty atom carbon molecule (C60).J Phys Chem. 1991; 95: 11-12Crossref Scopus (1152) Google Scholar that is, the 3C60* efficiently transfers its energy to molecular oxygen to give singlet oxygen (1O2). In spite of these potential photoinduced biological activities of C60, its extremely low solubility and poor dispersibility in water have significantly impeded pharmaceutical applications.17.Ruoff R.S. Tse D.S. Malhotra R. Lorents D.C. Solubility of fullerene (C60) in a variety of solvents.J Phys Chem. 1993; 97: 3379-3383Crossref Scopus (1070) Google ScholarAlthough several water-soluble fullerene derivatives are reported,18.Friedman S.H. DeCamp D.L. Sijbesma R.P. Srdanov G. Wudl F. Kenyon G.L. Inhibition of the HIV-1 protease by fullerene derivatives: Model building studies and experimental verification.J Am Chem Soc. 1993; 115: 6506-6509Crossref Scopus (962) Google Scholar, 19.Tokuyama H. Yamago S. Nakamura E. Shiraki T. Sugiura Y. Photoinduced biochemical activity of fullerene carboxylic acid.J Am Chem Soc. 1993; 115: 7918-7919Crossref Scopus (624) Google Scholar, 20.Ikeda A. Yoshimura M. Shinkai S. Solution complexes formed from C60and calixarenes. On the importance of the preorganized structure for cooperative interactions.Tetrahedron Lett. 1997; 38: 2107-2110Crossref Scopus (86) Google Scholar, 21.Yoshida Z. Takekuma H. Takekuma S. Matsubara Y. Molecular recognition of C60with γ-cyclodextrin.Angew Chem Int Engl. 1994; 33: 1597-1599Crossref Scopus (242) Google Scholar, 22.Iwamoto Y. Yamakoshi Y. A highly water-soluble C60-NVP copolymer: A potential material for photodynamic therapy.Chem Commun. 2006; : 4805-4807Crossref PubMed Scopus (86) Google Scholar, 23.Xiao L. Takada H. Gan Xue h. Miwa N. The water-soluble fullerene derivative “Radical Sponge” exerts cytoprotective action against UVA irradiation but not visible-light-catalyzed cytotoxicity in human skin keratinocytes.Bioorg Med Chem Lett. 2006; 16: 1590-1595Crossref PubMed Scopus (109) Google Scholar chemical modifications of C60 usually decrease its photophysical properties.24.Hamano T. Okuda K. Mashino T. Hirobe M. Arakane K. Ryu A. Mashiko S. Nagano T. Singlet oxygen production from fullerene derivatives: Effect of sequential functionalization of the fullerene core.Chem Commun. 1997; : 21-22Crossref Scopus (116) Google Scholar25.Prat F. Stackow R. Bernstein R. Qian W. Rubin Y. Foote C.S. Triplet-state properties and singlet oxygen generation in a homologous series of functionalized fullerene derivatives.J Phys Chem A. 1999; 103: 7230-7235Crossref Scopus (98) Google Scholar Therefore, solubilization of C60 without chemical modifications is a better approach for pharmaceutical applications of C60. Furthermore, the large aggregation of C60 significantly accelerates the decay of excited triplet state C60, thus, reducing the photosensitizing ability of C60.26.Lee J. Yamakoshi Y. Hughes J.B. Kim J.-.H. Mechanism of C60photoreactivity in water: Fate of triplet state and radical anion and production of reactive oxygen species.Environ Sci Technol. 2008; 42: 3459-3464Crossref PubMed Scopus (87) Google Scholar In a previous study, we reported the formation of stable C60 nanoparticles, on whose surfaces hydrophilic 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) covered through the adsorption and weak interaction.27.Iohara D. Hirayama F. Higashi K. Yamamoto K. Uekama K. Formation of stable hydrophilic C60nanoparticles by 2-hydroxypropyl-β-cyclodextrin.Mol Pharmaceutics. 2011; 8: 1276-1284Crossref PubMed Scopus (30) Google Scholar In this study, we evaluated the ability of C60/HP-β-CyD naonparticles to generate ROS after photoirradiation by using spectroscopic methods including the electron spin resonance (ESR) spin-trapping method. In addition, we examined the photoinduced cytotoxicity of C60/HP-β-CyD nanoparticles using Hela cells, and these photodynamic activities were compared with those of C60/polyvinylpyrrolidone (PVP) particles.MATERIALS AND METHODSMaterialsC60 (nanom purple SUH) was obtained from Frontier Carbon Company (Tokyo, Japan). HP-β-CyD (degree of substitution of 2-hydroxypropyl group is 5.6) was donated by Nihon Shokuhin Kako Company. Ltd. (Tokyo, Japan). PVP (K-30) was obtained from BASF Japan Ltd. (Tokyo, Japan). 5,5-Dimethy-1-pyrroline N-oxide (DMPO) was purchased from Labotec Company Ltd. (Tokyo, Japan). 4-Hydroxy-2,2,6,6,-tetramethylpiperidine (TEMP-OH) was purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Superoxide dismutase (SOD) was purchased from Sigma-Aldrich Company. (Tokyo, Japan). Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium, and penicillin–streptomycin were purchased from GIBCO Invitrogen (Tokyo, Japan). Fetal calf serum was obtained from Nichirei (Tokyo, Japan). All other materials and solvents were of analytical reagent grade, and Milli-Q water was used throughout the study.Preparation of Hydrophilic C60 Nanoparticles and C60/PVP DispersionC60 (15 mg) was ground with HP-β-CyD (60 mg) in a mole ratio 1:2 (guest:host) using an automatic magnetic agitating mortar (MNV-01, AS ONE Company, Tokyo, Japan) for 3 h at 4°C under reduced pressure. The pulverized C60/HP-β-CyD powder was dispersed in water by ultrasonication for 5 min. C60/PVP particles were prepared in the same manner. The resulting dispersions of C60/HP-β-CyD and C60/PVP were syringe-filtered through a filter of 0.2 or 0.45 µm pore size, respectively. C60 was also dispersed in water and various solvents by ultrasonicating intact C60 for 1 h, then syringe-filtered through a filter of 0.8 µm pore size. Particle sizes of the C60 colloidal solutions were determined by a dynamic light scattering machine (DLS-8000HL, Otsuka Electronics Company Ltd., Tokyo, Japan) equipped with He–Ne laser (10 mW) operating at 632.8 nm. DLS measurements were performed at a scattering angle of 90°. The autocorrelation function was analyzed by the cumulant method to obtain the average particle diameter. Concentrations of C60 in the colloidal solutions were determined by the method reported previously.27.Iohara D. Hirayama F. Higashi K. Yamamoto K. Uekama K. Formation of stable hydrophilic C60nanoparticles by 2-hydroxypropyl-β-cyclodextrin.Mol Pharmaceutics. 2011; 8: 1276-1284Crossref PubMed Scopus (30) Google ScholarO2·− Generation Ability of C60/HP-β-CyD NanoparticlesO2·− generation from C60/HP-β-CyD nanoparticles by visible light irradiation was measured by the cytochrome c method, that is, aqueous cytochrome c solution (80 µM) was mixed with C60 colloidal solution (C60 = 80 µM). The resulting solutions were exposed to visible light supplied from a fluorescence lamp (3500 lux, 400–700 nm, 2 cm from the bottom). An increase in the absorbance at 548 nm of the reduced cytochrome c was measured with a spectrophotometer (U-2800A, Hitachi, Tokyo, Japan).·OH and 1O2 Generation Ability of C60/HP-β-CyD NanoparticlesHydroxyl radical (·OH) and 1O2 generations from C60/HP-β-CyD nanoparticles were measured by an X-band ESR spectrometer (JES-FA100, JEOL Ltd., Tokyo, Japan) under the following conditions: microwave frequency 9.417 GHz, microwave power 8 mW, field modulation 0.1 mT at 100 kHz, and sweep time 2 min. ·OH was detected using DMPO as a spin-trapping reagent. One hundred microliter of C60 solutions (80 µM) and 80 µL of milli-Q, 600 units SOD solution or 5.0 M sodium formate solution, and 20 µL of DMPO were mixed well under an aerobic condition. 1O2 was also detected by the ESR method using TEMP-OH as a spin-trapping reagent. One hundred microliter of C60 solutions (100 µM), 50 µL of milli-Q or NaN3 solution (400 mM), and 100 µL of TEMP-OH (160 mM) were mixed well under an aerobic condition. The mixed solutions were collected in a flat cell, then exposed to visible light supplied from a fluorescence lamp (3500 lux, 400–700 nm), and subjected immediately to ESR measurement. Generation efficiency of ·OH and 1O2 was evaluated by the relative intensity to an external reference of Mn2+.Phototoxicity of C60/HP-β-CyDs NanoparticlesHela cells were cultured in MEM supplemented with 10% fetal bovine serum containing 100 units/mL penicillin–streptomycin, at 37°C and 5% of CO2. The cells were seeded in 96-well plates at a density of 1.0 × 104 cells/well. After growing overnight, the cells were incubated with C60/HP-β-CyD nanoparticles for 48 h in the dark. The cells were washed with phosphate-buffered saline and replaced with a fresh culture medium. The treated cells were exposed to light (2 × 105 lux, 400–700 nm) for 30 min, using a high-pressure mercury lamp. To measure the viability of cells as a ratio (%) compared with cells, which were not treated with C60, WST-1 assay was carried out 24 h after the photoirradiation, using Cell Counting Kit (Dojindo Laboratories, Kumamoto, Japan).RESULTS AND DISCUSSIONROS Generation Ability of C60/HP-β-CyD NanoparticlesC60/HP-β-CyD nanoparticles were prepared by the method reported previously.27.Iohara D. Hirayama F. Higashi K. Yamamoto K. Uekama K. Formation of stable hydrophilic C60nanoparticles by 2-hydroxypropyl-β-cyclodextrin.Mol Pharmaceutics. 2011; 8: 1276-1284Crossref PubMed Scopus (30) Google Scholar28.Iohara D. Hirayama F. Kansui H. Aoshima H. Yamana S. Yano M. Kitaguchi J. Takashima S. Uekama K. Preparation of hydrophilic nanoparticles of C60with high resistance to aggregation during storage, using 2-hydroxypropyl-β-cyclodextrin.Chem Lett. 2009; 38: 1104-1105Crossref Scopus (7) Google Scholar C60 was also dispersed in water using PVP, an effective solubillizer for C60.22.Iwamoto Y. Yamakoshi Y. A highly water-soluble C60-NVP copolymer: A potential material for photodynamic therapy.Chem Commun. 2006; : 4805-4807Crossref PubMed Scopus (86) Google Scholar29.Yamakoshi Y.N. Yagami T. Fukuhara K. Sueyoshi S. Miyata N. Solubilization of fullerenes into water with poly(vinylpyrrolidone) applicable to biological tests.J Chem Soc Chem Commun. 1994; : 517-518Crossref Google Scholar Figure 2 shows the appearances and the mean particle diameters of C60 colloidal solutions prepared with or without solubillizing agents. The C60/HP-β-CyD colloidal solution was a transparent dark brown-colored solution with 90 nm of the mean particle diameter. In the case of C60/PVP, the brown-colored solution was obtained and the mean particle diameter was 215 nm. When C60 alone was dispersed in water by ultrasonication, the suspensions with mean particle diameter of 427 nm was obtained, indicating that C60 formed large aggregates in water. The particle size distribution of C60/HP-β-CyD and C60/PVP measured by DLS showed one narrow population group, but the C60 alone showed a wide population group. The amount (i.e., yield) of C60 in the filtrates was almost 100% for the C60/HP-β-CyD colloidal solution indicating that it changed to small-sized particles and passed completely through the 0.2 µm filter.27.Iohara D. Hirayama F. Higashi K. Yamamoto K. Uekama K. Formation of stable hydrophilic C60nanoparticles by 2-hydroxypropyl-β-cyclodextrin.Mol Pharmaceutics. 2011; 8: 1276-1284Crossref PubMed Scopus (30) Google Scholar On the contrary, C60/PVP and C60 alone gave the yields of 10% and 1.8%, respectively, due to the filtration of large aggregate. These results suggested that HP-β-CyD is much more useful for dispersing C60 in water, when compared with PVP.Figure 2Mean particle diameters and appearances of C60 water dispersion prepared with or without solubilizing agent. Each value represents the mean ± S.E. of three experiments.View Large Image Figure ViewerDownload (PPT)To evaluate the photosensitizing ability of C60/HP-β-CyD nanoparticles, the generation of superoxide anion (O2·−) with the irradiation of visible light was measured by the cytochrome c method. In the presence of C60/HP-β-CyD nanoparticles, the absorbance at 548 nm of the reduced cytochrome c form increased with the photoirradiation time. The effect of C60/HP-β-CyD nanoparticles on the O2·− generation was significantly higher than that of the C60/PVP and C60 alone, as shown in Figure 3. Such an increase in the absorbance was not observed for CyD and PVP solutions alone and without light irradiation (date not shown). The increase of the absorbance was completely suppressed by the addition of SOD. These results indicate that C60/HP-β-CyD nanoparticles effectively generate superoxide anions, reacting with cytochrome c to form its reduced form. Furthermore, the ESR spin-trapping method was used for detection of O2·−. Figure 4a shows ESR spectra of C60/HP-β-CyD colloidal solutions under the photoirradiation. A four-line ESR signal with hyperfine splitting constants corresponding to those of DMPO-OH (aN = 1:49 mT, aH = 1:49 mT)30.Buettner GR. Spin trapping: ESR parameters of spin adducts.Free Radical Biol Med. 1987; 3: 259-303Crossref PubMed Scopus (1505) Google Scholar was detected on ESR spectra and increased with elapse of the photoirradiation time. In the presence of C60/HP-β-CyD nanoparticles, the relative intensity of the DMPO-OH to an external reference (Mn2+) increased with the irradiation time. This increase of the ESR signals was suppressed by the addition of a quencher specific to ·OH radical, sodium formate, and the ESR signal of the carbon dioxide radical anion (CO2·−) adduct of DMPO was observed. These results indicate that ·OH was generated in these solutions by the visible light irradiation. The addition of SOD, a O2·− quencher, also reduced the ESR signal increment. Because ·OH radicals are known to be generated from O2·− via the Fenton reaction under reducing conditions (Fig. 1),31.Prousek J. Fenton chemistry in biology and medicine.Pure Appl Chem. 2007; 79: 2325-2338Crossref Scopus (303) Google Scholar it is probable that O2·− generated from C60 is rapidly converted to ·OH, which reacts with DMPO. Consequently, the DMPO-OH adduct was observed in the ESR spectra. However, the suppression of the DMPO-OH signal by the addition of sodium formate was not complete, as shown in Figure 4. This may be due to the reaction of sodium formate with ·OH to give CO2·− radicals, which reacts with O2 reproducing O2·− radicals. Therefore, it is apparent that the ·OH generation ability of C60/HP-β-CyD nanoparticles was significantly higher than that of the C60/PVP and C60 alone, as shown in Figure 4b.Figure 3Absorbance increment of reduced cytochrome c with visible light irradiation in the presence of C60/HP-β-CyD nanopartlcles, C60/PVP, and C60 alone (C60 = 40 µM). ○: C60/HP-β-CyD nanoparticles; Δ: C60/PVP dispersion; ⋄: C60 alone; ▪: C60/HP-β-CyD nanoparticles with SOD. Each point represents the mean ± S.E. of three to four experiments.View Large Image Figure ViewerDownload (PPT)Figure 4ESR spectra of DMPO-OH adduct generated in C60/HP-β-CyD colloidal solution after visible light irradiation (a) and ·OH generation ability of C60/HP-β-CyD nanoparticles, C60/PVP, and C60 alone (b) (C60 = 40 µM). ○: C60/HP-β-CyD nanoparticles; Δ: C60/PVP dispersion; ◊: C60 alone; •: C60/HP-β-CyD nanoparticles with HCOONa; ▪: C60/HP-β-CyD nanoparticles with SOD. Each point represents the mean ± S.E. of three to six experiments. Relative intensity to an external references (Mn2+).View Large Image Figure ViewerDownload (PPT)The ability of C60/HP-β-CyD nanoparticles to generate 1O2 through the type II pathway (Fig. 1) was also studied by the ESR spin-trapping method. As shown in Figure 5a, C60/HP-β-CyD nanoparticles gave three characteristic signals of TEMPO-OH32.Yamakoshi Y. Umezawa N. Ryu A. Arakane K. Miyata N. Goda Y. Masumizu T. Nagano T. Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2⋅−versus1O2.J Am Chem Soc. 2003; 125: 12803-12809Crossref PubMed Scopus (596) Google Scholar on ESR spectra, indicating the generation of 1O2 by the photoirradiation, and the relative intensity of the TEMPO-OH increased with the photoirradiation time. This increase of intensity was completely suppressed by the addition of high concentration of sodium azide (NaN3), a 1O2 quencher. However, NaN3 did not show any 1O2 quenching at low concentrations. It is reported that the microenvironment of C60 nanoparticles resists 1O2 quenchers, that is, the photoproduced 1O2 is mostly deactivated inside C60 nonoaggregates.33.Bilski P. Zhao B. Chignell C.F. Singlet oxygen phosphorescence photosensitized in nano-aggregates of C60buckminsterfullerene is insensitive to solvent and quenchers and strongly red-shifted indicating highly polarizable interior.Chem Phys Lett. 2008; 458: 157-160Crossref Scopus (22) Google Scholar In fact, we have confirmed no deuterium oxide effect under the experimental conditions. These results indicate that C60/HP-β-CyD nanoparticles effectively generate not only O2·−, but also 1O2 by the photoirradiation. In sharp contrast, the C60/PVP dispersion and C60 alone hardly generated 1O2. Therefore, it can be concluded that C60/HP-β-CyD nanoparticles have a high potential for generating the ROS, including O2·−, ·OH and 1O2 with photoirradiation.Figure 5ESR spectra of TEMPO-OH adduct generated in C60/HP-β-CyD colloidal solution after visible light irradiation (a) and 1O2 generation ability of C60/HP-β-CyD nanoparticles, C60/PVP, and C60 alone (b) (C60 = 40 µM). ○: C60/HP-β-CyD nanoparticles; Δ: C60/PVP dispersion; ◊: C60 alone; ▪: C60/HP-β-CyD nanoparticles with NaN3. Each point represents the mean ± S.E. of three to four experiments. Relative intensity to an external references (Mn2+).View Large Image Figure ViewerDownload (PPT)Effect of C60 Particle Size on ROS Generation of C60/HP-β-CyD NanoparticlesC60/HP-β-CyD nanoparticles generated ROS with the photoirradiation, that is, O2·− and ·OH species through the type I pathway and 1O2 species through the type II pathway, respectively. It should be noted that C60 alone with the large particle size hardly generated O2·− and 1O2 species, indicating that particle size of C60 significantly affects the generation of ROS. It has been reported that the aggregation of C60 accelerates the decay of 3C60*, resulting in short interactions between 3C60* and oxygen molecule, thus, reducing the generation of ROS.26.Lee J. Yamakoshi Y. Hughes J.B. Kim J.-.H. Mechanism of C60photoreactivity in water: Fate of triplet state and radical anion and production of reactive oxygen species.Environ Sci Technol. 2008; 42: 3459-3464Crossref PubMed Scopus (87) Google Scholar Furthermore, the increase in particle size reduces surface areas to be exposed to light, which may reduce the photoexcitation reaction of C60. Therefore, we investigated the effect of the size of C60/HP-β-CyD nanoparticles on the generation of ROS, by means of the ESR spin-trapping method (Fig. 6). C60 particles with different sizes were obtained by changing in the cogrinding time with HP-β-CyD.27.Iohara D. Hirayama F. Higashi K. Yamamoto K. Uekama K. Formation of stable hydrophilic C60nanoparticles by 2-hydroxypropyl-β-cyclodextrin.Mol Pharmaceutics. 2011; 8: 1276-1284Crossref PubMed Scopus (30) Google Scholar The relative intensity of DMPO-OH signals increased with a decrease in the mean particle diameter of C60, and a high generation of ·OH was observed when the particle size was smaller than 150 nm. The generation ability of 1O2 was also size dependent and the high generation of 1O2 was achieved at the smaller particle size than 150 nm, as shown in Figure 6b. These results indicate that particle size of C60 significantly affects the generation of ROS, that is, to produce ROS efficiently, the mean particle size of C60 should be smaller than 200 nm, whereas a particle size over 300 nm barely generates ROS. Thus, the C60/PVP system and C60 alone in largely aggregated states (215 and 427 nm, respectively) showed the low generation of ROS. C60/HP-β-CyD nanoparticles may be efficiently excited due to their high surface-to-mass ratio by the photoirradiation and maintained the excited state for longer times, leading to the high ROS generation. Furthermore, it was interesting to note that the effect of C60/HP-β-CyD nanoparticles on the 1O2 generation was much greater than those of the PVP system and C60 alone (Fig. 5b), and it generated 1O2 even though the particle size of C60 became as large as C60/PVP system (Fig. 6b). It is suggested that 1O2 is preferably produced in the nonpolar solvents.34.Scurlock R.D. Ogilby P.R. Effect of solvent on the rate constant for the radiative deactivation of singlet molecular oxygen (1ΔgO2).J Phys Chem. 1987; 91: 4599-4602Crossref Scopus (119) Google Scholar35.Wessels J.M. Rodgers M.A.J. Effect of solvent polarizability on the forbidden1Δg→3Σg−transition in molecular oxygen: A Fourier transform near-infrared luminescence stu" @default.
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