Kinetic Analysis of Superoxide Anion Radical-Scavenging and

Jun 14, 2008 - Department of Genetic Resources Technology, Kyushu University. , ‡. Nihon Trim Co. Ltd. , §. Department of Chemistry and Biochemistr...
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7354

Langmuir 2008, 24, 7354-7364

Kinetic Analysis of Superoxide Anion Radical-Scavenging and Hydroxyl Radical-Scavenging Activities of Platinum Nanoparticles Takeki Hamasaki,† Taichi Kashiwagi,† Toshifumi Imada,† Noboru Nakamichi,‡ Shinsuke Aramaki,† Kazuko Toh,† Shinkatsu Morisawa,‡ Hisashi Shimakoshi,§ Yoshio Hisaeda,§ and Sanetaka Shirahata*,† Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu UniVersity, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, Nihon Trim Co. Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka 531-0076, Japan, and Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu UniVersity, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan ReceiVed December 28, 2007. ReVised Manuscript ReceiVed April 12, 2008 There are few reports on the physiological effects of metal nanoparticles (nps), especially with respect to their functions as scavengers for superoxide anion radical (O2 · -) and hydroxyl radical ( · OH). We tried to detect the scavenging activity of Pt nps using a hypoxanthine-xanthine oxidase system for O2 · - and using a Fenton and a UV/H2O2 system for · OH. Electron spin resonance analysis revealed that 2 nm particle size Pt nps have the ability to scavenge O2 · - and · OH. The calculated rate constant for the O2 · --scavenging reaction was 5.03 ( 0.03 × 107 M-1 s-1. However, the analysis of the Fenton and UV/H2O2 system in the presence of Pt nps suggested that the · OH-scavenging reaction cannot be determined in both systems. Among particle sizes tested from 1 to 5 nm, 1 nm Pt nps showed the highest O2 · --scavenging ability. Almost no cytotoxicity was observed even after adherent cells (TIG-1, HeLa, HepG2, WI-38, and MRC-5) were exposed to Pt nps at concentrations as high as 50 mg/L. Pt nps scavenged intrinsically generated reactive oxygen species (ROS) in HeLa cells. Additionally, Pt nps significantly reduced the levels of intracellular O2 · - generated by UVA irradiation and subsequently protected HeLa cells from ROS damage-induced cell death. These findings suggest that Pt nps may be a new type of antioxidant capable of circumventing the paradoxical effects of conventional antioxidants.

1. Introduction Reactive oxygen species (ROS) such as superoxide anion radical (O2 · -), hydrogen peroxide (H2O2), the hydroxyl radical ( · OH) are generated as a result of normal intracellular metabolism.1–3 As well, external factors4 can also trigger the production of ROS. Excessive levels of ROS can damage intracellular molecules, resulting in various diseases and aging.3 The superoxide anion radical (O2 · -) is generated in large amounts in the body from mitochondria5,6 and converted to hydrogen peroxide (H2O2) and hydroxyl radicals ( · OH).3 The body has an O2 · - defense system regulated by superoxide dismutase (SOD) and a catalase,7 which helps to counteract ROS and regulate overall ROS levels to maintain physiological homeostasis.4,8 Controlling excess O2 · - will contribute to a reduction of ROS damage in the living body. The hydroxyl radical is known as one of the most highly reactive species of all ROS because of an unpaired electrons (second-order rate constants with organic compounds range from 108-1010 M-1 s-1).9 It has an average lifetime of 10-9 s and can react with nearly all biomolecules * To whom correspondence should be addressed. Telephone: +81-92642-3045. Fax: +81-92-642-3052. E-mail: [email protected] (S.S.); [email protected] (T.H.). † Department of Genetic Resources Technology, Kyushu University. ‡ Nihon Trim Co. Ltd. § Department of Chemistry and Biochemistry, Kyushu University.

(1) Finkel, T. Curr. Opin. Cell Biol. 2003, 15, 247–254. (2) Allen, R. G.; Tresini, M. Free Radical Biol. Med. 2000, 28, 463–499. (3) Dro¨ge, W. Physiol. ReV. 2002, 82, 47–95. (4) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239–247. (5) Aiken, J. D.; Finke, R. G. J. Mol. Catal. 1999, 145, 1–44. (6) Toshima, N.; Yonezawa, T. New. J. Chem. 1998, 22, 1179–1201. (7) MacInnes, D. A. J. Am. Chem. Soc. 1914, 36, 878–881. (8) Curtin, J. F.; Donovan, M.; Cotter, T. G. J. Immunol. Methods 2002, 265, 49–72. (9) Einschlag, F. S. G.; Carlos, L.; Capparelli, A. L. Chemoshpere 2003, 53, 1–7.

including DNA, proteins, and membrane lipids.3,10 To date, a scavenging system for · OH has not been identified, and thus, there is a strong demand for antioxidants which could act against such a harmful species. To aid cellular defenses against ROS, it is important to develop efficient methods and/or materials that are useful for prevention and/or treatment. One such approach is to make use of naturally available antioxidants, many of which possess potential health benefits, as revealed from human epidemiological studies.11–14 Metal nanoparticles (nps) have been extensively studied because of their unique functions.15–21 Metal nps consist of a metal, but, because of their characteristic ultrafine particle size, they can be homogeneously dispersed in solution15,17,18 so that their catalytic ability and molecular adsorption activities are expected to work in biological systems.22,23 Most of the earlier studies using nanometer-sized metal particles have focused on (10) Fang, Y. Z.; Yang, S.; Wu, G. Nutrition 2002, 18, 872–879. (11) Agarwal, A.; Saleh, R. A.; Mohamed, A. Fertil. Steril. 2003, 79, 829– 843. (12) Banerjee, S. K.; Mukherjee, P. K.; Maulik, S. K. Phytother. Res. 2003, 17, 97–106. (13) Kaugars, G. E.; Silverman, S., Jr.; Lovas, J. G.; Thompson, J. S.; Brandt, R. B.; Singh, V. N. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 1996, 81, 5–14. (14) Fuhrman, B.; Volkova, N.; Coleman, R.; Aviram, M. J. Nutr. 2005, 13, 722–728. (15) Lewis, L. N. Chem. ReV. 1993, 93, 2693–2730. (16) Link, S.; Ei-Sayed, M. A. Annu. ReV. Phys. Chem. 2003, 54, 331–366. (17) Roucoux, A.; Schulz, J.; Patin, H. Chem. ReV. 2002, 102, 3757–3778. (18) Bo¨nnemann, H.; Richards, R. M. Eur. J. Inorg. Chem. 2001, 10, 2455– 2480. (19) Heath, J. R.; Shiang, J. J. Chem. Soc. ReV. 1998, 27, 65–71. (20) Schmid, G. Polyhedron 1988, 7, 2321–2329. (21) Kubo, R. Ann. ReV. Mater. Sci. 1984, 14, 49–66. (22) Oberdo¨rster, G.; Sharp, Z.; Atudorei, V.; Elder, A.; Gelein, R.; Kreyling, W.; Cox, C. Inhalation Toxicol. 2004, 16, 437–445. (23) Warheit, D. B.; Webb, T. R.; Sayes, C. M.; Colvin, V. L.; Reed, K. L. Toxicol. Sci. 2006, 91, 227–236.

10.1021/la704046f CCC: $40.75  2008 American Chemical Society Published on Web 06/14/2008

AntioxidatiVe ActiVity of Pt nps

biological safety and potential hazards.23–30 A recent review summarized compelling evidence that np activities depend on a greater surface area per mass of nps relative to larger particles, which have inflammatory and pro-oxidant functions as well as antioxidative activity.25 Recently, it has been suggested that hydroxylated metallofullerenes regulate production of reactive oxygen species (ROS) in ViVo.31 However, only a limited number of reports are available regarding the antioxidative function of nps.32–35 In these reports, the authors utilized an electron spin resonance (ESR) method to evaluate the antioxidative activity as a measure of the bioactivity of 5,5-dimethyl-1-pyrroline-noxide (DMPO) to be able to trap O2 · - and · OH radicals. This ESR coupled with the DMPO spin-trapping method is widely used to evaluate activities of endogenous antioxidative molecules.36,37 However, caution should be taken when attempting to directly apply this in Vitro evaluation system to nps because metal nps could act as either reductive or oxidative catalysts.15 Therefore, in the present study, we examined whether enhancement of the direct O2 · -- or · OH-scavenging reaction would occur. Second, the antioxidative activity was evaluated by determining a second-order rate constant for Pt nps, O2 · -, and · OH using the DMPO ESR spin-trapping method. It was then determined whether the radical-scavenging activities of Pt nps are dependent on their particle size by examining their properties in the 1-5 nm particle size range. Furthermore, the studies extended to an investigation of the ROS-scavenging activity and cytotoxicity of Pt nps using cultured cell lines.

2. Materials and Methods 2.1. Materials. Hydrogen hexachloroplatinate (H2PtCl6), Lascorbic acid (AsA), diethylenetriamine-N,N,N′,N′′,N′′′-pentaacetic acid (DETAPAC), 1,1-diphenyl-2-picrylhydrazyl (DPPH), dihydroethidium (HEt), and potassium superoxide (KO2) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). The spin trap, fine 5,5dimethyl-1-pyrroline-n-oxide (DMPO), and 4-hydroxy-2,2,6,6tetramethylpiperidine-N-oxyl (TEMPOL) were obtained from Labotec Co. (Tokyo, Japan). 5-(Diethoxyphosphoryl)-5-methyl-1pyrroline-N-oxide (DEPMPO) was obtained from Alexis Co., Ltd. Hypoxanthine (HPX) was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Xanthine oxidase (XOD) and bovine Mn-SOD were purchased from Roche Diagnostics (Tokyo, Japan). 2-(4-Iodophenyl)3-(4-nitrophenyl)-3-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) was obtained from Dojindo Laboratories Corp. (Tokyo, Japan). For cell culture, Eagle’s minimum essential medium (MEM) was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and fetal (24) Chang, C. C.; Chiu, H. F.; Wu, Y. S.; Li, Y. C.; Tsai, M. L.; Shen, C. K.; Yang, C. Y. EnViron. Health Perspect. 2005, 113, 454–460. (25) Oberdo¨rster, G.; Oberdo¨rster, E.; Oberdo¨rster, J. EnViron. Health Perspect. 2005, 113, 823–839. (26) Dick, C. A. J.; Brown, D. M.; Donaldson, K.; Stone, V. Inhalation Toxicol. 2003, 15, 39–52. (27) Dugan, L. L.; Gabrielsen, J. K.; Yu, S. P.; Lin, T. S.; Choi, D. W. Neurobiol. Dis. 1996, 3, 129–135. (28) Soto, K. F.; Carrasco, A.; Powell, T. G.; Murr, L. E.; Garza, K. M. Mater. Sci. Eng., C 2006, 26, 1421–1427. (29) Zhang, Q. W.; Kusaka, Y.; Zhu, X. Q.; Sato, K.; Mo, Y. Q.; Kluz, T.; Donaldson, K. J. Occup. Health 2003, 45, 23–30. (30) Rahman, Q.; Lohani, M.; Dopp, E.; Pemsel, H.; Jonas, L.; Weiss, D. G.; Schiffmann, D. EnViron. Health Perspect. 2002, 110, 797–800. (31) Wang, J. X.; Chen, C. Y.; Li, B.; Yu, H. W.; Zhao, Y. L.; Sun, J.; Li, Y. F.; Xing, G. M.; Yuan, H.; Tang, J.; Chen, Z.; Meng, H.; Gao, Y. X.; Ye, C.; Chai, Z. F.; Zhu, C. F.; Ma, B. C.; Fang, X. H.; Wan, L. J. Biochem. Pharmacol. 2006, 14, 782–881. (32) Kajita, M.; Hikosaka, K.; Iitsuka, M.; Kanayama, A.; Toshima, N.; Miyamoto, Y. Free Radical Res. 2007, 41, 615–626. (33) Esumi, K.; Takei, N.; Yoshimura, T. Colloids Surf., B 2003, 32, 117–123. (34) Esumi, K.; Houdatsu, H.; Yoshimura, T. Langmuir 2004, 20, 2536–2538. (35) Shukla, R.; Bansal, V.; Chaudhary, M.; Basu, A.; Bhonde, R. R.; Sastry, M. Langmuir 2005, 21, 10644–10654. (36) Olive, G.; Mercier, A.; Le Moigne, F.; Rockenbauer, A.; Tordo, P. Free Radical Biol. Med. 2000, 28, 403–408. (37) Zhang, H.; Joseph, J.; Vasquez-Vivar, J.; Karoui, H.; Nsanzumuhire, C.; Martasek, P.; Tordo, P.; Kalyanaraman, B. FEBS Lett. 2000, 473, 58–62.

Langmuir, Vol. 24, No. 14, 2008 7355 bovine serum (FBS) was from Invitrogen Japan (Tokyo, Japan). Hanks balanced salt solution (HBSS) was obtained from SigmaAldrich. A fluorescent dye, 2′,7′-dichlorofluorescein-diacetate (DCFHDA), and other reagents not detailed here were obtained from Wako Pure Chemical Inc. (Tokyo, Japan). All solutions were prepared with deionized water drawn from a Milli-Q synthesis system (Milli-Q water, Millipore, Tokyo, Japan). 2.2. Methods. 2.2.1. Preparation of Pt nps. Pt nps of ∼2.0 nm size were synthesized by a modified ethanol reduction method.38 Briefly, the reaction mixture containing 0.04% hydrogen hexachloroplatinate, 0.5% polyethylene glycol sorbitan monooleate (Tween 80, Tw80), 40 mM sodium phosphate buffer (pH 7.0), and 10% ethanol was incubated with stirring in a sealed bottle at 60 °C until the mixture became dark black (10-16 h). After incubation, the synthesized Pt np solution was desalted by adding 10 volumes of Milli-Q water to 1 volume of Pt np solution and concentrated by ultrafiltration with a 10 000 molecular weight limiting ultrafiltration membrane (Advantec MFS, Inc., CA). These desalting and concentration steps were repeated three times. The concentration of Pt nps was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500, Agilent Technologies, CA) at the Center for Advanced Instrumental Analysis, Kyushu University. The concentration of the synthesized Pt nps was adjusted to 200 mg/L, and the Pt nps were stored at 4 °C. Pt nps of ∼1 and 3 nm were synthesized by a previously reported sodium citrate reduction method.39 Pt nps with particle sizes of 3-5 nm were synthesized using glycerol and based on another method which has been described previously.40 Briefly, a reaction mixture containing 0.04% H2PtCl6, 40 mM sodium phosphate buffer (pH 7.0), and 90% glycerol was incubated with stirring for 9-18 h at 60 °C. After the reaction mixture turned dark black, the reaction was terminated by cooling on ice. Concentration and purification were performed, and gold (Au) nps were synthesized using the ethanol reduction method described above. The sizes of the nps were analyzed by electron microscopy (TEMEDX; JEM 2000 FX, JEOL Tokyo, Japan) at the Research Laboratory for High Voltage Electron Microscopy (the HVEM Laboratory), Kyushu University. About 100 nps were chosen at random from the image and then used to obtain a measure of the distribution and average particle size of the synthetic nps. 2.2.2. ESR Analysis of O2 · --ScaVenging ActiVity. Production of O2 · - via the hypoxanthine-xanthine oxidase (HPX-XOD) and KO2 systems was confirmed using the ESR (JES-FR30, JEOL, Tokyo, Japan) spin-trapping method with DMPO as described by Mitsuta.41,42 The O2 · --scavenging activity of Pt nps was evaluated by determining the second-order rate constant against O2 · -. The reaction mixture (200 µL) used to measure DMPO-O2 · - spin adducts specific to O2 · - contained 0.2 mM hypoxanthine (HPX), 100 µM DETAPAC, 40 mM sodium phosphate buffer (pH 7.8), 20 U/L XOD, 0.50 M DMPO, and 150 µL of sample. The reaction mixture (198 µL) without XOD solution was mixed in a tube, and then 2 µL of the XOD solution was added into the tube and immediately stirred using a vortex mixer. After 60 s, the signal intensity was measured in an aqueous quartz flat cell (inner size 60 mm × 10 mm × 0.31 mm, Labotec Co., Tokyo, Japan). Under these conditions, DMPO (0.45 M) traps almost all of the O2 · -, and thus, the spontaneous dismutation can be ignored (Supporting Information Figure 1A). ESR analysis using DEPMPO was performed according to a previously reported procedure.43 Data acquisition parameters were as follows: magnetic field, 336 ( 10 mT; microwave power, 8 mW; modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; microwave frequency, 9.4271 GHz; sweep time, 2 min; and time constant, 0.1 s. For the (38) Hirai, H.; Nakano, Y.; Toshima, N. J. Macromol. Sci. Chem. 1979, A13, 727–750. (39) Turkevich, J.; Miner, R. S.; Babenkova, L. J. Phys. Chem. 1986, 90, 4765–4767. (40) Wang, Y.; Ren, J. W.; Deng, K.; Gui, L. L.; Tang, Y. Q. Chem. Mater. 2000, 12, 1622–1627. (41) Mitsuta, K.; Mizuta, Y.; Kohno, M.; Hiramatsu, M.; Mori, A. Bull. Chem. Soc. Jpn. 1990, 63, 187–191. (42) Mitsuta, K.; Hiramatsu, M.; Ohyanishiguchi, H.; Kamada, H.; Fujii, K. Bull. Chem. Soc. Jpn. 1994, 67, 529–538. (43) Va´squez-Vivar, J.; Whitsett, J.; Marta´sek, P.; Hogg, N.; Kalyanaraman, B. Free Radical Biol. Med. 2001, 31, 975–985.

7356 Langmuir, Vol. 24, No. 14, 2008 value of VDMPO-O2 · -/VSA, rates of reaction of DMPO-O2 · - (VDMPOand O2 · - (VSA) of 0.2 or less and 0.8 or greater were excluded when calculating rate constants. The intensity of each radical was taken at the height of the Mn2+ signal, and concentrations were determined by double integration of the ESR signal using TEMPOL as a standard. Fitting of all the experimental data was carried out using an exponential decay equation included in Sigma Plot 8.0 for Windows (SPSS Inc. Chicago, IL). Measurements of XOD activity by uric acid formation were performed as previously reported.44 2.2.3. ESR Analysis of · OH-ScaVenging ActiVity. We used two methods for an · OH production system for analyzing · OHscavenging activity: a Fenton reaction system and a UV/H2O2 system. The typical procedure for using a Fenton system is as follows: the standard reaction mixture (200 µL) contained 50 µM FeSO4, 40 mM phosphate buffer (pH 7.8), 178 mM DMPO, 100 µM H2O2, and sample. The reaction mixture containing all the reactants except the H2O2 solution was prepared in a tube. A volume of 10 µL of 10 mM H2O2 solution was added into the tube, and the ESR intensity was measured after 60 s. The change in the intensity in the presence of sample was measured in the Fenton reaction system to evaluate the scavenging activity of · OH. The procedure for detecting competition kinetics using the UV/H2O2 system was as follows: the reaction mixture (standard) contained 40 mM phosphate buffer (pH 7.4), 178 mM DMPO, 1 M H2O2, and sample. The intensity of the reaction mixture except for the H2O2 solution was prepared in the tube. Ultraviolet ray irradiation was executed after adding H2O2 solution into the flat cell, and the intensity was measured after 60 s. The degree of UV irradiation was adjusted so the production of · OH was 1.4 µM at 60 s. The data acquisition parameters were as follows: magnetic field, 336 ( 10 mT; microwave power, 8 mW; modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; microwave frequency, 9.4271 GHz; sweep time, 2 min; and time constant, 0.03 s. 2.2.4. Cell Culture, Cytotoxicity, and Cellular Uptake. Normal human cell lines including human diploid embryonic lung fibroblasts (TIG-1; JCRB0501), human diploid fibroblasts (WI-38; IFO50075), and human diploid embryonic lung cell lines (MRC-5; JCRB9008); cervical carcinoma cells (HeLa; JCRB9004); and human hepatocellular carcinoma cell lines (HepG2; JCRB1054) were obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan). Cells were cultured in MEM medium supplemented with 10% FBS at 37 °C in a 95% air, 5% CO2 atmosphere. Cells were seeded (2.5 × 104 cells/cm2) in 24- or 96-well plates. Cultures were incubated with different concentrations of Pt nps for 24 h. Untreated cultures were included as controls. Cytotoxicity was determined using the WST-1 assay and reported as the percent (%) reduction of absorption at 450 nm of treated versus untreated control cultures. The cellular uptake measurement for Pt nps was done via a modified dry ash method.45 Briefly, cells were seeded (1 × 105 cells) in 90 mm dishes. Cells were then rinsed three times and recovered after incubation with Pt nps. Samples were dried in a forced air drying oven at 75-180 °C. After drying, samples were placed in an electrical furnace and the temperature was gradually increased (100 °C per h) to 700 °C. Dry ashing was conducted overnight (until a gray to white powder was observed). After cooling, the ash was dissolved in 5 mL of aqua regalis, digested on a hotplate for 5 h, cooled, and then brought to a 2 mL volume with 0.1% (v/v) HNO3. The concentration of Pt nps was determined by ICP-MS. 2.2.5. Intracellular ROS-ScaVenging ActiVities by Flow Cytometry. The amounts of intracellular ROS, and of intracellular H2O2 and O2 · - in particular, were determined using DCFH-DA46 and HEt.47 Cells were precultured for 60 min in a Ca2+, Mg2+-free HBSS buffer with a given amount of Pt nps. After incubation, H2O2 and HPX-XOD were added to the MEM medium, or irradiation was performed by UVA treatment (Cross-linker model NCL-100, Upland, O2 · - )

(44) Nukatsuka, M.; Sakurai, H.; Kawada, J. Biochem. Biophys. Res. Commun. 1989, 165, 278–283. (45) Hall, G. E. M.; Rencz, A. N.; Maclaurin Rencz, A. I. J. Geochem. Explor. 1991, 41, 291–307. (46) LeBel, C. P.; Ishiropoulos, H.; Bondy, S. C. Chem. Res. Toxicol. 1992, 5, 227–231. (47) Zhao, H.; Kalivendi, S.; Zhang, H.; Joseph, J.; Nithipatikom, K.; VasquezVivar, J.; Kalyanaraman, B. Free Radical Biol. Med. 2003, 34, 1359–1368.

Hamasaki et al. CA) in HBSS and cells were incubated for 60 min. The energy of UVA used in the experiments was 50 kJ/m2. The distance between the UV lamps and the surface of the cells was approximately 2 cm. Cells were then rinsed with HBSS, and 5 µM DCFH-DA was added. Cells were then incubated for 10 min, or 3 µM HEt was added and then cells were incubated for 30 min. After resuspending in HBSS, the intracellular redox state of the cells was analyzed immediately using a flow cytometer (EPICS XL ADC system, Beckman Coulter, Inc., CA). Measurements of intracellular O2 · - using HEt was performed as previously reported.47 2.2.6. Expression of Data and Statistics. All measurements were taken at least three times. Data are presented as mean ( SD, and p values were calculated using the Student’s t-test and analysis of variance (ANOVA) where appropriate.

3. Results 3.1. Preparation of Pt nps. The investigation was initiated by synthesizing Pt nps of the desired sizes in the presence or absence of stabilizers, as Pt nps in the specific size ranges appropriate for this study are not commercially available. A modified ethanol reduction method using Tween 80 as a stabilizer was used to synthesize smaller and more stable Pt nps. Using this method, nps were prepared within a 1.2-2.6 nm size range, with a main particle size of 2.0 nm, and an average diameter of 1.8 nm, with 93% of particles distributed within a range of 1.5-2.3 nm (Figure 1A). Pt nps with a main particle size of 1 nm, with an average diameter of 1.2 nm, and of which 90% of particles were 0.5-1.5 nm were synthesized using the sodium citrate reduction method (Figure 1B). Pt nps with a main particle size of 3 nm, with an average diameter of 2.4 nm, and of which 85% of particles distributed within a range of 2.2-3.3 nm were synthesized using the same method (Figure 1C). Polymer organic compounds were not used to protect the synthetic Pt nps prepared via the sodium citrate reduction method (Figure 1B and C). Pt nps with a main particle size of 3-5 nm, with an average particle diameter of 4.0 nm, and of which 78% of particles distributed within a range of 3.5-5.0 nm were synthesized using glycerol as a stabilizer as well as a reducing agent (Figure 1D). To compare the catalytic activities of Pt nps and another metal, Au, nps with a main particle size of 6 nm, with an average diameter of 6.1 nm, and of which 82% of particles distributed within a range of 4.5-7.5 nm were synthesized using the modified ethanol reduction method as described above (Figure 1E). Whether the agglomerated Pt nps exert catalytic activity on both in Vitro and in ViVo environments is not known. Agglomeration of Pt nps is known to be caused by factors such as high temperature, high salt concentration, or strong reducing agents.48 Therefore, an autoclaving method was used to create agglomerated Pt nps and confirm the formation of agglomerated Pt nps by visual observation. As expected, dozens of single Pt nps connected to each other in a chainlike form were detected (Figure 1F-H). Autoclaving induced the formation of Pt nps with agglomerated chainlike structures (100 atm, 170 °C). Agglomerated sizes were larger with increased autoclaving times (Figure 1F, 20 min; Figure 1G, 40 min; Figure 1H, 60 min). These agglomerated Pt nps were used to determine if catalytic activity is influenced by agglomeration levels. The purified Pt nps (200-500 mg/L) solutions contained the following impurities: Tween 80, < ∼20 mg/L; H2PtCl6,